- Email: [email protected]
Immune responsiveness of splenocytes after chronic daily melatonin administration in male syrian hamsters
ELSEVIER
Immunology
Letters
58 (1997) 95-100
Immune responsiveness of splenocytes after chronic daily melatonin administration in male Syrian hamsters Thomas
H. Champney
a,*, Jessica Prado a,b, Tonya David N. McMurray
Youngblood a*b
a-b, Kevin Appel a,b,
a Department of Human Anatomy and Medical Neurobiology, College of Medicine, Texas A&M University Health Science Centre, College Station, TX 77 843- 1114, USA b Department of Medical Microbiology and Immunology, College of Medicine, Texas A&M University Health Science Center. College Station, TX 77843-l 114, USA
Received
14 November
1996; accepted
3 March
1997
Abstract The interrelationships between the immune system and the pineal hormone, melatonin, have been explored recently. The present studies investigated the effects of daily melatonin injections on reproductive and spleen function in male Syrian hamsters. Testes weights and serum testosterone levels were depressed after 8- 10 weeks of daily melatonin injections. Melatonin-treated hamsters exhibited increased splenic lymphoproliferative responses to a polyclonal T-cell mitogen (concanavalin A (Con-A)), but decreased proliferation following stimulation with a polyclonal B-cell mitogen (lipopolysaccharide). It appears that daily melatonin injections in male hamsters increase the T-cell-mediated immune capacity while reducing the antibody-mediated immune potential. These data suggest that chronic, daily melatonin alters immune system responsiveness in hamsters by shifting the balance of cellular and humoral reactivity. 0 1997 Elsevier Science B.V. Keywords:
Melatonin;
Immune: Spleen; Hamster; Lymphoproliferation
1. Introduction The pineal hormone, melatonin (MEL, 5-methoxyN-acetyltryptamine), provides organ systems with a signal of the changing seasons [l]. These systems can utilize this signal to prepare for upcoming seasons. For example, the signal can produce a decline in reproductive activity [2,3], an increase in fur density [4] and an alteration in metabolism in preparation for winter [5]. Immune responsiveness displays seasonal differences in humans [6,7] and other mammalian species [8,9] with the pineal and melatonin implicated as regulatory agents [lo-121. The relationship of the pineal gland and the immune
system has been previously examined by numerous investigators [12-161. It appears that the pineal hormone, melatonin, has an important immunomodulatory role [10,15]. A melatonin-immune system interaction in humans has also been investigated and both stimulatory and depressive effects of melatonin have been observed, depending on the type of immune functions tested [13,17-231. However, none of the current investigators have utilized a highly melatonin responsive species (Syrian hamsters) combined with specific immunologic assays.
2. Materials *Corresponding 8450790; e-mail:
author. Tel.: [email protected]
+
0165-2478/97/$17.00 0 1997 Elsevier PII SOI 65-2478(97)00039-4
1 409 8454963;
fax:
Science B.V. All rights
+
1 409
reserved.
and methods
Adult male Syrian hamsters were purchased from Harlan Sprague-Dawley and maintained according to
96
T.H. Champney
et al. /Immunology
the NIH Guide for the Care and Use of Laboratory Animals. The hamsters received food and water ad libitum in temperature (21-22’C) controlled rooms. After specified treatment periods, the hamsters were killed by decapitation while under metofane (methoxyflurane, Mallinckrodt Veterinary, Mundelein, IL) anesthesia. The hamsters’ spleens were collected aseptically and weighed. The testes and trunk blood were also collected to confirm that the MEL treatments were effective. Serum was isolated from trunk blood by chilling at 4°C centrifuging (2000 x g) for 30 min at 4°C and freezing ( - 20°C) the resulting serum. In the first experiment, male hamsters were placed in long photoperiod (14L:lOD, lights on at 05:OO h) and received either 1 week (n = 10) or 8 weeks (n = 10) of daily MEL (melatonin, # M-5250, Sigma, St. Louis, MO) injections (25 pug, SC, 16:45 h, dissolved in ethanol and diluted 1:lO in 0.9% saline). Control hamsters (n = 10 for each control group) received daily vehicle injections (VEH, 1: 10 ethanolic saline) at the same time. At the end of the treatment period, the hamsters were killed between 08:30 and 12:15 h and their tissues collected as described. In the second experiment, MEL (25 pg, SC, 17:OOh) or VEH was injected daily into long photoperiod-exposed, male hamsters for 10 weeks (n = 10 per group). Ten weeks after beginning MEL injections, the hamsters were killed between 09:OO and 1l:OO h and their tissues were collected as described. Free testosterone levels were determined by radioimmunoassay using a commercial kit (Diagnostic Products, Los Angeles, CA). The minimum detectable dose for this assay was 1.31 ng/dl with an intra-assay variability of 1.95%. Immune response studies involved establishing short-term in vitro lymphocyte cultures from a splenocyte suspension that was prepared by gently homogenizing the spleen in a glass-glass homogenizer in complete tissue medium (RPM1 1640 supplemented with 10% fetal bovine serum, HEPES buffer, 100 lug/ml streptomycin, 100 U/ml penicillin and 0.02 mM 2-mercaptoethanol). The resulting suspension was filtered through a fine stainless steel wire mesh to remove clumps and cell viability was determined by trypan blue exclusion. Aliquots (2 x 10’ cells in 100 ~1) were placed into the wells of a 96-well microtiter plate and triplicate wells treated with one of three concentrations of either the lectin, concanavalin A (Con-A, C0412, Sigma, St. Louis, MO) (1.25, 0.625 or 0.3125 pug/ml) or bacterial lipopolysaccharide (LPS, L-2654, Sigma, St. Louis, MO) (1.25, 0.625 or 0.3125 pg/ml). These concentrations have been determined in preliminary experiments to be in the linear dose response range for stimulation of hamster lymphocytes. The plates were incubated at 37°C and 5% CO, in air atmosphere for a total of 96 h with 1 PCi of tritiated thymidine per well added for the last 6 h. The cells were
Letters
58 (1997) 95-100
harvested onto glass fiber filter disks and counted in a liquid scintillation counter [24-261. Statistical significance was determined by two-way analysis of variance for all of the data from the first experiment and for the immune data from the second experiment. The remaining data from the second experiment were compared by t-test.
