0013-7227/90/1266-3028$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 6 Printed in U.S.A.
In Vitro 0-Adrenergic Stimulation of Lymphocytes Induces the Release of Immunoreactive /8-Endorphin ANNEMIEKE KAVELAARS, RUDY E. BALLIEUX, AND COBIJ. HEIJNEN Department of Pediatric Immunology (A.K., C.J.H.), University Hospital for Children and Youth "Het Wilhelmina Kinderziekenhuis", Utrecht, The Netherlands; and Department of Clinical Immunology (R.E.B.), University Hospital, Utrecht, The Netherlands
ABSTRACT. The immune system and the neuroendocrine system have been shown to be functionally interactive. The neuroendocrine system can modulate the immune response and immune mediators can influence the neuroendocrine system. The present paper focuses on the capacity of lymphocytes to produce and secrete neuroendocrine substances. Lymphocytes can secrete the neuropeptide /3-endorphin in response to activation with mitogen or antigen. Moreover, mediators that are involved in the adaptation to stress have also been shown to induce the release of immunoreactive-jS-endorphin by lymphocytes. It is shown here that stimulation of human peripheral blood mononuclear cells with the /3-adrenergic agonist isoprenaline induces /3-endorphin secretion. The effect of isoprenaline can be mimicked by elevation of the intracellular concentration
T
HE IMMUNE system and the central nervous system can interact via shared mediators and receptors (1, 2). It has now been demonstrated that various neuropeptides, e.g. the POMC-derived peptides /3-endorphin and ACTH, can be synthesized by cells of the immune system (1, 2). Production of neuropeptides by lymphoid cells can be stimulated by mitogenic or antigenic activation. Antibody directed against the T cell receptorCD3 complex, Newcastle disease virus, lipopolysaccharide, interleukin-1, or the T cell mitogens phytohaemagglutinin and Concanavalin A (1, 3-5) have been shown to increase neuropeptide secretion by lymphocytes. It is of interest that the immune system does not only produce neuropeptides in response to classical immune stimulation. Mediators that are released after exposure to a stressor, such as CRF, can also trigger neuropeptide secretion by cells of the immune system. Administration of CRF to peripheral blood mononuclear cells (PBMC) in vitro or to intact rats has been shown to induce the secretion of ^-endorphin and ACTH by lymphocytes (57).
of cAMP with forskolin or (Bu)2cAMP. Inhibition of cAMPdependent protein kinase PKA by the antagonist iV-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide abrogates isoprenaline-induced secretion of immunoreactive-/3-endorphin by peripheral blood mononuclear cells. The present data give evidence that, /3-adrenergic activation activation of lymphocytes stimulates the secretion of ir-/3-endorphin via a protein kinase Adependent mechanism. Both j8-adrenergic agonists as well as (3-endorphin have been shown to modulate the immune response. The data presented here are indicative for a role of /3-endorphin in the modulation of the immune response after /3-adrenergic activation. (Endocrinology 126: 3028-3032,1990)
Another important element of the stress reaction is the sympatho-adrenomedullary response. In vivo cells of the immune system are in the immediate vicinity of adrenergic nerve endings in primary and secondary lymphoid organs, e.g. the splenic white pulp, the subcapsular zone of the lymph node, and the cortico-medullary boundary of the thymus (8). Triggering of ^-adrenergic receptors on PBMC (1013) induces a transient rise in the intracellular cAMP concentration. Addition of /3-adrenergic agonists such as catecholamines to lymphocytes has been shown to influence cell proliferation as well as antibody formation (1416). In the present paper we demonstrate that /3-adrenergic stimulation of PBMC can induce production of immunoreactive /?-endorphin (ir-/3-endorphin) by these cells. Furthermore we show evidence that a rise in intracellular cAMP concentration and the subsequent activation of the cAMP-dependent protein kinase A (PKA) mediate the induction of ir-/?-endorphin secretion by PBMC after /3-adrenergic stimulation.
Materials and Methods Received January 19, 1990. Address correspondence and reprint requests to: Annemieke Kavelaars, University Hospital for Children and Youth "Het Wilhelmina Kinderziekenhuis," P.O. Box 18009, 3501 CA Utrecht, The Netherlands.
Isolation of peripheral blood cells The isolation of PBMC, T cells, and non-T cells has been described extensively elsewhere (5). Cells staining positively
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/3-ADRENERGIC ACTIVATION AND /3-ENDORPHIN SECRETION BY LYMPHOID CELLS with the monoclonal antibody Leu M3 (Becton and Dickinson, Mountain View, CA) were separated from the non-T cell fraction by rosetting with ox erythrocytes (Ox E) coated with goat anti-mouse antibody (Tago, Burlingame, CA). This Leu M3positive fraction, which will be referred to here as monocytes, consisted of 92 ± 5% nonspecific esterase-positive cells. To isolate B cells, the monocyte-depleted fraction was incubated with the monoclonal antibody Bl (Coulter Immunology, Hialeah, FL) and rosetted with Ox E coated with goat anti-mouse antibody.
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7OOO
Cell culture In a total volume of 0.5 ml, 107 cells/ml were cultured for 18 h in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 100 U/ml penicillin, 100 Mg/ml streptomycin, 2 mM glutamine, 5 x 10~5 M 2-mercapto-ethanol and 5% heatinactivated fetal calf serum (Hyclone, Logan, UK). Reagents Isoprenaline, forskolin, and (Bu)2cAMP were obtained from Sigma (St. Louis, MO). Rabbit antiserum to /3-endorphin (17) was a gift from Prof. Dr. V. M. Wiegant of the Rudolf Magnus Institute for Pharmacology (Utrecht, the Netherlands).
