Developmentaland ComparativeImmunology,Vol. 14, pp. 85-93, 1990 Printed in the USA. All rights reserved.
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
MONOAMINES ALTER IN VITRO MIGRATION OF CHICKEN LEUKOCYTES Fred M. M c C o r k l e , Robert L. Taylor, Jr.,* Kelly M. Denno, and J. Mike Jabe Department of Biology, Central Michigan University, Mt. Pleasant, MI 48859 and *Department of Animal and Nutritional Sciences, University of New Hampshire, Durham, NH 03824 (Submitted August 1988;Accepted September 1988) DAbstract--The effect of the biogenic amines Introduction serotonin (5-HT), dopamine (DA), and norepinephrine (NE) on peripheral blood leukocyte Neurotransmitters and hormones regu(PBL) migration was studied in two populalate physiologic processes throughout tions derived from line UNH 105 New Hampthe body (1). In mammals or birds, adshire chickens. Maximum migration from capministration of androgenic, estrogenic, illary tube migration chambers was achieved in and adrenocortical steroid hormones 1 hr. An age effect in both populations was incause acute involution of the lymphoid dicated by significantly larger migration areas organs which alters both antibody and found in leukocytes from 7-week-old chickens cell-mediated immune responses (2,3). compared to those of 4-week-old chicks. Thirty min after intravenous monoamine injection, Bliznakov (4) demonstrated that mulline UNH 105 PBL migration was unaffected tiple doses of serotonin (5-HT) depress by exogenous monoamines. In the second popT-cell dependent, primary, humoral imulation, B24/B~ chicks, NE enhanced migramune responses in mice. Administration tion at 4 weeks of age but DA suppressed miof 5-HT reduces both IgM and IgG gration at 7 weeks of age. In vitro exposure of plaque-forming cell (PFC) response to PBL to the biogenic amines also affected leusheep red blood cells (SRBC) in CBA kocyte migration. Migration was augmented m i c e (5,6). N o r e p i n e p h r i n e (NE) by 100 ng 5-HT but suppressed by 1 p.g 5-HT strongly suppresses the in vitro antiin UNH 105 chicks. Furthermore, DA supSRBC response of murine spleen cells pressed PBL migration and NE enhanced mibut dopamine (DA) enhances PFC folgration in the same population. PBL from B24/B~ chicks were not affected by in vitro exlowing immunization (7). posure to 5-HT, however, DA enhanced miBoth antibody and cellular immunity gration whereas NE suppressed migration. in chickens are affected by exogenous Specific antagonists for 5-HT, DA, and NE monoamines. Chicken IgM and IgG PFC blocked the effects of each monoamine sugagainst SRBC were lowered significantly gesting that receptors are present on chicken when 5-HT or DA was injected 30 min leukocytes. These receptors mediate action of prior to antigen (8,9) in agreement with the monoamines on leukocyte migration acthe mammalian data. The phytohemagtivity.
glutinin (PHA) wattle response, a T-cellmediated response (10), was also depressed by the same single dose monoamine treatment. Either 5-HT, DA, or NE given 30 min prior to PHA significantly reduced subsequent wattle swelling (11). Just as monoamine administration reduced immune responses, pathologic sequelae which stimulated an immune re-
[]Keywords--Biogenic amines; Chicken, Leukocyte migration; Serotonin; Dopamine; Norepinephrine.
Address correspondence to Dr. Robert L. Taylor, Jr., Department of Animal and Nutritional Sciences, University of New Hampshire, Kendall Hall, Durham, NH 03824. 85
86
sponse reduced monoamine concentrations. The levels of 5-HT, DA, and NE were d e p r e s s e d significantly in the brain, spleen, thymus, and bursa of Fabricius of turkey poults infected with Bordetella avium, a mild upper respiratory pathogen that causes rhinotracheitis (12,13). Furthermore, in vitro leukocyte migration from B. avium-infected poults was enhanced compared to uninfected poults (14). This enhanced migration which occurred in the context of a complex disease process may be attributed to primary or secondary effects of the bacterial infection. Since immune stimulation increased leukocyte migration and lowered monoamine levels, the influence of increased monoamine concentrations on migration was questioned. This study was designed to determine whether in vivo and in vitro 5-HT, DA, and NE would disturb in vitro migration of chicken peripheral blood leukocytes (PBL).