3. Results In the first experiment, all of the hamsters that received 8 weeks of MEL had reduced testes weights and testosterone levels (P < 0.05) as well as increased spleen weights (P < 0.05) when compared to the VEH injected or short term (1 week) treated animals (Table 1). In contrast to the increase in spleen weight seen in the hamsters treated with melatonin for 8 weeks, the total number of splenocytes recovered from those spleens was significantly less (P < 0.05) compared to the 1 week vehicle control. The reduced cellularity of spleens from melatonin-treated animals was even more obvious when the data were expressed as number of splenocytes per mg of spleen (Table 1). Thus, the cellular density of spleens from 8 week, melatonintreated hamsters was significantly less than both the one week, vehicle (P < 0.01) and the one week, melatonin-treated (P < 0.05) groups. Although the cellular density of the melatonin group at 8 weeks was nearly 50% less than the 8 week, vehicle group, this difference was not statistically significant. Spleen blastogenesis induced by different doses of Con-A was increased (P < 0.01) in the long term, MEL treated hamsters (Fig. 1). The enhanced proliferation of T-lymphocytes following 8 weeks of melatonin treatment was most striking at the lowest dose of Con A (0.3125 pg/ml), which induced only minimal mitosis in the other treatment groups. As the dose of Con-A was increased, the responses of all the groups increased. However, the 8-week, melatonin-treated animals continued to exhibit the highest degree of proliferation. In the second experiment, testes weights and testosterone levels of MEL treated hamsters were reduced (P < 0.01) in six of the ten treated hamsters. The remaining four animals had testes weights and testosterone values similar to control animals, but all animals were included in the analyses (Table 2). Inclusion of the MEL non-responsive animals (n = 4) with the MEL responsive animals (n = 6) reduces the level of significance between treatment groups, but preserves the initial experimental design. Once again, the cellular density (number of splenocytes per mg spleen) was reduced by about 20% in the melatonin-treated hamsters, although this difference was not statistically different. Spleen blastogenesis induced by LPS (using the same doses as Con-A) was depressed in MEL treated
groups.
161 + 6 150* 5 166k5 155+6
10 10 IO 10
Values are means k S.E. * P~0.05 vs. 8 weeks-vehicle. ** P~0.05 vs. I week treatment
8 weeks vehicle 8 weeks melatonin
Body weight
(g)
splenic cellularity,
n
spleen weights,
1 week melatonin
1 week vehicle
Treatment
Table 1 Body weights,
191 * 8 173+7 164&8 225 + 19*
Spleen weight
testes weights (mg) 63.4 54.6 46.5 35.1
Total + k f +
6.4 6.2 9.2 6.0**
33.9 29.6 25.7 14.4
* k k k
4.2 4.8 2.5 2.3**
Splenocytes/mg 4053 3712 3944 965
* + f f
140 122 118 204*,**
Testes weight
(mg)
261 k 35 185k26 235 k 36 136 _+ 32*+*
(ng/dl)
in male Syrian hamsters Testosterone
(25 fig, SC, 16:45 h) or vehicle injections
spleen (x lo‘+)
1 or 8 weeks of daily melatonin
( x 106)
levels after
splenocytes
and serum testosterone
T.H. Champney et al. /Immunology Letters 58 (1997) 95-100
98
16’
-
0
VEHICLE
m
MELATONIN
m
VEHICLE
140 -
-
1 WEEK
-
8 WEEKS
-
MELATONIN
40
-
20
-
-
1 WEEK
0
VEHICLE
m
MELATONIN
8 WEEKS
O-
0
0.625
0.3125 CONCANAVALIN
1.25
0
A
0.3125
0.625
1.25
LIPOPOLYSACHARIDE
k/m0
(w/ml)
Fig. 1. Spleen blastogenesis (cpm x 103) induced by addition of various doses of Con-A to splenocyte cultures from male Syrian hamsters that previously received daily vehicle injections for 1 or 8 weeks or daily melatonin (25 fig, sc, 16:45 h) injections for 1 or 8 weeks. Values are means k S.E. with ten animals per group.
Fig. 2. Spleen blastogenesis (cpm x 103) induced by addition of various doses of lipopolysaccharide to splenocyte cultures from male Syrian hamsters that previously received daily vehicle or melatonin (25 fig, SC, 17:00 h) injections for 10 weeks. Values are means k S.E. with ten animals per group.
hamsters when compared with the vehicle group (Fig. 2). Splenocytes from both groups responded in a dose dependent fashion to LPS with the cells from the melatonin-treated animals proliferating less than control cells at all three doses. This depressed proliferative response reached statistical significance (P < 0.05) at a LPS dose of 0.3125 pgg/ml.
unable to block melatonin’s direct effect on blood mononuclear cells [28]. Melatonin could also indirectly mediate immune function by its well known effects on circulating steroids [3]. Steroids are known to affect immune function [29,30] and melatonin can modify steroid levels. Therefore, melatonin’s alteration of other endocrine systems could indirectly affect immune function. In contrast, a direct effect of melatonin on immune function has been observed in lymphocyte cultures. Melatonin was able to directly inhibit blastogenesis and natural killer cell activity [ 13,17,20,28]. These results suggest that melatonin has an acute, direct effect on lymphocyte activity in humans. In addition, melatonin receptors have been found on peripheral lymphocytes [18,31] and hamster spleen cells [32]. Melatonin can also activate cGMP and inhibit stimulated CAMP levels in lymphocytes [23,33]. Therefore, melatonin may be able to regulate immune function by both direct and indirect means.