O
10
For the detection of ir-/3-endorphin secreting cells we used an RPFC assay specific for /3-endorphin described extensively elsewhere (5). Briefly, sheep red blood cells were coated with protein A (Pharmacia, Uppsala, Sweden) and a rabbit anti-/3endorphin serum (SRBC-protein A-antibody). Cultured cells were washed twice and resuspended in Dulbecco's Modified Eagle's medium (GIBCO) supplemented with 15% heat-inactivated SRBC-absorbed horse serum. Cunningham chambers were filled with a suspension of 20% SRBC-protein A-antibody, 2-4 x 106 cells/ml and 5% SRBC-absorbed guinea pig serum as a complement source. After 1-h incubation at 37 C, /3endorphin-RPFC were enumerated using an inverted microscope. Controls consisted of chambers filled with a suspension which contained either heat-inactivated complement or SRBC coated with preimmune serum instead of the antiserum or with a suspension devoid of lymphocytes. The control assays with preimmune serum gave rise to 200 ± 20 RPFC per 106 cells for both unstimulated cells and cells cultured in the presence of isoprenaline.
3.10"7
1 0 * 3 . 1 0 * !•• 3 . 1 0 " 1O" 4
[isoprenalin], M FIG. 1. Effect of the /3-adrenergic agonist isoprenaline on ir-/3-endorphin secretion by human PBMC. PBMC (107/ml) were cultured for 18 h in the presence of various concentrations of isoprenaline and tested for ir-/?-endorphin secretion as described in Materials and Methods. (Representative experiment out of three; data points represent duplicates with SD < 1%). TABLE 1. Effect of propanolol on the isoprenaline-induced secretion of ir-/?-endorphin by PBMC /3E-RPFC/105 cells Propanolol
Reverse plaque-forming cell assay (RPFC)
T
(M)
Control
Isoprenaline (lO"6 M)
0
266 ± 23° 5692 ± 272 2500 ± 103 138 ± 18 10"9 313 ± 23 1026 ± 45 10~ 7 289 ± 17 1037 ± 73 10~ 6 301 ± 25 800 ± 67 /3E-RPFC, /3-endorphin RPFC (see Table 2). PBMC (107/ml) were cultured for 18 h with 10"5 M isoprenaline in the presence of various concentrations of propanolol and were then tested for ir-/3-endorphin secretion. 0 Data points represent duplicates ± SD of one representative experiment out of three.
io- n
number of ir-/3-endorphin-secreting lymphocytes is observed. Higher concentrations of isoprenaline inhibit the response. The response to isoprenaline can be blocked by addition of the /3-adrenergic receptor antagonist propanolol to the cultures (Table 1). The total population of PBMC can be separated into three major subsets of cells: T lymphocytes, B lymphocytes, and monocytes. Subpopulations of PBMC were isolated (see Materials and Methods), cultured with 10"5 M isoprenaline and Results after 18 h tested for ir-/3-endorphin secretion. Every Effect of {3-adrenergic activation on ir-(3-endorphin secre- major subset of PBMC responds to /?-adrenergic stimulation with an increase in the number of ir-/5-endorphin tion by PBMC secreting cells (Table 2). Human PBMC were cultured with different concentrations of the /3-adrenergic agonist isoprenaline and Effect of cAMP accumulation on ir-(3-endorphin secretion tested for ir-/3-endorphin secretion by means of a RPFC by PBMC assay. The results depicted in Fig. 1 demonstrate that 18 h after the addition of 10~7-10~5 M isoprenaline to culThe 0-adrenergic receptor is coupled to adenylate cytures of human PBMC a dose-dependent increase in the clase which implies that triggering of the 0-adrenergic
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/3-ADRENERGIC ACTIVATION AND 0-ENDORPHIN SECRETION BY LYMPHOID CELLS
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Endo.1990 Voll26«No6
TABLE 2. Secretion of ir-/3-endorphin by various subsets of PBMC 0E-RPFC/1O6 cells Cells
Control
Isoprenaline
T cells B cells Monocytes
957 ± 73° 762 ± 58 1750 ± 140
6950 ± 320 5913 ± 307 4320 ± 360
I
7
Purified fractions of T cells, B cells and monocytes (10 /ml) were cultured for 18 h in the presence or absence of isoprenaline (10~5 M) and tested for ir-/3-endorphin secretion by means of a /3E-RPFC assay. 0 Data points represent duplicates ± SD of one representative experiment out of three.
T3
20
10
0 O
O.O1 O.I
1
[db-cAMP], mM
10
0.1
1
10
100
[forskolin],
FIG. 2. Effect of (Bu)2cAMP and forskolin on the secretion of ir-/3endorphin by PBMC. PBMC were cultured for 18 h with various concentrations of either (Bu)2cAMP or forskolin and tested for ir-/3endorphin secretion. (Representative experiment out of three; data points represent duplicates with SD < 7%).
receptor leads to an increase in the intracellular concentration of cAMP in PBMC (10, 18). The effect of isoprenaline on intracellular cAMP concentration can be mimicked by directly activating the catalytic subunit of adenylate cyclase with forskolin or by addition of the cAMP analog (Bu)2cAMP to the cells. Figure 2 shows that addition of either (Bu)2cAMP or forskolin to cultures of human PBMC can stimulate ir-0-endorphin secretion by these cells. The maximal increase in the number of j8-endorphin-RPFC was 20-100 times for both forskolin and (Bu)2cAMP. The kinetics of the response to forskolin, (Bu)2cAMP and isoprenaline are comparable, suggesting that these compounds induce ir-/3endorphin secretion via a shared mechanism (Fig. 3).