Materials and Methods Animals Chicks of mixed sexes from line U N H 105, a noninbred line of New Hampshires, were used throughout this study. The line, originally obtained from a commercial breeder, is maintained at the University of New Hampshire Poultry Research Farm. Two populations were studied. The first, U N H 105, segregates for four B blood group haplotypes B 22, B 23, B 24, and B 26. The B blood group locus is a marker for the chicken major histocompatibility complex (15). These four haplotypes have approximate frequencies of 0.09, 0.20, 0.39, and 0.32, respectively, within line U N H 105. The second population derived from U N H 105, was homozygous for the B24/B24 genotype and was designated B24/B 24. Chicks were vaccinated for Marek's disease at hatching and housed in brooder
F.M. McCorkle et al.
batteries with free access to feed and water.
Monoamines 5-hydroxytryptamine (5-HT), 3-hydroxytyramine (DA), and arterenol (NE) were obtained from Sigma Chemical Company, St. Louis, Missouri. Monoamines were dissolved in physiologic saline and injected at the following doses: 5-HT, 100 I~g/kg of body weight; DA and NE, I mg/kg of body weight.
Leukocyte Collection In each of two trials, either a monoamine solution or a saline control was injected into separate groups of 10 birds of each population at 4 or 7 weeks of age. Thirty min after injection, heparinized blood was collected by cardiac puncture. Leukocytes, a heterogeneous population of lymphocytes, granulocytes, and monocytes, were separated by centrifugation (15 rain, 100 x g), removed as the buffy coat and washed three times in RPMI 1640 cell-culture medium (pH 7.4). Cell viability was assessed by the trypan-blue exclusion (16) after which cell numbers were adjusted to approximately 2 × 108 cells/ml. Cytochalasin B, a known inhibitor of cell membrane motility, was added to the reaction mixture to demonstrate inhibition of cell migration (14).
Cell Migration Nonheparinized capillary tubes (1.25 x 75 mm) were filled with the cell suspension and sealed with a clay-type tube sealer. The capillary tubes were centrifuged at 500 × g for 3 min and were cut evenly at the cell-fluid interface. The cell packs were placed into 24-well plates
Monoamines and leukocyte migration
87
supported by a small amount of vacuum grease (Dow Corning Corp., Midland, MI). The wells were filled with RPMI 1640, pH 7.4, and incubated at 37°C for 1 h. Three replicate capillary tubes were used per bird (14). The length and width of each migration zone was measured using an ocular scale on a dissecting microscope. The relative area of cell migration was calculated by the formula [(length x width)/10,000].
amine) and antagonist 2 (concentration I/I0 & 1/100 of biogenic amine). Leukocytes were treated with the antagonist in vitro for 30 min. After the cells were washed, all groups except controls were treated with the biogenic amine (5-HT, DA or NE) in vitro for 30 min followed by a second wash. Leukocytes were placed into capillary tubes for migration assay. This procedure was repeated in a second trial.
In Vitro Exposure
Statistical Analysis
Two trials were conducted. Leukocytes were collected as described above and s a m p l e s w e r e a l i q u o t e d into 3 groups: control, 5-HT--100 ng and 1 ~g or D A - - 1 Ixg and 10 I~g or N E - - 1 I~g and 10 Ixg. A control and two doses of a single monoamine were tested simultaneously. Leukocytes were exposed for 30 min on ice, washed 3 times, and placed in capillary tubes for cell migration.
The data were evaluated by two-way analysis of variance (21). Differences between monoamine treatments and controls within ages were assessed by Dunnett's test at the 0.05 level of significance. Means in the antagonist study were compared by orthogonal contrasts.