4. Discussion The mechanism(s) by which melatonin produces its effects on the immune system are not well understood. It appears that melatonin has both direct and indirect effects. For example, Maestroni [lo] has repeatedly demonstrated that melatonin’s effects on the mouse immune system are indirectly mediated by an endogenous opiate system. Naloxone, an opiate blocker, was able to block melatonin’s immune stimulating activity in mice [l&27]. However, naloxone was
Table 2 Body weights, spleen weights, splenic cellularity, testes weights and serum testosterone levels after ten weeks of daily melatonin (25 ,ug, SC, 17:00 h) or vehicle injections in male Syrian hamsters Treatment
n
Body weight (g)
Spleen weight (mg)
Total splenocytes (x 106)
Splenocytes/mg spleen (x 104)
Testes weight (mg)
Testosterone (ng/dl)
Vehicle Melatonin
10 10
137+4 143&S
141 k 6 154&5
27.9 + 3.7 25.6 f 2.6
19.7 + 2.2 16.7 & 1.6
4219 1.59 2032 + 397*
268 + 39 158 +46*
Values are means f S.E. * P< 0.05 vs. vehicle.
T.H. Champney et al. /Immunology
Only four studies to date have utilized the Syrian hamster to examine photoperiodic or pineal regulation of immune function [25,34-361. Brainard and coworkers [34,35] found that spleen weight, total number of splenic lymphocytes and the total number of splenic macrophages were increased in short photoperiod exposed hamsters. Antibody production after antigenic stimulation was unaltered by photoperiod exposure in these studies. Vaughan and colleagues [36] used histologic and morphologic measures to determine anatomic changes in hamster spleens. In that study, spleen weight and extramedullary hematopoiesis were increased after short photoperiod or melatonin injections [36]. These results were confirmed by Champney and McMurray [25] who also demonstrated that melatonin treated hamster splenocytes were more responsive to Con A stimulation than vehicle controls. These anatomic results indicate that chronic melatonin (injected or increased naturally by short photoperiod exposure) increases both spleen size and immunologic potential. In contrast to the findings of Brainard and colleagues [34], the present experiments report an inverse relationship between spleen size (weight) and splenic cellularity (number of cells per mg spleen). Melatonin treatment for 8- 10 weeks resulted in increased spleen size, as has been reported in the past [34,36]. However, cellular density was reduced in the melatonin-treated hamsters in the first experiment (Table l), suggesting that the increase in spleen weight was not due to hyperplasia. This conclusion is also supported by the fact that no increase in baseline (unstimulated) lymphoproliferation was observed in melatonin-treated hamsters (Figs. 1 and 2), as might have been expected if the spleens were in a hyperplastic state. Melatonin treated male hamsters exhibited an increased responsiveness to Con-A stimulation, especially to suboptimal doses of the mitogen, but a decreased responsiveness to LPS stimulation compared to vehicle treated animals. Con-A is a specific mitogen for T-cell proliferation that has little to no B-cell activity [37,38]. Lipopolysaccharide, on the other hand, is a specific mitogen for B-cell proliferation with limited T-cell activity [39]. A differential effect of photoperiod on Tand B-cell functions was also reported in humans, where seasonal effects on T- and B-cell mitogenic responses were uncoupled [7]. In that study, however, human T-lymphocytes proliferated less well to mitogens (Con-A, phytohemagglutinin) during the winter season, while the cellular response to a T-cell-dependent B-cell mitogen (poke weed) was unaffected. Since the cultures from both melatonin-treated and vehicle-treated spleens contained equal numbers of viable lymphocytes, these results can be interpreted in one of two ways. Melatonin treatment may have resulted in a disproportionate increase in the T-cell fraction (response to Con-A) at the expense of the B-cell fraction (response to LPS).
Letters 58 (1997) 95-100
99
Alternatively, chronic melatonin exposure may have modified the intrinsic mitogenic reactivity of the lymphocytes on a per cell basis, increasing the responsiveness of T-cells and decreasing the responsiveness of B-cells. Discrimination between these two hypotheses must await the development of antibodies with which to separate and identify hamster T- and B-cells. The present results indicate that long term (8- 10 weeks) melatonin treatment can differentially alter Tand B-cell lymphocyte proliferation in male hamsters, while short term (1 week) melatonin treatment is ineffective. This provides further evidence for the parallel between melatonin’s effects on the reproductive system and the immune system; suggesting that melatonin’s effects on the immune system may be mediated by changes in the reproductive system. These results are in contrast to the reviews of Grossman 129,401 that indicate that decreased testosterone levels increase both cell-mediated and antibody- mediated immune function. Therefore, melatonin’s effects on spleen blastogenesis may not be exclusively due to its effects on reproductive hormone levels. Since the T-cell and B-cell lymphocyte proliferation tests were run in different experiments and, therefore, in different groups of hamsters, the difference between the responses could be due to differences in experimental protocols. However, both experiments used the same dosage and time of day of melatonin injections and produced the same decline in the reproductive system. This suggests that the animals were responding to the melatonin injections similarly across experiments and that the differences in the T-cell and B-cell proliferation tests are not due to differences between the experiments. It should be noted that, at the time of death, the hamsters had low levels of melatonin in their circulation. Therefore, the immune effects of melatonin were not due to an acute effect of melatonin on the splenocytes, but to some prolonged aspect of melatonin administration that altered the inherent sensitivity of the splenocytes to mitogenic stimulation or the relative proportions of T- and B-cells in the spleens. Whether these changes are a direct effect of melatonin on the splenocytes or an indirect effect of melatonin on other systems is one goal of future studies. In conclusion, these results suggest that the pineal hormone, melatonin, can modify splenic immune function in the hamster. In general, it appears that T-cell function is increased, while B-cell function is decreased by daily melatonin injections for 8 10 weeks. The precise mechanisms and consequences of immune regulation by melatonin are important to determine. Future studies will examine other T-cell (delayed hypersensitivity, cytokine production) and B-cell (antibody production) functions, as well as an assessment of the effect of melatonin on the relative proportions and individual
100
T.H. Champney et al. /Immunology
reactivity of T- and B-cell populations in the spleen and other lymphoid organs. If a significant degree of regulation is found, then the use of melatonin could be contemplated for the treatment of immunodeficiency diseases or the down-regulation of intense hypersensitivity or autoimmunity.