3
6
9
12
15
18
21
24
time, hours FIG. 3. Kinetics of the isoprenaline (Bu)2cAMP- or forskolin-induced secretion of ir-/3-endorphin by PBMC. PBMC were cultured for the time periods indicated with 10"6 M isoprenaline (+)> 1 mM (Bu)2cAMP (•), or 1 fiM forskolin (A). After the culture period the cells were washed and assayed for ir-/3-endorphin secretion. (Representative experiment out of two; data points represent duplicates with SD < 1%).
isoquinoline-sulfonamide (H8) (19). We investigated whether the PKA inhibitor H8 can interfere with the isoprenaline-induced secretion of ir-/3-endorphin by PBMC. The results depicted in Fig. 4 clearly demonstrate that H8 efficiently inhibits isoprenaline-induced ir-/3endorphin secretion. The protein kinase C (PKC) inhibitor l-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine(H7) only inhibited the effect of isoprenaline at a 30 times higher concentration. The inhibitory effect of H8 on the isoprenaline-induced secretion on ir-/?-endorphin cannot be attributed to a decrease in cell viability. Recovery of the cells as determined by trypan-blue exclusion was not affected by 18 h of culture with 2.5-40 /uM of H8.
Discussion Effect of inhibition of PKA A rise in intracellular cAMP concentration is known to activate the cAMP-dependent protein kinase PKA. PKA activation has been shown to stimulate the production of various factors by lymphocytes, e.g. immunoglobulins and interleukin-2. The activity of PKA can be blocked by the antagonist JV-[2-[methylamino)ethyl]-5-
The results depicted in the present paper describe a novel pathway for the induction of POMC-peptide secretion by lymphocytes. /?-Adrenergic activation of human PBMC in vitro induces ir-/3-endorphin secretion as determined by a 0-endorphin-RPFC-assay. The results described here were obtained using a polyclonal rabbit anti/3-endorphin serum recognizing predominantly the mid-
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/3-ADRENERGIC ACTIVATION AND /3-ENDORPHIN SECRETION BY LYMPHOID CELLS
4000
I
-•-•x
3000
I1000
10
20
30
40
[inhibitor], \M FIG. 4. The effect of H8 and H7 on isoprenaline-induced ir-/3-endorphin secretion. PBMC were cultured for 18 h with 10~5 M isoprenaline and different concentrations of the PKA inhibitor H8 (•) or the PKC inhibitor H7 (•). The number of ir-/3-endorphin-secreting cells was determined. (Representative experiment out of three; data points represent duplicates with SD < 7%).
portion of /3-endorphin (17). Comparable results were found when a mouse monoclonal antibody (gift of Dr. G. Riethmiiller, Institiit fur Immunologie, Universitat Miinchen, Munich, West Germany) against the N-terminal part of /3-endorphin was used (results not shown). These data support the hypothesis that the immunoreactivity is caused by /3-endorphin. Expression of /3-adrenergic receptors on lymphocytes is well documented. B cells, T cells as well as monocytes express /3-adrenergic receptors that are coupled to adenylate cyclase (10-13). The effect of 0-adrenergic activation on ir-/3-endorphin secretion by the different subsets of PBMC can be mimicked with forskolin or (Bu)2cAMP (results not shown) suggesting that in all subsets the mechanism for induction of /?-endorphin secretion is the same. (Bu)2cAMP and forskolin are more potent inducers of /3-endorphin secretion than isoprenaline (Figs. 1 and 2). Apparently isoprenaline does not fully activate cells that are capable of ir-/3-endorphin secretion after a rise in cAMP level. The fact that H8 efficiently inhibits ir-/3-endorphin secretion after 0-adrenergic activation indicates that a PKA-dependent pathway is involved. We have evidence that PKA activation is not the only
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mechanism by which PBMC can be stimulated to secrete jS-endorphin. Secretion of ir-/3-endorphin by PBMC in response to stimulation with, e.g. an antibody directed against the CD3 complex can be blocked by the addition of the PKC antagonist H7 exclusively (Kavelaars, A., R. E. Ballieux, and C. J. Heijnen, submitted). Moreover, it has been shown by Farrar et al. (1) that stimulation of PKC with a phorbol-ester induces the expression of POMC-mRNA in lymphocytes. These data suggest that both PKA and PKC can stimulate the secretion of POMC peptides by lymphoid cells in vitro. The regulation of the secretion of POMC peptides in the immune system is to a certain extent comparable to the situation in the pituitary. POMC gene expression as well as peptide production in pituitary cells can be stimulated by /3-adrenergic activation (9). /3-Adrenergic agonists stimulate the release of POMC-derived peptides from pituitary cells in culture through elevation of intracellular cAMP and activation of PKA (20). In the pituitary tumor cell line AtT 20, inhibition of PKA blocks the effect of /3-adrenergic agonists as well as of cAMP analogs on POMC-peptide secretion (21). A second pathway for the induction of POMC peptide secretion by the pituitary is via activation of PKC. In contrast to the effect of PKC activation in lymphocytes, activation of PKC in pituitary cells only induces POMC peptide secretion without any increase in POMC gene expression (1, 22). As is mentioned in the introduction /3-adrenergic agonists can modulate immune functioning, presumably via a direct effect on the intracellular concentration of cAMP. The data presented in this report demonstrate that the rise in cAMP concentration after triggering the /3-adrenergic receptor induces the secretion of ir-/?-endorphin by PBMC. /3-Endorphin has been shown to be a potent immunomodulatory peptide (23-29). Therefore, the possibility should be considered that the effect of 0adrenergic activation on the immune response is mediated directly via an effect of cAMP on the lymphocyte as well as indirectly via the induction of secretion of the immunomodulatory substance /3-endorphin.