Antagonists Pindolol (Sigma) is a 5-HTI antagonist while ketanserin (Janssen Pharmaceutica Inc., Beerse, Belgium) is a 5-HT2 antagonist (17). Apomorphine (Sigma) and metoclopramide (Sigma) are D1 and D2 receptor antagonists (18), respectively. Propranolol (Sigma) is a 13-receptor antagonist (19) and phenoxybenzamine (Ciba-Giegy, Summit, N J) is an a-receptor antagonist (20).
In Vitro Exposure with Antagonists Peripheral blood leukocytes were collected from ten 7-week-old birds and washed. Leukocytes were aliquoted into 6 groups: c o n t r o l , biogenic amine treated in vitro for 30 min, antagonist 1 (Concentration 1/10 & 1/100 of biogenic
Results Peripheral blood leukocytes of 7week-old chickens of both populations had significantly larger migration areas than those of 4-week-old birds (Figs. 1 and 2). None of the monoamines tested (5-HT, DA and NE) significantly affected migration of leukocytes in line U N H 105 chicks (Fig. I). In contrast, NE increased significantly the migration area in 4-week-old B24/B24 chicks (Fig. 2), over that of the control. 5-HT and DA did not affect migration. DA suppressed significantly the migration area of PBL relative to that of the control (Fig. 2) in 7-week-old B24/B24 chicks but 5-HT and NE had no effect. With in vitro exposure of peripheral blood leukocytes to the biogenic amines, cell migration was significantly enhanced by 100 ng 5-HT and significantly suppressed by 1 ~g 5-HT in U N H 105 chicks (Fig. 3). No significant differences were found with in vitro exposure to 5-HT in BZ4/B24 chicks (Fig. 4). DA
88
F.M. McCorkle et al.
I
CONTROL 5-HT DA NE
12.0 lO.O 8.0
/
4.0 r~
2.0 0.0
4 WEEKS
7 WEEKS AGE
Figure 1. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vivo exposure to monoamines for 30 min. Values are expressed as mean standard error.
s i g n i f i c a n t l y s u p p r e s s e s UNH 105 chicks but enhances significantly cell migration in B24/B 24 (Figs. 3 and 4). Conversely, NE enhances significantly migration area in UNH 105 but is significantly suppressed in B24/B 24 (Figs. 3 and 4). 5-HT suppressed leukocyte migration at a dose of 1 Ixg. Both 100 ng and 10 ng pindolol, a 5-HT Z antagonist, blocked
Ill
12.0
"T ~u ~: 10.o
the suppressive effect of 5-HT as evidenced by the significant differences between migration areas (Fig. 5). Ketanserin, a 5-HT 2 receptor antagonist, arr e s t e d 5-HT s u p p r e s s i o n of P B L migration. Ten ng ketanserin produced significantly higher migration while I00 ng of the antagonist resulted in a migration area between those of the control and the 5-HT treatment (Fig. 5). The en-
CONTROL 5-HT
~DA ~ NE
"T 8.o s.o
i,., 'q:
4,0
r~
2.0 0.0
i
i
4 WEEKS
7 WEEKS
AGE Figure 2. Relative migration area of peripheral blood leukocytes from line UNH 105 B24/B24 chicks 1 hr following in vivo exposure to monoamines for 30 min. Values are expressed as mean standard error. Stars indicate significant differences from the control within an age (p < 0.05).
Monoamines and leukocyte migration
89
CONTROL
I
LOW
HIGH 10.0 1
1
L
l
I
6.0-
I,.,.
4.o-
r~ 2.00.0
SEROTONIN
DOPAMINE
NOREPINEPHRINE
TREA TMEN T Figure 3. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to m o n o a m i n e s for 30 rain. Values are expressed as mean standard error. (Low = 100 ng for 5-HT, 1 p.g for DA and NE; HIGH = 1 p~g for 5-HT, 10 p.g for DA and NE). Stars indicate significant differences from the control within a m o n o a m i n e treatment (p < 0.05).
hancing concentration 5-HT (I00 ng) was not blocked by pindolol. In fact, 1 ng pindolol further enhanced migration above that of the stimulatory dose of 5-HT (Fig. 6). K e t a n s e r i n (10 ng) blocked 5-HT enhancement of migration since the migration area in the presence of antagonist was between the control and 5-HT treatment (Fig. 6).