Acknowledgements
The authors express their thanks for the excellent technical assistance of Susan Phalen and Daniel Ramirez.
References [1] H.S. Yu, R.J. Reiter, Melatonin. Biosynthesis, Physiological Effects and Clinical Applications, CRC Press, Boca Raton, 1993. [2] L.J. Petterborg, B.A. Richardson, M.K. Vaughan, R.J. Reiter, J. Neural Transm. 59 (1984) 299-307. [3] R.J. Reiter, Endocr. Rev. 12 (1991) 151-180. [4] B. Gunduz, M.H. Stetson, Biol. Reprod. 51 (1994) 1181-1187. [S] T.J. Bartness, G.N. Wade, Endocrinology 114 (1984) 492-498. [6] B. Bjorksten, A. Gamkrelidze, T. Vanto, M. Kjellman, Allergy 45 (1990) 572-576. [7] F.N. Boctor, R.A. Charmy, E.L. Cooper, Immunol. Invest. 18 (1989) 775-784. [8] R.L. Davis, R.L. Lochmiller, Biol. Rhythm Res. 26 (1995) 20-31. [9] M. Giordano, M. Vermeullen, M.S. Palermo, J. FASEB 7 (1993) 1052- 1054. [lo] G.J.M. Maestroni, J. Pineal. Res. 14 (1993) l-10. [ll] G.J.M. Maestroni, A. Conti, in: H-S. Yu, R.J. Reiter (Eds.), Melatonin: Biosynthesis, Physiological Effects and Clinical Applications, CRC Press, Boca Raton, 1993, pp. 289-310. [12] R.J. Nelson, G.E. Demas, S.L. Klein, L.J. Kriegsfeld, J. Pineal Res. 19 (1995) 149-165. [13] F. Fraschini, R.J. Reiter, Role of Melatonin and Pineal Peptides in Neuroimmunomodulation, Plenum Press, New York, 1991. [14] L.C. Hofbauer, A.E. Heufelder, Eur. J. Endocrinol. 134 (1996) 424-425. [15] G.J.M. Maestroni, J. Pineal Res. 18 (1995) 84-89.
Letters 58 (1997) 95-100
[16] K. Skwarlo-Sonta, Acta Neurobiol. Exp. 56 (1996) 341-357. [17] ES. Arzt, S. Femandez-Castelo, L.M.E. Finocchiaro, M.E. Criscuolo, A. Diaz, S. Finkielman, V.E. Nahmod, J. Clin. Immunol. 8 (1988) 513-520. [18] J.R. Calvo, M. Rafii-El-Idrissi, D. Pozo, J.M. Guerrero, J. Pineal Res. 18 (1995) 119-126. [191 V. de1 Gobbo, V. Libri, N. Villani, R. Callio, G. Nistico, Int. J. Immunopharmacol. 11 (1989) 567-573. I201R. Konakchieva, S. Kyurkchiev, I. Kehayov, P. Taushanova, L. Kanchev, J. Neuroimmunol. 63 (1995) 125-132. WI A. Lewinski, P. Zelazowski, E. Sewerynek, G. Zerek-Melen, M. Szkudlinski, E. Zelazowska, J. Pineal Res. 7 (1989) 153-164. LW P. Lissoni, S. Pittalis, F. Rovelli, M.G. Roselli, A. Ardizzoia, Oncol. Rep. 3 (1996) 541-543. 1231 M. Rafii-El-Idrissi, J.R. Calvo, D. Pozo, A. Harmouch. J.M. Guerrero, J. Neuroimmunol. 57 (1995) 171-178. v41 R.A. Bartow, D.N. McMurray, Infect. Immunol. 57 (1989) 1374-1379. 1251T.H. Champney, D.N. McMurray, in: F. Fraschini, R.J. Reiter (Eds.), Role of Melatonin and Pineal Peptides in Neuroimmunomodulation, Plenum Press, New York, 1991, pp. 219-223. WI D.W. McGee, D.N. McMurray, Immunology 64 (1988) 697702. 1271G.J.M. Maestroni, A. Conti, J. Neuroimmunol. 28 (1990) 167176. WI A. Di Stefano, L. Paulesu, J. Pineal Res. 17 (1994) 164-169. PI C.J. Grossman, Endocr. Rev. 5 (1984) 435-455. [301 G.F. Paciotti, R.G. Skwerer, L. Tamarkin, J. Neuroimmunol. 16 (1987) 253-259. [311 A.M.S. Poon, E.A. Ayre, Y. Song, S.F. Pang, Biol. Signals 3 (1994) 278-287. [321 L.P. Niles, Med. Sci. Res. 17 (1989) 179-180. 1331M.A. Lopez-Gonzalez, J.R. Calvo, C. Osuna, J.M. Guerrero, J. Pineal Res. 12 (1992) 97-104. 1341G.C. Brainard, R.L. Knobler, P.L. Podolin, M. Lavasa, F.D. Lublin, Life Sci. 40 (1987) 1319-1326. t351 G.C. Brainard, M. Watson-Whitmeyer, R.L. Knobler, F.D. Lublin, Ann. NY Acad. Sci. 540 (1988) 704-706. t361 M.K. Vaughan, G.B. Hubbard, T.H. Champney, G.M. Vaughan, J.C. Little, R.J. Reiter, Am. J. Anat. 179 (1987) 131-136. [371 F. Palacios, Immunol. Rev. 63 (1982) 72- 110. t381 A. Cogoli, J. Leukocyte Biol. 54 (1993) 259-268. [391 T. Hofstad, N. Skaug, K. Sveen, Clin. Infect. Dis. 16 (4) (1993) s2Ol- s202. [401 C.J. Grossman. J. Steroid Biochem. 34 (1989) 241-251.