References 1. Farrar WL, Hill JM, Havel-Belland A, Vinocour M 1987 The immune logical brain. Immunol Rev 100:361 2. Weigant DA, Blalock JE 1987 Interactions between the neuroendocrine and immune systems: common hormones and receptors. Immunol Rev 100:79 3. Westly HJ, Kleiss AJ, Kelley KW, Wong PKY, Yuen PH 1986 Newcastle disease virus-infected splenocytes express the pro-opiomelanocortin gene. J Exp Med 163:1589 4. Harbour-McMenamin D, Smith EM, Blalock JE 1985 Bacterial lipopolysaccharide induction of leukocyte-derived corticotropin and endorphins. Infect Immun 48:813 5. Kavelaars A, Ballieux RE, Heijnen CJ 1989 The role of interleukin1 in the CRF- and AVP-induced secretion of ir-/?-endorphin by human peripheral blood mononuclear cells. J Immunol 142:2338
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/3-ADRENERGIC ACTIVATION AND /3-ENDORPHIN SECRETION BY LYMPHOID CELLS
6. Smith EM, Morrill AC, Meyer WJ, Blalock JE 1986 Corticotropin releasing factor induction of leukocyte-derived immunoreactive ACTH and endorphins. Nature 321:881 7. Kavelaars A, Berkenbosch F, Croiset G, Ballieux RE, Heijnen CJ 1990 Induction of /3-endorphin secretion by lymphocytes after subcutaneous administration of CRF. Endocrinology 126:759 8. Felten DL, Felten SY, Bellinger DL, Carlson SL, Ackerman KD, Madden KS, Olschowki JA, Livnat S 1987 Noradrenergic sympathetic neural interactions with the immune system: structure and function. Immunol Rev 110:225 9. Lundblad JR, Roberts JL 1988 Regulation of proopiomelanocortin gene expression in pituitary. Endocr Rev 9:135 10. Stahelin M, Muller P, Portenier M, Harris AW 1985 Adrenergic receptors and adenylate cyclase activity in murine lymphoid cell lines. J Cyclic Nucleotide Protein Phosphoryl Res 10:55 11. Pochet R, Delespese G, Gausset PW, Collet HD 1979 Distribution of beta-adrenergic receptors on human lymphocytes subpopulations. Clin Exp Immunol 38:578 12. Khan MN, Sansoni P, Silverman ED, Engleman EG, Melmaon KL 1986 Beta-adrenergic receptors on human suppressor, helper and cytolytic lymphocytes. Biochem Pharmacol 35:1137 13. Bisphoric NH, Cohen HJ, Lefkowitz RJ 1980 Beta-adrenergic receptors in lymphocytes subpopulations. J Allergy Clin Immunol 65:29 14. Hallengren BA, Forsgren A, Melander A 1982 Influence of /3adrenergic blocking agents on lymphocyte function in vitro. Br J Clin Pharmacol 13:543 15. Sanders VM, Munson AE 1985 Norepinephrine and the antibody response. Pharmacol Rev 37:229 16. Feldman RD, Hunningcake GW, McArdle WL 1987 /3-Adrenergic receptor-mediated suppression of interleukin-2 receptors in human lymphocytes. J Immunol 139:3355 17. Barna I, Sweep CGJ, Veldhuis HD, Wiegant VM 1988 Differential effects of cisterna magna cannulation on 0-endorphin levels in rat plasma and cerebrospinal fluid. Acta Endocrinol (Copenh) 117:517 18. Benovic JL, Bouvier M, Caron MG, Lefkowitz RJ 1988 Regulation of adenylyl cyclase coupled /3-adrenergic receptors. Annu Rev Cell Biol 4:405
Endo • 1990 Vol 126 • No 6
19. Hidaka H, Ignagaki M, Kawamoto S, Sasaki Y 1984 Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5836 20. Miyazki K, Reisine T, Kebabian JW 1984 Adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in rodent pituitary tissue: possible role in cAMP-dependent hormone secretion. Endocrinology 115:1933 21. Reisine T, Rougon G, Barbet J, Affolter HU 1985 Corticotropinreleasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes. Proc Natl Acad Sci USA 82:8261 22. Suda T, Tozawa F, Ushiyama T, Tomori N, Sumitomo T, Nakagami Y, Yamada M, Demura H, Shizume K 1989 Effects of protein kinase-C-related adrenocorticotropin secretagogues and Interleukin-1 on proopiomelanocortin gene expression in rat anterior pituitary cells. Endocrinology 124:1444 23. Fischer EG, Falke NE 1984 /3-endorphin modulates immune functions. Psychother Psychosom 42:195 24. Gilman SC, Schwarz JM, Milner RJ, Bloom FE, Feldman JD 1982 Beta-endorphin enhances lymphocyte proliferative responses. Proc Natl Acad Sci USA 79:4226 25. Heijnen CJ, Bevers C, Kavelaars A, Ballieux RE 1985 Effect of aendorphin on the antigen-induced primary antibody. J Immunol 136:213 26. Heijnen CJ, Zijlstra J, Kavelaars A, Croiset G, Ballieux RE 1987 Modulation of the immune response by POMC-derived peptides: I. Influence on proliferation of human lymphocytes. Brain Behav Immun 1:284 27. Kay N, Allen J, Morley JE 1984 Endorphins stimulate normal human peripheral blood lymphocyte natural killer activity. Life Sci 35:53 28. Brown SL, Van Epps DE 1986 Opioid peptides modulate production of interferon-7 by human mononuclear cells. Cell Immunol 103:19 29. Van Epps DE, Saland L 1984 /3-endorphin and met-enkephalin stimulate human peripheral blood mononuclear cell chemotaxis. J Immunol 132:3046
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Vol. 126, No. 6 Printed in U.S.A.