I
Antagonists for DA and NE reduced their respective monoamine effects. Apomorphine (a D~ antagonist) at 300 ng and 30 ng and metoclopramide (a D 2 antagonist) at 1 p~g and 100 ng blocked the inhibitory effects of DA (Fig. 7). Both concentrations of the [~-receptor antagonist, propranolol, (I p,g and 100 ng) reduced the NE suppression of leukocyte
CONTROL LOW
HIGH
10.0
~k
¢c 8.0 i
¢b
6.o
I
4.0 ~
2.O 0.0
i
SEROTONIN
DOPAMINE
NOREPINEPHRINE
TREA TMENT Figure 4. Relative migration area of peripheral blood leukocytes from line UNH 105 B24/B24 chicks 1 hr following in vitro exposure to m o n o a m i n e s for 30 min. Values are expressed as mean standard error. (Low = 100 ng for 5-HT, 1 p.g for DA and NE; HIGH = 1 I~g for 5-HT, 10 p.g for DA and NE). Stars indicate significant differences from the control within a m o n o a m i n e treatment (p < 0.05).
F . M . M c C o r k l e et al.
90
ab
ab
b
10.0 a
8.0
;~ 0.o I,~ 4.0 e:
2.0 0.0
CONTROL
5-HT
100 ng 10 ng PINDOLOL
100 ng 10 ng KETANSERIN
TREA TMEN T Figure 5. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to an inhibitory concentration of 5-HT or 5-HT + antagonists for 30 rain. Values are expressed as mean standard error, a = value significantly different from the control (p < 0.05); b = value significantly different from the 5-HT treatment (p < 0.05).
migration (Fig. 8). Only 1 Ixg phenoxybenzamine, an a-receptor antagonist, blocked the suppression induced by NE (Fig. 8).
PBL, although McCorkle and Simmons (14) reported its use in turkeys. Leukocytes achieved maximum migration in 1 hr. Addition of cytocholasin B to the cell mixture almost completely inhibited leukocyte migration demonstrating that the cells leave the capillary tube by innate cellular activity rather than by Brownian movement or diffusion. PBL from 7-week-old chicks migrated
Discussion This is the first report using a rapid in vitro migration technique on chicken
ab
10.0
a
a
~
a
I~ 8.0 'IT
i
6.0
NN
4.0 ~
2.0 0.0
iiiiiii ;-:.:.:.:.:.:;:
CONTROL
5-HT
10 n g
ng
PINDOLOL TREA TMEN T
10 n g
1 ng
KETANSERIN
Figure 6. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to a stimulatory concentration of 5-HT or 5-HT + antagonists for 30 min. Values are expressed as mean standard error, a = value significantly different from the control (/9 < 0.05); b = value significantly different from the 5-HT treatment (p < 0.05).
Monoamines and leukocyte migration
91
qq~ 10.0
ab
b e~ 8.o
b
s.o
~
b
~ii!i!
4.0
i!iiil
2.o.
~i~ i~!i
0.0
CONTROL
DA
300 ng 30 ng APOMORPHINE
iii!iiii
.... ili!iil
1 ug 100 ng METOCLOPRAMIDE
TREA TMEN T Figure 7. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to DA or DA + antagonists for 30 min. Values are expressed as mean standard error, a = value significantly different from the control (p < 0.05); b = value significantly different from the DA treatment (p < 0.05).
into significantly larger areas than those of 4-week-old chicks in both populations tested (Figs. 1 and 2), indicating that migration ability has matured. In vivo exposure to monoamines did not affect PBL migration in line U N H 105 chicks. The genetic heterogeneity of this line which segregates for many genes including the B complex may have masked the influence of monoamines. NE treatment resulted in a significant enhancement of PBL migration in 4week-old B24/B ~ chicks after a 30 min in vivo exposure (Fig. 2). 5-HT and DA had no significant effect on PBL migration in 4-week-old B24/B 24 chicks. In
contrast, DA significantly suppressed the leukocyte migration in 7-week-old B24/B 24 chicks. Direct action of NE and DA on leukocytes in vivo seems plausible since none of the monoamines tested cross the blood-brain barrier (26,27) to exert effects through the hypothalamic-pituitary-adrenal axis. This possibility does not exclude direct action of the monoamines on the pituitary or the adrenal since they lie outside the blood-brain barrier. Capillary tube migration represents a method of evaluating an early leukocyte response to compounds which may utlimately alter important immune functions
10.0
kl '¢
8.0
a
b
b ......~......