Immunology
Letters
58 (1997) 95-100
Immune responsiveness of splenocytes after chronic daily melatonin administration in male Syrian hamsters Thomas
H. Champney
a,*, Jessica Prado a,b, Tonya David N. McMurray
Youngblood a*b
a-b, Kevin Appel a,b,
a Department of Human Anatomy and Medical Neurobiology, College of Medicine, Texas A&M University Health Science Centre, College Station, TX 77 843- 1114, USA b Department of Medical Microbiology and Immunology, College of Medicine, Texas A&M University Health Science Center. College Station, TX 77843-l 114, USA
Received
14 November
1996; accepted
3 March
1997
Abstract The interrelationships between the immune system and the pineal hormone, melatonin, have been explored recently. The present studies investigated the effects of daily melatonin injections on reproductive and spleen function in male Syrian hamsters. Testes weights and serum testosterone levels were depressed after 8- 10 weeks of daily melatonin injections. Melatonin-treated hamsters exhibited increased splenic lymphoproliferative responses to a polyclonal T-cell mitogen (concanavalin A (Con-A)), but decreased proliferation following stimulation with a polyclonal B-cell mitogen (lipopolysaccharide). It appears that daily melatonin injections in male hamsters increase the T-cell-mediated immune capacity while reducing the antibody-mediated immune potential. These data suggest that chronic, daily melatonin alters immune system responsiveness in hamsters by shifting the balance of cellular and humoral reactivity. 0 1997 Elsevier Science B.V. Keywords:
Melatonin;
Immune: Spleen; Hamster; Lymphoproliferation
1. Introduction The pineal hormone, melatonin (MEL, 5-methoxyN-acetyltryptamine), provides organ systems with a signal of the changing seasons [l]. These systems can utilize this signal to prepare for upcoming seasons. For example, the signal can produce a decline in reproductive activity [2,3], an increase in fur density [4] and an alteration in metabolism in preparation for winter [5]. Immune responsiveness displays seasonal differences in humans [6,7] and other mammalian species [8,9] with the pineal and melatonin implicated as regulatory agents [lo-121. The relationship of the pineal gland and the immune
system has been previously examined by numerous investigators [12-161. It appears that the pineal hormone, melatonin, has an important immunomodulatory role [10,15]. A melatonin-immune system interaction in humans has also been investigated and both stimulatory and depressive effects of melatonin have been observed, depending on the type of immune functions tested [13,17-231. However, none of the current investigators have utilized a highly melatonin responsive species (Syrian hamsters) combined with specific immunologic assays.
2. Materials *Corresponding 8450790; e-mail:
author. Tel.: [email protected]
+
0165-2478/97/$17.00 0 1997 Elsevier PII SOI 65-2478(97)00039-4
1 409 8454963;
fax:
Science B.V. All rights
+
1 409
reserved.
and methods
Adult male Syrian hamsters were purchased from Harlan Sprague-Dawley and maintained according to
96
T.H. Champney
et al. /Immunology
the NIH Guide for the Care and Use of Laboratory Animals. The hamsters received food and water ad libitum in temperature (21-22’C) controlled rooms. After specified treatment periods, the hamsters were killed by decapitation while under metofane (methoxyflurane, Mallinckrodt Veterinary, Mundelein, IL) anesthesia. The hamsters’ spleens were collected aseptically and weighed. The testes and trunk blood were also collected to confirm that the MEL treatments were effective. Serum was isolated from trunk blood by chilling at 4°C centrifuging (2000 x g) for 30 min at 4°C and freezing ( - 20°C) the resulting serum. In the first experiment, male hamsters were placed in long photoperiod (14L:lOD, lights on at 05:OO h) and received either 1 week (n = 10) or 8 weeks (n = 10) of daily MEL (melatonin, # M-5250, Sigma, St. Louis, MO) injections (25 pug, SC, 16:45 h, dissolved in ethanol and diluted 1:lO in 0.9% saline). Control hamsters (n = 10 for each control group) received daily vehicle injections (VEH, 1: 10 ethanolic saline) at the same time. At the end of the treatment period, the hamsters were killed between 08:30 and 12:15 h and their tissues collected as described. In the second experiment, MEL (25 pg, SC, 17:OOh) or VEH was injected daily into long photoperiod-exposed, male hamsters for 10 weeks (n = 10 per group). Ten weeks after beginning MEL injections, the hamsters were killed between 09:OO and 1l:OO h and their tissues were collected as described. Free testosterone levels were determined by radioimmunoassay using a commercial kit (Diagnostic Products, Los Angeles, CA). The minimum detectable dose for this assay was 1.31 ng/dl with an intra-assay variability of 1.95%. Immune response studies involved establishing short-term in vitro lymphocyte cultures from a splenocyte suspension that was prepared by gently homogenizing the spleen in a glass-glass homogenizer in complete tissue medium (RPM1 1640 supplemented with 10% fetal bovine serum, HEPES buffer, 100 lug/ml streptomycin, 100 U/ml penicillin and 0.02 mM 2-mercaptoethanol). The resulting suspension was filtered through a fine stainless steel wire mesh to remove clumps and cell viability was determined by trypan blue exclusion. Aliquots (2 x 10’ cells in 100 ~1) were placed into the wells of a 96-well microtiter plate and triplicate wells treated with one of three concentrations of either the lectin, concanavalin A (Con-A, C0412, Sigma, St. Louis, MO) (1.25, 0.625 or 0.3125 pug/ml) or bacterial lipopolysaccharide (LPS, L-2654, Sigma, St. Louis, MO) (1.25, 0.625 or 0.3125 pg/ml). These concentrations have been determined in preliminary experiments to be in the linear dose response range for stimulation of hamster lymphocytes. The plates were incubated at 37°C and 5% CO, in air atmosphere for a total of 96 h with 1 PCi of tritiated thymidine per well added for the last 6 h. The cells were
Letters
58 (1997) 95-100
harvested onto glass fiber filter disks and counted in a liquid scintillation counter [24-261. Statistical significance was determined by two-way analysis of variance for all of the data from the first experiment and for the immune data from the second experiment. The remaining data from the second experiment were compared by t-test.