In Vitro 0-Adrenergic Stimulation of Lymphocytes Induces the Release of Immunoreactive /8-Endorphin ANNEMIEKE KAVELAARS, RUDY E. BALLIEUX, AND COBIJ. HEIJNEN Department of Pediatric Immunology (A.K., C.J.H.), University Hospital for Children and Youth "Het Wilhelmina Kinderziekenhuis", Utrecht, The Netherlands; and Department of Clinical Immunology (R.E.B.), University Hospital, Utrecht, The Netherlands
ABSTRACT. The immune system and the neuroendocrine system have been shown to be functionally interactive. The neuroendocrine system can modulate the immune response and immune mediators can influence the neuroendocrine system. The present paper focuses on the capacity of lymphocytes to produce and secrete neuroendocrine substances. Lymphocytes can secrete the neuropeptide /3-endorphin in response to activation with mitogen or antigen. Moreover, mediators that are involved in the adaptation to stress have also been shown to induce the release of immunoreactive-jS-endorphin by lymphocytes. It is shown here that stimulation of human peripheral blood mononuclear cells with the /3-adrenergic agonist isoprenaline induces /3-endorphin secretion. The effect of isoprenaline can be mimicked by elevation of the intracellular concentration
T
HE IMMUNE system and the central nervous system can interact via shared mediators and receptors (1, 2). It has now been demonstrated that various neuropeptides, e.g. the POMC-derived peptides /3-endorphin and ACTH, can be synthesized by cells of the immune system (1, 2). Production of neuropeptides by lymphoid cells can be stimulated by mitogenic or antigenic activation. Antibody directed against the T cell receptorCD3 complex, Newcastle disease virus, lipopolysaccharide, interleukin-1, or the T cell mitogens phytohaemagglutinin and Concanavalin A (1, 3-5) have been shown to increase neuropeptide secretion by lymphocytes. It is of interest that the immune system does not only produce neuropeptides in response to classical immune stimulation. Mediators that are released after exposure to a stressor, such as CRF, can also trigger neuropeptide secretion by cells of the immune system. Administration of CRF to peripheral blood mononuclear cells (PBMC) in vitro or to intact rats has been shown to induce the secretion of ^-endorphin and ACTH by lymphocytes (57).
of cAMP with forskolin or (Bu)2cAMP. Inhibition of cAMPdependent protein kinase PKA by the antagonist iV-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide abrogates isoprenaline-induced secretion of immunoreactive-/3-endorphin by peripheral blood mononuclear cells. The present data give evidence that, /3-adrenergic activation activation of lymphocytes stimulates the secretion of ir-/3-endorphin via a protein kinase Adependent mechanism. Both j8-adrenergic agonists as well as (3-endorphin have been shown to modulate the immune response. The data presented here are indicative for a role of /3-endorphin in the modulation of the immune response after /3-adrenergic activation. (Endocrinology 126: 3028-3032,1990)
Another important element of the stress reaction is the sympatho-adrenomedullary response. In vivo cells of the immune system are in the immediate vicinity of adrenergic nerve endings in primary and secondary lymphoid organs, e.g. the splenic white pulp, the subcapsular zone of the lymph node, and the cortico-medullary boundary of the thymus (8). Triggering of ^-adrenergic receptors on PBMC (1013) induces a transient rise in the intracellular cAMP concentration. Addition of /3-adrenergic agonists such as catecholamines to lymphocytes has been shown to influence cell proliferation as well as antibody formation (1416). In the present paper we demonstrate that /3-adrenergic stimulation of PBMC can induce production of immunoreactive /?-endorphin (ir-/3-endorphin) by these cells. Furthermore we show evidence that a rise in intracellular cAMP concentration and the subsequent activation of the cAMP-dependent protein kinase A (PKA) mediate the induction of ir-/?-endorphin secretion by PBMC after /3-adrenergic stimulation.
Materials and Methods Received January 19, 1990. Address correspondence and reprint requests to: Annemieke Kavelaars, University Hospital for Children and Youth "Het Wilhelmina Kinderziekenhuis," P.O. Box 18009, 3501 CA Utrecht, The Netherlands.
Isolation of peripheral blood cells The isolation of PBMC, T cells, and non-T cells has been described extensively elsewhere (5). Cells staining positively
3028
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2015. at 01:04 For personal use only. No other uses without permission. . All rights reserved.
/3-ADRENERGIC ACTIVATION AND /3-ENDORPHIN SECRETION BY LYMPHOID CELLS with the monoclonal antibody Leu M3 (Becton and Dickinson, Mountain View, CA) were separated from the non-T cell fraction by rosetting with ox erythrocytes (Ox E) coated with goat anti-mouse antibody (Tago, Burlingame, CA). This Leu M3positive fraction, which will be referred to here as monocytes, consisted of 92 ± 5% nonspecific esterase-positive cells. To isolate B cells, the monocyte-depleted fraction was incubated with the monoclonal antibody Bl (Coulter Immunology, Hialeah, FL) and rosetted with Ox E coated with goat anti-mouse antibody.
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Cell culture In a total volume of 0.5 ml, 107 cells/ml were cultured for 18 h in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 100 U/ml penicillin, 100 Mg/ml streptomycin, 2 mM glutamine, 5 x 10~5 M 2-mercapto-ethanol and 5% heatinactivated fetal calf serum (Hyclone, Logan, UK). Reagents Isoprenaline, forskolin, and (Bu)2cAMP were obtained from Sigma (St. Louis, MO). Rabbit antiserum to /3-endorphin (17) was a gift from Prof. Dr. V. M. Wiegant of the Rudolf Magnus Institute for Pharmacology (Utrecht, the Netherlands).