a
iiiiiiiiiiilil
s.o C) 4.o
~
2.o 0.0 CONTROL
NE
1 ug
100 ng
PROPANOLOL
1 ug 100 ng PHENOXYBENZAMINE
TREA TMEN T Figure 8. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to NE or NE + antagonists for 30 min. Values are expressed as mean standard error, a = value significantly different from the control (p < 0.05); b = value significantly different from the NE treatment (p < 0.05).
92
F.M. McCorkle et al.
such as antigen recognition or phagocytosis of foreign material. The study suggests that modulated monoamine levels affect immune responses of birds in agreement with reports from the mammalian literature (4-7). Age and genetic composition of the bird also influence these monoamine effects. No evidence of sex effects have been found. The increased migration area in older birds indicates a maturation of the immune system. Differential effects between line UNH 105 chicks and B24/B 24 homozygotes suggest that the latter birds may have a higher concentration of monoamine receptors, or a greater receptor sensitivity due to their different genetic composition. Additional studies are in progress to assess whether this effect is associated with the B complex or with other background genes. While the in vivo tests demonstrated an effect of the biogenic amines on leukocyte migration, the result could be due to the activation of a second messenger such as cAMP, IL-1, IL-2, or release of a hormone. In vitro exposure of leukocytes to the biogenic amines demonstrated significant alterations of leukocyte migration in both UNH 105 and B24/B24 chicks which is suggestive evidence for receptors on the leukocytes. Additional evidence for the presence of amine receptors comes from the antagonist studies. Pindolol (a mammalian 5-HT 1 receptor antagonist) blocked the suppressive but not the enhancing effects of 5-HT. Ketanserin (a mammalian 5-HT2 receptor antagonist), blocked both enhancing and suppressing effects of 5-HT. Therefore, these results suggest that 5-HTI and 5-HT2 receptors are present on chicken leukocytes. Similar consequences were found with DA an-
tagonists. The in vitro DA response, suppressed leukocyte migration, was blocked by apomorphine (a mammalian D I receptor antagonist and'by metoclopramide (a mammalian D2 receptor antagonist), suggesting that chicken leukocytes possess D 1 and D z dopamine receptors. The presence of ot and 13 NE receptors on chicken leukocytes is demonstrated by the blockage of NE suppression in the presence of phenoxybenzamine and propanolol respectively. Monoamine receptors have been found on mammalian lymphocyte and monocyte surface membranes (22-25). The present study suggests that there may be avian receptors similar to both types of known mammalian receptors for each amine (5-HT, DA, and NE) on avian leukocytes. Monoamine effects may be mediated by these leukocyte cell surface receptors to influence the synthesis of rate-limiting enzymes as suggested in mice by Besedovsky and coworkers (7). The present study does not explain if the biogenic amines are altering leukocyte migration by direct action following attachment to the receptors or by indirect action through cAMP, IL-1, IL-2, or other intercellular messengers. These modes of action could be the basis for genetic differences observed between the line UNH 105 and B24/B 24 populations.
Acknowledgements--The
authors
thank
Jackie Packard for clerical assistance and Tom Thorpe for animal care. Critical reviews of the manuscript by P. J. Battista, W. M. Collins, and D. G. Simmons are gratefuly acknowledged. Scientific Contribution No. 1457 of the New Hampshire Agricultural Experiment Station.
References 1. Krulich, L.; Fawcett, C. P. The hypothalamic h y p o p h y s i o t r o p i c h o r m o n e s . I n t . Rev. Physiol. 16:35-92; 1977.