3. Results In the first experiment, all of the hamsters that received 8 weeks of MEL had reduced testes weights and testosterone levels (P < 0.05) as well as increased spleen weights (P < 0.05) when compared to the VEH injected or short term (1 week) treated animals (Table 1). In contrast to the increase in spleen weight seen in the hamsters treated with melatonin for 8 weeks, the total number of splenocytes recovered from those spleens was significantly less (P < 0.05) compared to the 1 week vehicle control. The reduced cellularity of spleens from melatonin-treated animals was even more obvious when the data were expressed as number of splenocytes per mg of spleen (Table 1). Thus, the cellular density of spleens from 8 week, melatonintreated hamsters was significantly less than both the one week, vehicle (P < 0.01) and the one week, melatonin-treated (P < 0.05) groups. Although the cellular density of the melatonin group at 8 weeks was nearly 50% less than the 8 week, vehicle group, this difference was not statistically significant. Spleen blastogenesis induced by different doses of Con-A was increased (P < 0.01) in the long term, MEL treated hamsters (Fig. 1). The enhanced proliferation of T-lymphocytes following 8 weeks of melatonin treatment was most striking at the lowest dose of Con A (0.3125 pg/ml), which induced only minimal mitosis in the other treatment groups. As the dose of Con-A was increased, the responses of all the groups increased. However, the 8-week, melatonin-treated animals continued to exhibit the highest degree of proliferation. In the second experiment, testes weights and testosterone levels of MEL treated hamsters were reduced (P < 0.01) in six of the ten treated hamsters. The remaining four animals had testes weights and testosterone values similar to control animals, but all animals were included in the analyses (Table 2). Inclusion of the MEL non-responsive animals (n = 4) with the MEL responsive animals (n = 6) reduces the level of significance between treatment groups, but preserves the initial experimental design. Once again, the cellular density (number of splenocytes per mg spleen) was reduced by about 20% in the melatonin-treated hamsters, although this difference was not statistically different. Spleen blastogenesis induced by LPS (using the same doses as Con-A) was depressed in MEL treated
groups.
161 + 6 150* 5 166k5 155+6
10 10 IO 10
Values are means k S.E. * P~0.05 vs. 8 weeks-vehicle. ** P~0.05 vs. I week treatment
8 weeks vehicle 8 weeks melatonin
Body weight
(g)
splenic cellularity,
n
spleen weights,
1 week melatonin
1 week vehicle
Treatment
Table 1 Body weights,
191 * 8 173+7 164&8 225 + 19*
Spleen weight
testes weights (mg) 63.4 54.6 46.5 35.1
Total + k f +
6.4 6.2 9.2 6.0**
33.9 29.6 25.7 14.4
* k k k
4.2 4.8 2.5 2.3**
Splenocytes/mg 4053 3712 3944 965
* + f f
140 122 118 204*,**
Testes weight
(mg)
261 k 35 185k26 235 k 36 136 _+ 32*+*
(ng/dl)
in male Syrian hamsters Testosterone
(25 fig, SC, 16:45 h) or vehicle injections
spleen (x lo‘+)
1 or 8 weeks of daily melatonin
( x 106)
levels after
splenocytes
and serum testosterone
T.H. Champney et al. /Immunology Letters 58 (1997) 95-100
98
16’
-
0
VEHICLE
m
MELATONIN
m
VEHICLE
140 -
-
1 WEEK
-
8 WEEKS
-
MELATONIN
40
-
20
-
-
1 WEEK
0
VEHICLE
m
MELATONIN
8 WEEKS
O-
0
0.625
0.3125 CONCANAVALIN
1.25
0
A
0.3125
0.625
1.25
LIPOPOLYSACHARIDE
k/m0
(w/ml)
Fig. 1. Spleen blastogenesis (cpm x 103) induced by addition of various doses of Con-A to splenocyte cultures from male Syrian hamsters that previously received daily vehicle injections for 1 or 8 weeks or daily melatonin (25 fig, sc, 16:45 h) injections for 1 or 8 weeks. Values are means k S.E. with ten animals per group.
Fig. 2. Spleen blastogenesis (cpm x 103) induced by addition of various doses of lipopolysaccharide to splenocyte cultures from male Syrian hamsters that previously received daily vehicle or melatonin (25 fig, SC, 17:00 h) injections for 10 weeks. Values are means k S.E. with ten animals per group.
hamsters when compared with the vehicle group (Fig. 2). Splenocytes from both groups responded in a dose dependent fashion to LPS with the cells from the melatonin-treated animals proliferating less than control cells at all three doses. This depressed proliferative response reached statistical significance (P < 0.05) at a LPS dose of 0.3125 pgg/ml.
unable to block melatonin’s direct effect on blood mononuclear cells [28]. Melatonin could also indirectly mediate immune function by its well known effects on circulating steroids [3]. Steroids are known to affect immune function [29,30] and melatonin can modify steroid levels. Therefore, melatonin’s alteration of other endocrine systems could indirectly affect immune function. In contrast, a direct effect of melatonin on immune function has been observed in lymphocyte cultures. Melatonin was able to directly inhibit blastogenesis and natural killer cell activity [ 13,17,20,28]. These results suggest that melatonin has an acute, direct effect on lymphocyte activity in humans. In addition, melatonin receptors have been found on peripheral lymphocytes [18,31] and hamster spleen cells [32]. Melatonin can also activate cGMP and inhibit stimulated CAMP levels in lymphocytes [23,33]. Therefore, melatonin may be able to regulate immune function by both direct and indirect means.