O
10
For the detection of ir-/3-endorphin secreting cells we used an RPFC assay specific for /3-endorphin described extensively elsewhere (5). Briefly, sheep red blood cells were coated with protein A (Pharmacia, Uppsala, Sweden) and a rabbit anti-/3endorphin serum (SRBC-protein A-antibody). Cultured cells were washed twice and resuspended in Dulbecco's Modified Eagle's medium (GIBCO) supplemented with 15% heat-inactivated SRBC-absorbed horse serum. Cunningham chambers were filled with a suspension of 20% SRBC-protein A-antibody, 2-4 x 106 cells/ml and 5% SRBC-absorbed guinea pig serum as a complement source. After 1-h incubation at 37 C, /3endorphin-RPFC were enumerated using an inverted microscope. Controls consisted of chambers filled with a suspension which contained either heat-inactivated complement or SRBC coated with preimmune serum instead of the antiserum or with a suspension devoid of lymphocytes. The control assays with preimmune serum gave rise to 200 ± 20 RPFC per 106 cells for both unstimulated cells and cells cultured in the presence of isoprenaline.
3.10"7
1 0 * 3 . 1 0 * !•• 3 . 1 0 " 1O" 4
[isoprenalin], M FIG. 1. Effect of the /3-adrenergic agonist isoprenaline on ir-/3-endorphin secretion by human PBMC. PBMC (107/ml) were cultured for 18 h in the presence of various concentrations of isoprenaline and tested for ir-/?-endorphin secretion as described in Materials and Methods. (Representative experiment out of three; data points represent duplicates with SD < 1%). TABLE 1. Effect of propanolol on the isoprenaline-induced secretion of ir-/?-endorphin by PBMC /3E-RPFC/105 cells Propanolol
Reverse plaque-forming cell assay (RPFC)
T
(M)
Control
Isoprenaline (lO"6 M)
0
266 ± 23° 5692 ± 272 2500 ± 103 138 ± 18 10"9 313 ± 23 1026 ± 45 10~ 7 289 ± 17 1037 ± 73 10~ 6 301 ± 25 800 ± 67 /3E-RPFC, /3-endorphin RPFC (see Table 2). PBMC (107/ml) were cultured for 18 h with 10"5 M isoprenaline in the presence of various concentrations of propanolol and were then tested for ir-/3-endorphin secretion. 0 Data points represent duplicates ± SD of one representative experiment out of three.
io- n
number of ir-/3-endorphin-secreting lymphocytes is observed. Higher concentrations of isoprenaline inhibit the response. The response to isoprenaline can be blocked by addition of the /3-adrenergic receptor antagonist propanolol to the cultures (Table 1). The total population of PBMC can be separated into three major subsets of cells: T lymphocytes, B lymphocytes, and monocytes. Subpopulations of PBMC were isolated (see Materials and Methods), cultured with 10"5 M isoprenaline and Results after 18 h tested for ir-/3-endorphin secretion. Every Effect of {3-adrenergic activation on ir-(3-endorphin secre- major subset of PBMC responds to /?-adrenergic stimulation with an increase in the number of ir-/5-endorphin tion by PBMC secreting cells (Table 2). Human PBMC were cultured with different concentrations of the /3-adrenergic agonist isoprenaline and Effect of cAMP accumulation on ir-(3-endorphin secretion tested for ir-/3-endorphin secretion by means of a RPFC by PBMC assay. The results depicted in Fig. 1 demonstrate that 18 h after the addition of 10~7-10~5 M isoprenaline to culThe 0-adrenergic receptor is coupled to adenylate cytures of human PBMC a dose-dependent increase in the clase which implies that triggering of the 0-adrenergic
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/3-ADRENERGIC ACTIVATION AND 0-ENDORPHIN SECRETION BY LYMPHOID CELLS
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Endo.1990 Voll26«No6
TABLE 2. Secretion of ir-/3-endorphin by various subsets of PBMC 0E-RPFC/1O6 cells Cells
Control
Isoprenaline
T cells B cells Monocytes
957 ± 73° 762 ± 58 1750 ± 140
6950 ± 320 5913 ± 307 4320 ± 360
I
7
Purified fractions of T cells, B cells and monocytes (10 /ml) were cultured for 18 h in the presence or absence of isoprenaline (10~5 M) and tested for ir-/3-endorphin secretion by means of a /3E-RPFC assay. 0 Data points represent duplicates ± SD of one representative experiment out of three.
T3
20
10
0 O
O.O1 O.I
1
[db-cAMP], mM
10
0.1
1
10
100
[forskolin],
FIG. 2. Effect of (Bu)2cAMP and forskolin on the secretion of ir-/3endorphin by PBMC. PBMC were cultured for 18 h with various concentrations of either (Bu)2cAMP or forskolin and tested for ir-/3endorphin secretion. (Representative experiment out of three; data points represent duplicates with SD < 7%).
receptor leads to an increase in the intracellular concentration of cAMP in PBMC (10, 18). The effect of isoprenaline on intracellular cAMP concentration can be mimicked by directly activating the catalytic subunit of adenylate cyclase with forskolin or by addition of the cAMP analog (Bu)2cAMP to the cells. Figure 2 shows that addition of either (Bu)2cAMP or forskolin to cultures of human PBMC can stimulate ir-0-endorphin secretion by these cells. The maximal increase in the number of j8-endorphin-RPFC was 20-100 times for both forskolin and (Bu)2cAMP. The kinetics of the response to forskolin, (Bu)2cAMP and isoprenaline are comparable, suggesting that these compounds induce ir-/3endorphin secretion via a shared mechanism (Fig. 3).