2. Gisler, R. H. Stress and the hormonal regulation of the immune response in mice. Psychother. Psychosom. 23:197-208; 1974.
Monoamines and leukocyte migration
3. Solomon, G. E; Ammkraut, A. A.; Kasper, P. Immunity, emotions and stress. Ann. Clin. Res. 6:313-319; 1974. 4. Bliznakov, E. G. Serotonin and its precursors as modulators of the immunological responsiveness in mice. J. Med. 11:81-105; 1980. 5. Devoino, L. V.; Ilyutchenok, R. Influence of some drugs on the immune response II. Effects of serotonin, 5-hydroxytryptophan, reserpine, and iproniazid on delayed hypersensitivity. Eur. J. Pharmacol. 4:449-456; 1968. 6. Jackson, J. C.; Cross, R. J.; Walker, R. F.; Markesbery, W. R.; Brooks, W. H.; Roszman, T. L. Influence of serotonin on the immune response. Immunology 54:505-512; 1985. 7. Besedovsky, H. O.; Del Rey, A.; Sorkin, E.; DaPrada, M.; Keller; H. H. Immunoregulation mediated by the sympathetic nervous system. Cell. Immunol. 48:346-355; 1979. 8. Gray, R.; McCorkle, E M.; Taylor, R. L., Jr. Effect of serotonin on plaque-forming cells in chickens. Poultry Sci. 65(Suppl. 1):50, 1986. 9. McCorkle, F. M.; Gray, R.; Lukacs, N.; Taylor, R. L., Jr. Effect of dopamine on plaque-forming cells and delayed hypersensitivity in chickens. Proc. 6th Int. Cong. Immunol. 476; 1986. 10. McCorkle, E M.; Stinson, R.; Glick, B. A biphasic graft vs. host response in aging chickens. Cell. Immunol. 46:208-212; 1979. I1. Lukacs, N.; McCorkle, E M.; Taylor, R. L., Jr. Suppression of the phytohemagglutinin wattle response by biogenic amines. Dev. Comp. Immunol. 11:759-768; 1987. 12. Edens, E W.; McCorkle, E M.; Simmons, D. G.; Yersin, A. G. Effects of Bordetella avium on lymphoid tissue monoamine concentrations in turkey poults. Avian Dis. 31:746751; 1987. 13. Edens, E W.; McCorkle, F. M.; Simmons, D. G.; Yersin, A. G. Brain monoamine concentrations in turkey poults infected with Bordetella avium. Avian Dis. 31:504-508; 1987. 14. McCorkle, E M.; Simmons, D. G. In vitro migration of leukocytes from turkey poults infected with Alcaligenes faecalis. Avian Dis. 28:853-857; 1984. 15. Briles, W. E.; Briles, R. W. Identification of haplotypes of the chicken major histocompati-
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16. 17.
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20. 21. 22.
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bility complex (B). Immunogenetics 15:449459; 1982. Tennant, J. R. Evaluation of the trypan blue technique determination of cell viability. Transplantation 2:685-694; 1964. Tricklebank, M. D. The behavioral response to 5-HT receptor agonists and subtypes of the central 5-HT receptor. Trends Pharmacol. Sci. 6:403-407; 1985. Kilpatrick, G. J.; E1 Tayar, N.; Van De Watrebeemd, H.; Jenner, P.; Testa, B.; Marsden, C. D. The thermodynamics of agonist and antagonist binding to dopamine D-2 receptors. Mol. Pharmacol. 30:226-234; 1986. Fitzgerald, J. D. 13-adrenoceptor antagonists. In: van Zwieten, P. A., ed. Handbook of hypertension, vol. 3: pharmacology of antihypertensive drugs. Amsterdam: Elsevier, 1984: p. 249-260. Goth, A. Medical pharmacology, 7th ed. St. Louis: C. V. Mosby; 1974: p. 149-159. Steel, R. G. D.; Torrie, J. H. Principles and procedures of statistics, New York: McGrawHill; 1980. Elisieva, L. S.; Stefanovich, L. E. Specific binding of serotonin by blood leukocytes and peritoneal cells in the mouse. Biokhimica 47:810-815; 1982. Fleminger, S.; Jenner, P.; Marsden, C. D. Are dopamine receptors present on human lymphocytes? J. Pharm. Pharmacot. 34:658-663; 1982. Loveland, B. E.; Jannott, B.; McKenzie, I. E The detection of 13-adrenoceptors on immune lymphocytes. Int. J. lmmunopharmacol. 3:45-51; 1981. Maslinski, W.; Grabczewska, E.; Ryzewski, J. Acetylcholine receptors of rat lymphocytes. Biochem. Biophys. Acta. 633:269-273; 1980. Axelrod, J.; Weil-Malherbe, H.; Tomchick, R. The physiological disposition of 3H-epinephrine and its metabolite metanephrine. J. Pharmacol. Exp. Ther. 137:251-256; 1959. Douglas, W. W. Histamine and antihistamine; 5-hydroxytryptamine and antagonists. In: Goodman, L. S.; Gilman, A., eds. The pharmacological basis of therapeutics. New York: Macmillan; 1970:645-662.