4. Discussion The mechanism(s) by which melatonin produces its effects on the immune system are not well understood. It appears that melatonin has both direct and indirect effects. For example, Maestroni [lo] has repeatedly demonstrated that melatonin’s effects on the mouse immune system are indirectly mediated by an endogenous opiate system. Naloxone, an opiate blocker, was able to block melatonin’s immune stimulating activity in mice [l&27]. However, naloxone was
Table 2 Body weights, spleen weights, splenic cellularity, testes weights and serum testosterone levels after ten weeks of daily melatonin (25 ,ug, SC, 17:00 h) or vehicle injections in male Syrian hamsters Treatment
n
Body weight (g)
Spleen weight (mg)
Total splenocytes (x 106)
Splenocytes/mg spleen (x 104)
Testes weight (mg)
Testosterone (ng/dl)
Vehicle Melatonin
10 10
137+4 143&S
141 k 6 154&5
27.9 + 3.7 25.6 f 2.6
19.7 + 2.2 16.7 & 1.6
4219 1.59 2032 + 397*
268 + 39 158 +46*
Values are means f S.E. * P< 0.05 vs. vehicle.
T.H. Champney et al. /Immunology
Only four studies to date have utilized the Syrian hamster to examine photoperiodic or pineal regulation of immune function [25,34-361. Brainard and coworkers [34,35] found that spleen weight, total number of splenic lymphocytes and the total number of splenic macrophages were increased in short photoperiod exposed hamsters. Antibody production after antigenic stimulation was unaltered by photoperiod exposure in these studies. Vaughan and colleagues [36] used histologic and morphologic measures to determine anatomic changes in hamster spleens. In that study, spleen weight and extramedullary hematopoiesis were increased after short photoperiod or melatonin injections [36]. These results were confirmed by Champney and McMurray [25] who also demonstrated that melatonin treated hamster splenocytes were more responsive to Con A stimulation than vehicle controls. These anatomic results indicate that chronic melatonin (injected or increased naturally by short photoperiod exposure) increases both spleen size and immunologic potential. In contrast to the findings of Brainard and colleagues [34], the present experiments report an inverse relationship between spleen size (weight) and splenic cellularity (number of cells per mg spleen). Melatonin treatment for 8- 10 weeks resulted in increased spleen size, as has been reported in the past [34,36]. However, cellular density was reduced in the melatonin-treated hamsters in the first experiment (Table l), suggesting that the increase in spleen weight was not due to hyperplasia. This conclusion is also supported by the fact that no increase in baseline (unstimulated) lymphoproliferation was observed in melatonin-treated hamsters (Figs. 1 and 2), as might have been expected if the spleens were in a hyperplastic state. Melatonin treated male hamsters exhibited an increased responsiveness to Con-A stimulation, especially to suboptimal doses of the mitogen, but a decreased responsiveness to LPS stimulation compared to vehicle treated animals. Con-A is a specific mitogen for T-cell proliferation that has little to no B-cell activity [37,38]. Lipopolysaccharide, on the other hand, is a specific mitogen for B-cell proliferation with limited T-cell activity [39]. A differential effect of photoperiod on Tand B-cell functions was also reported in humans, where seasonal effects on T- and B-cell mitogenic responses were uncoupled [7]. In that study, however, human T-lymphocytes proliferated less well to mitogens (Con-A, phytohemagglutinin) during the winter season, while the cellular response to a T-cell-dependent B-cell mitogen (poke weed) was unaffected. Since the cultures from both melatonin-treated and vehicle-treated spleens contained equal numbers of viable lymphocytes, these results can be interpreted in one of two ways. Melatonin treatment may have resulted in a disproportionate increase in the T-cell fraction (response to Con-A) at the expense of the B-cell fraction (response to LPS).
Letters 58 (1997) 95-100
99
Alternatively, chronic melatonin exposure may have modified the intrinsic mitogenic reactivity of the lymphocytes on a per cell basis, increasing the responsiveness of T-cells and decreasing the responsiveness of B-cells. Discrimination between these two hypotheses must await the development of antibodies with which to separate and identify hamster T- and B-cells. The present results indicate that long term (8- 10 weeks) melatonin treatment can differentially alter Tand B-cell lymphocyte proliferation in male hamsters, while short term (1 week) melatonin treatment is ineffective. This provides further evidence for the parallel between melatonin’s effects on the reproductive system and the immune system; suggesting that melatonin’s effects on the immune system may be mediated by changes in the reproductive system. These results are in contrast to the reviews of Grossman 129,401 that indicate that decreased testosterone levels increase both cell-mediated and antibody- mediated immune function. Therefore, melatonin’s effects on spleen blastogenesis may not be exclusively due to its effects on reproductive hormone levels. Since the T-cell and B-cell lymphocyte proliferation tests were run in different experiments and, therefore, in different groups of hamsters, the difference between the responses could be due to differences in experimental protocols. However, both experiments used the same dosage and time of day of melatonin injections and produced the same decline in the reproductive system. This suggests that the animals were responding to the melatonin injections similarly across experiments and that the differences in the T-cell and B-cell proliferation tests are not due to differences between the experiments. It should be noted that, at the time of death, the hamsters had low levels of melatonin in their circulation. Therefore, the immune effects of melatonin were not due to an acute effect of melatonin on the splenocytes, but to some prolonged aspect of melatonin administration that altered the inherent sensitivity of the splenocytes to mitogenic stimulation or the relative proportions of T- and B-cells in the spleens. Whether these changes are a direct effect of melatonin on the splenocytes or an indirect effect of melatonin on other systems is one goal of future studies. In conclusion, these results suggest that the pineal hormone, melatonin, can modify splenic immune function in the hamster. In general, it appears that T-cell function is increased, while B-cell function is decreased by daily melatonin injections for 8 10 weeks. The precise mechanisms and consequences of immune regulation by melatonin are important to determine. Future studies will examine other T-cell (delayed hypersensitivity, cytokine production) and B-cell (antibody production) functions, as well as an assessment of the effect of melatonin on the relative proportions and individual
100
T.H. Champney et al. /Immunology
reactivity of T- and B-cell populations in the spleen and other lymphoid organs. If a significant degree of regulation is found, then the use of melatonin could be contemplated for the treatment of immunodeficiency diseases or the down-regulation of intense hypersensitivity or autoimmunity.