3
6
9
12
15
18
21
24
time, hours FIG. 3. Kinetics of the isoprenaline (Bu)2cAMP- or forskolin-induced secretion of ir-/3-endorphin by PBMC. PBMC were cultured for the time periods indicated with 10"6 M isoprenaline (+)> 1 mM (Bu)2cAMP (•), or 1 fiM forskolin (A). After the culture period the cells were washed and assayed for ir-/3-endorphin secretion. (Representative experiment out of two; data points represent duplicates with SD < 1%).
isoquinoline-sulfonamide (H8) (19). We investigated whether the PKA inhibitor H8 can interfere with the isoprenaline-induced secretion of ir-/3-endorphin by PBMC. The results depicted in Fig. 4 clearly demonstrate that H8 efficiently inhibits isoprenaline-induced ir-/3endorphin secretion. The protein kinase C (PKC) inhibitor l-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine(H7) only inhibited the effect of isoprenaline at a 30 times higher concentration. The inhibitory effect of H8 on the isoprenaline-induced secretion on ir-/?-endorphin cannot be attributed to a decrease in cell viability. Recovery of the cells as determined by trypan-blue exclusion was not affected by 18 h of culture with 2.5-40 /uM of H8.
Discussion Effect of inhibition of PKA A rise in intracellular cAMP concentration is known to activate the cAMP-dependent protein kinase PKA. PKA activation has been shown to stimulate the production of various factors by lymphocytes, e.g. immunoglobulins and interleukin-2. The activity of PKA can be blocked by the antagonist JV-[2-[methylamino)ethyl]-5-
The results depicted in the present paper describe a novel pathway for the induction of POMC-peptide secretion by lymphocytes. /?-Adrenergic activation of human PBMC in vitro induces ir-/3-endorphin secretion as determined by a 0-endorphin-RPFC-assay. The results described here were obtained using a polyclonal rabbit anti/3-endorphin serum recognizing predominantly the mid-
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/3-ADRENERGIC ACTIVATION AND /3-ENDORPHIN SECRETION BY LYMPHOID CELLS
4000
I
-•-•x
3000
I1000
10
20
30
40
[inhibitor], \M FIG. 4. The effect of H8 and H7 on isoprenaline-induced ir-/3-endorphin secretion. PBMC were cultured for 18 h with 10~5 M isoprenaline and different concentrations of the PKA inhibitor H8 (•) or the PKC inhibitor H7 (•). The number of ir-/3-endorphin-secreting cells was determined. (Representative experiment out of three; data points represent duplicates with SD < 7%).
portion of /3-endorphin (17). Comparable results were found when a mouse monoclonal antibody (gift of Dr. G. Riethmiiller, Institiit fur Immunologie, Universitat Miinchen, Munich, West Germany) against the N-terminal part of /3-endorphin was used (results not shown). These data support the hypothesis that the immunoreactivity is caused by /3-endorphin. Expression of /3-adrenergic receptors on lymphocytes is well documented. B cells, T cells as well as monocytes express /3-adrenergic receptors that are coupled to adenylate cyclase (10-13). The effect of 0-adrenergic activation on ir-/3-endorphin secretion by the different subsets of PBMC can be mimicked with forskolin or (Bu)2cAMP (results not shown) suggesting that in all subsets the mechanism for induction of /?-endorphin secretion is the same. (Bu)2cAMP and forskolin are more potent inducers of /3-endorphin secretion than isoprenaline (Figs. 1 and 2). Apparently isoprenaline does not fully activate cells that are capable of ir-/3-endorphin secretion after a rise in cAMP level. The fact that H8 efficiently inhibits ir-/3-endorphin secretion after 0-adrenergic activation indicates that a PKA-dependent pathway is involved. We have evidence that PKA activation is not the only
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mechanism by which PBMC can be stimulated to secrete jS-endorphin. Secretion of ir-/3-endorphin by PBMC in response to stimulation with, e.g. an antibody directed against the CD3 complex can be blocked by the addition of the PKC antagonist H7 exclusively (Kavelaars, A., R. E. Ballieux, and C. J. Heijnen, submitted). Moreover, it has been shown by Farrar et al. (1) that stimulation of PKC with a phorbol-ester induces the expression of POMC-mRNA in lymphocytes. These data suggest that both PKA and PKC can stimulate the secretion of POMC peptides by lymphoid cells in vitro. The regulation of the secretion of POMC peptides in the immune system is to a certain extent comparable to the situation in the pituitary. POMC gene expression as well as peptide production in pituitary cells can be stimulated by /3-adrenergic activation (9). /3-Adrenergic agonists stimulate the release of POMC-derived peptides from pituitary cells in culture through elevation of intracellular cAMP and activation of PKA (20). In the pituitary tumor cell line AtT 20, inhibition of PKA blocks the effect of /3-adrenergic agonists as well as of cAMP analogs on POMC-peptide secretion (21). A second pathway for the induction of POMC peptide secretion by the pituitary is via activation of PKC. In contrast to the effect of PKC activation in lymphocytes, activation of PKC in pituitary cells only induces POMC peptide secretion without any increase in POMC gene expression (1, 22). As is mentioned in the introduction /3-adrenergic agonists can modulate immune functioning, presumably via a direct effect on the intracellular concentration of cAMP. The data presented in this report demonstrate that the rise in cAMP concentration after triggering the /3-adrenergic receptor induces the secretion of ir-/?-endorphin by PBMC. /3-Endorphin has been shown to be a potent immunomodulatory peptide (23-29). Therefore, the possibility should be considered that the effect of 0adrenergic activation on the immune response is mediated directly via an effect of cAMP on the lymphocyte as well as indirectly via the induction of secretion of the immunomodulatory substance /3-endorphin.