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
MONOAMINES ALTER IN VITRO MIGRATION OF CHICKEN LEUKOCYTES Fred M. M c C o r k l e , Robert L. Taylor, Jr.,* Kelly M. Denno, and J. Mike Jabe Department of Biology, Central Michigan University, Mt. Pleasant, MI 48859 and *Department of Animal and Nutritional Sciences, University of New Hampshire, Durham, NH 03824 (Submitted August 1988;Accepted September 1988) DAbstract--The effect of the biogenic amines Introduction serotonin (5-HT), dopamine (DA), and norepinephrine (NE) on peripheral blood leukocyte Neurotransmitters and hormones regu(PBL) migration was studied in two populalate physiologic processes throughout tions derived from line UNH 105 New Hampthe body (1). In mammals or birds, adshire chickens. Maximum migration from capministration of androgenic, estrogenic, illary tube migration chambers was achieved in and adrenocortical steroid hormones 1 hr. An age effect in both populations was incause acute involution of the lymphoid dicated by significantly larger migration areas organs which alters both antibody and found in leukocytes from 7-week-old chickens cell-mediated immune responses (2,3). compared to those of 4-week-old chicks. Thirty min after intravenous monoamine injection, Bliznakov (4) demonstrated that mulline UNH 105 PBL migration was unaffected tiple doses of serotonin (5-HT) depress by exogenous monoamines. In the second popT-cell dependent, primary, humoral imulation, B24/B~ chicks, NE enhanced migramune responses in mice. Administration tion at 4 weeks of age but DA suppressed miof 5-HT reduces both IgM and IgG gration at 7 weeks of age. In vitro exposure of plaque-forming cell (PFC) response to PBL to the biogenic amines also affected leusheep red blood cells (SRBC) in CBA kocyte migration. Migration was augmented m i c e (5,6). N o r e p i n e p h r i n e (NE) by 100 ng 5-HT but suppressed by 1 p.g 5-HT strongly suppresses the in vitro antiin UNH 105 chicks. Furthermore, DA supSRBC response of murine spleen cells pressed PBL migration and NE enhanced mibut dopamine (DA) enhances PFC folgration in the same population. PBL from B24/B~ chicks were not affected by in vitro exlowing immunization (7). posure to 5-HT, however, DA enhanced miBoth antibody and cellular immunity gration whereas NE suppressed migration. in chickens are affected by exogenous Specific antagonists for 5-HT, DA, and NE monoamines. Chicken IgM and IgG PFC blocked the effects of each monoamine sugagainst SRBC were lowered significantly gesting that receptors are present on chicken when 5-HT or DA was injected 30 min leukocytes. These receptors mediate action of prior to antigen (8,9) in agreement with the monoamines on leukocyte migration acthe mammalian data. The phytohemagtivity.
glutinin (PHA) wattle response, a T-cellmediated response (10), was also depressed by the same single dose monoamine treatment. Either 5-HT, DA, or NE given 30 min prior to PHA significantly reduced subsequent wattle swelling (11). Just as monoamine administration reduced immune responses, pathologic sequelae which stimulated an immune re-
[]Keywords--Biogenic amines; Chicken, Leukocyte migration; Serotonin; Dopamine; Norepinephrine.