Acknowledgements
The authors express their thanks for the excellent technical assistance of Susan Phalen and Daniel Ramirez.
References [1] H.S. Yu, R.J. Reiter, Melatonin. Biosynthesis, Physiological Effects and Clinical Applications, CRC Press, Boca Raton, 1993. [2] L.J. Petterborg, B.A. Richardson, M.K. Vaughan, R.J. Reiter, J. Neural Transm. 59 (1984) 299-307. [3] R.J. Reiter, Endocr. Rev. 12 (1991) 151-180. [4] B. Gunduz, M.H. Stetson, Biol. Reprod. 51 (1994) 1181-1187. [S] T.J. Bartness, G.N. Wade, Endocrinology 114 (1984) 492-498. [6] B. Bjorksten, A. Gamkrelidze, T. Vanto, M. Kjellman, Allergy 45 (1990) 572-576. [7] F.N. Boctor, R.A. Charmy, E.L. Cooper, Immunol. Invest. 18 (1989) 775-784. [8] R.L. Davis, R.L. Lochmiller, Biol. Rhythm Res. 26 (1995) 20-31. [9] M. Giordano, M. Vermeullen, M.S. Palermo, J. FASEB 7 (1993) 1052- 1054. [lo] G.J.M. Maestroni, J. Pineal. Res. 14 (1993) l-10. [ll] G.J.M. Maestroni, A. Conti, in: H-S. Yu, R.J. Reiter (Eds.), Melatonin: Biosynthesis, Physiological Effects and Clinical Applications, CRC Press, Boca Raton, 1993, pp. 289-310. [12] R.J. Nelson, G.E. Demas, S.L. Klein, L.J. Kriegsfeld, J. Pineal Res. 19 (1995) 149-165. [13] F. Fraschini, R.J. Reiter, Role of Melatonin and Pineal Peptides in Neuroimmunomodulation, Plenum Press, New York, 1991. [14] L.C. Hofbauer, A.E. Heufelder, Eur. J. Endocrinol. 134 (1996) 424-425. [15] G.J.M. Maestroni, J. Pineal Res. 18 (1995) 84-89.
Letters 58 (1997) 95-100
[16] K. Skwarlo-Sonta, Acta Neurobiol. Exp. 56 (1996) 341-357. [17] ES. Arzt, S. Femandez-Castelo, L.M.E. Finocchiaro, M.E. Criscuolo, A. Diaz, S. Finkielman, V.E. Nahmod, J. Clin. Immunol. 8 (1988) 513-520. [18] J.R. Calvo, M. Rafii-El-Idrissi, D. Pozo, J.M. Guerrero, J. Pineal Res. 18 (1995) 119-126. [191 V. de1 Gobbo, V. Libri, N. Villani, R. Callio, G. Nistico, Int. J. Immunopharmacol. 11 (1989) 567-573. I201R. Konakchieva, S. Kyurkchiev, I. Kehayov, P. Taushanova, L. Kanchev, J. Neuroimmunol. 63 (1995) 125-132. WI A. Lewinski, P. Zelazowski, E. Sewerynek, G. Zerek-Melen, M. Szkudlinski, E. Zelazowska, J. Pineal Res. 7 (1989) 153-164. LW P. Lissoni, S. Pittalis, F. Rovelli, M.G. Roselli, A. Ardizzoia, Oncol. Rep. 3 (1996) 541-543. 1231 M. Rafii-El-Idrissi, J.R. Calvo, D. Pozo, A. Harmouch. J.M. Guerrero, J. Neuroimmunol. 57 (1995) 171-178. v41 R.A. Bartow, D.N. McMurray, Infect. Immunol. 57 (1989) 1374-1379. 1251T.H. Champney, D.N. McMurray, in: F. Fraschini, R.J. Reiter (Eds.), Role of Melatonin and Pineal Peptides in Neuroimmunomodulation, Plenum Press, New York, 1991, pp. 219-223. WI D.W. McGee, D.N. McMurray, Immunology 64 (1988) 697702. 1271G.J.M. Maestroni, A. Conti, J. Neuroimmunol. 28 (1990) 167176. WI A. Di Stefano, L. Paulesu, J. Pineal Res. 17 (1994) 164-169. PI C.J. Grossman, Endocr. Rev. 5 (1984) 435-455. [301 G.F. Paciotti, R.G. Skwerer, L. Tamarkin, J. Neuroimmunol. 16 (1987) 253-259. [311 A.M.S. Poon, E.A. Ayre, Y. Song, S.F. Pang, Biol. Signals 3 (1994) 278-287. [321 L.P. Niles, Med. Sci. Res. 17 (1989) 179-180. 1331M.A. Lopez-Gonzalez, J.R. Calvo, C. Osuna, J.M. Guerrero, J. Pineal Res. 12 (1992) 97-104. 1341G.C. Brainard, R.L. Knobler, P.L. Podolin, M. Lavasa, F.D. Lublin, Life Sci. 40 (1987) 1319-1326. t351 G.C. Brainard, M. Watson-Whitmeyer, R.L. Knobler, F.D. Lublin, Ann. NY Acad. Sci. 540 (1988) 704-706. t361 M.K. Vaughan, G.B. Hubbard, T.H. Champney, G.M. Vaughan, J.C. Little, R.J. Reiter, Am. J. Anat. 179 (1987) 131-136. [371 F. Palacios, Immunol. Rev. 63 (1982) 72- 110. t381 A. Cogoli, J. Leukocyte Biol. 54 (1993) 259-268. [391 T. Hofstad, N. Skaug, K. Sveen, Clin. Infect. Dis. 16 (4) (1993) s2Ol- s202. [401 C.J. Grossman. J. Steroid Biochem. 34 (1989) 241-251.