References 1. Farrar WL, Hill JM, Havel-Belland A, Vinocour M 1987 The immune logical brain. Immunol Rev 100:361 2. Weigant DA, Blalock JE 1987 Interactions between the neuroendocrine and immune systems: common hormones and receptors. Immunol Rev 100:79 3. Westly HJ, Kleiss AJ, Kelley KW, Wong PKY, Yuen PH 1986 Newcastle disease virus-infected splenocytes express the pro-opiomelanocortin gene. J Exp Med 163:1589 4. Harbour-McMenamin D, Smith EM, Blalock JE 1985 Bacterial lipopolysaccharide induction of leukocyte-derived corticotropin and endorphins. Infect Immun 48:813 5. Kavelaars A, Ballieux RE, Heijnen CJ 1989 The role of interleukin1 in the CRF- and AVP-induced secretion of ir-/?-endorphin by human peripheral blood mononuclear cells. J Immunol 142:2338
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/3-ADRENERGIC ACTIVATION AND /3-ENDORPHIN SECRETION BY LYMPHOID CELLS
6. Smith EM, Morrill AC, Meyer WJ, Blalock JE 1986 Corticotropin releasing factor induction of leukocyte-derived immunoreactive ACTH and endorphins. Nature 321:881 7. Kavelaars A, Berkenbosch F, Croiset G, Ballieux RE, Heijnen CJ 1990 Induction of /3-endorphin secretion by lymphocytes after subcutaneous administration of CRF. Endocrinology 126:759 8. Felten DL, Felten SY, Bellinger DL, Carlson SL, Ackerman KD, Madden KS, Olschowki JA, Livnat S 1987 Noradrenergic sympathetic neural interactions with the immune system: structure and function. Immunol Rev 110:225 9. Lundblad JR, Roberts JL 1988 Regulation of proopiomelanocortin gene expression in pituitary. Endocr Rev 9:135 10. Stahelin M, Muller P, Portenier M, Harris AW 1985 Adrenergic receptors and adenylate cyclase activity in murine lymphoid cell lines. J Cyclic Nucleotide Protein Phosphoryl Res 10:55 11. Pochet R, Delespese G, Gausset PW, Collet HD 1979 Distribution of beta-adrenergic receptors on human lymphocytes subpopulations. Clin Exp Immunol 38:578 12. Khan MN, Sansoni P, Silverman ED, Engleman EG, Melmaon KL 1986 Beta-adrenergic receptors on human suppressor, helper and cytolytic lymphocytes. Biochem Pharmacol 35:1137 13. Bisphoric NH, Cohen HJ, Lefkowitz RJ 1980 Beta-adrenergic receptors in lymphocytes subpopulations. J Allergy Clin Immunol 65:29 14. Hallengren BA, Forsgren A, Melander A 1982 Influence of /3adrenergic blocking agents on lymphocyte function in vitro. Br J Clin Pharmacol 13:543 15. Sanders VM, Munson AE 1985 Norepinephrine and the antibody response. Pharmacol Rev 37:229 16. Feldman RD, Hunningcake GW, McArdle WL 1987 /3-Adrenergic receptor-mediated suppression of interleukin-2 receptors in human lymphocytes. J Immunol 139:3355 17. Barna I, Sweep CGJ, Veldhuis HD, Wiegant VM 1988 Differential effects of cisterna magna cannulation on 0-endorphin levels in rat plasma and cerebrospinal fluid. Acta Endocrinol (Copenh) 117:517 18. Benovic JL, Bouvier M, Caron MG, Lefkowitz RJ 1988 Regulation of adenylyl cyclase coupled /3-adrenergic receptors. Annu Rev Cell Biol 4:405
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19. Hidaka H, Ignagaki M, Kawamoto S, Sasaki Y 1984 Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5836 20. Miyazki K, Reisine T, Kebabian JW 1984 Adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in rodent pituitary tissue: possible role in cAMP-dependent hormone secretion. Endocrinology 115:1933 21. Reisine T, Rougon G, Barbet J, Affolter HU 1985 Corticotropinreleasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes. Proc Natl Acad Sci USA 82:8261 22. Suda T, Tozawa F, Ushiyama T, Tomori N, Sumitomo T, Nakagami Y, Yamada M, Demura H, Shizume K 1989 Effects of protein kinase-C-related adrenocorticotropin secretagogues and Interleukin-1 on proopiomelanocortin gene expression in rat anterior pituitary cells. Endocrinology 124:1444 23. Fischer EG, Falke NE 1984 /3-endorphin modulates immune functions. Psychother Psychosom 42:195 24. Gilman SC, Schwarz JM, Milner RJ, Bloom FE, Feldman JD 1982 Beta-endorphin enhances lymphocyte proliferative responses. Proc Natl Acad Sci USA 79:4226 25. Heijnen CJ, Bevers C, Kavelaars A, Ballieux RE 1985 Effect of aendorphin on the antigen-induced primary antibody. J Immunol 136:213 26. Heijnen CJ, Zijlstra J, Kavelaars A, Croiset G, Ballieux RE 1987 Modulation of the immune response by POMC-derived peptides: I. Influence on proliferation of human lymphocytes. Brain Behav Immun 1:284 27. Kay N, Allen J, Morley JE 1984 Endorphins stimulate normal human peripheral blood lymphocyte natural killer activity. Life Sci 35:53 28. Brown SL, Van Epps DE 1986 Opioid peptides modulate production of interferon-7 by human mononuclear cells. Cell Immunol 103:19 29. Van Epps DE, Saland L 1984 /3-endorphin and met-enkephalin stimulate human peripheral blood mononuclear cell chemotaxis. J Immunol 132:3046
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