Address correspondence to Dr. Robert L. Taylor, Jr., Department of Animal and Nutritional Sciences, University of New Hampshire, Kendall Hall, Durham, NH 03824. 85
86
sponse reduced monoamine concentrations. The levels of 5-HT, DA, and NE were d e p r e s s e d significantly in the brain, spleen, thymus, and bursa of Fabricius of turkey poults infected with Bordetella avium, a mild upper respiratory pathogen that causes rhinotracheitis (12,13). Furthermore, in vitro leukocyte migration from B. avium-infected poults was enhanced compared to uninfected poults (14). This enhanced migration which occurred in the context of a complex disease process may be attributed to primary or secondary effects of the bacterial infection. Since immune stimulation increased leukocyte migration and lowered monoamine levels, the influence of increased monoamine concentrations on migration was questioned. This study was designed to determine whether in vivo and in vitro 5-HT, DA, and NE would disturb in vitro migration of chicken peripheral blood leukocytes (PBL).
Materials and Methods Animals Chicks of mixed sexes from line U N H 105, a noninbred line of New Hampshires, were used throughout this study. The line, originally obtained from a commercial breeder, is maintained at the University of New Hampshire Poultry Research Farm. Two populations were studied. The first, U N H 105, segregates for four B blood group haplotypes B 22, B 23, B 24, and B 26. The B blood group locus is a marker for the chicken major histocompatibility complex (15). These four haplotypes have approximate frequencies of 0.09, 0.20, 0.39, and 0.32, respectively, within line U N H 105. The second population derived from U N H 105, was homozygous for the B24/B24 genotype and was designated B24/B 24. Chicks were vaccinated for Marek's disease at hatching and housed in brooder
F.M. McCorkle et al.
batteries with free access to feed and water.
Monoamines 5-hydroxytryptamine (5-HT), 3-hydroxytyramine (DA), and arterenol (NE) were obtained from Sigma Chemical Company, St. Louis, Missouri. Monoamines were dissolved in physiologic saline and injected at the following doses: 5-HT, 100 I~g/kg of body weight; DA and NE, I mg/kg of body weight.
Leukocyte Collection In each of two trials, either a monoamine solution or a saline control was injected into separate groups of 10 birds of each population at 4 or 7 weeks of age. Thirty min after injection, heparinized blood was collected by cardiac puncture. Leukocytes, a heterogeneous population of lymphocytes, granulocytes, and monocytes, were separated by centrifugation (15 rain, 100 x g), removed as the buffy coat and washed three times in RPMI 1640 cell-culture medium (pH 7.4). Cell viability was assessed by the trypan-blue exclusion (16) after which cell numbers were adjusted to approximately 2 × 108 cells/ml. Cytochalasin B, a known inhibitor of cell membrane motility, was added to the reaction mixture to demonstrate inhibition of cell migration (14).
Cell Migration Nonheparinized capillary tubes (1.25 x 75 mm) were filled with the cell suspension and sealed with a clay-type tube sealer. The capillary tubes were centrifuged at 500 × g for 3 min and were cut evenly at the cell-fluid interface. The cell packs were placed into 24-well plates
Monoamines and leukocyte migration
87
supported by a small amount of vacuum grease (Dow Corning Corp., Midland, MI). The wells were filled with RPMI 1640, pH 7.4, and incubated at 37°C for 1 h. Three replicate capillary tubes were used per bird (14). The length and width of each migration zone was measured using an ocular scale on a dissecting microscope. The relative area of cell migration was calculated by the formula [(length x width)/10,000].
amine) and antagonist 2 (concentration I/I0 & 1/100 of biogenic amine). Leukocytes were treated with the antagonist in vitro for 30 min. After the cells were washed, all groups except controls were treated with the biogenic amine (5-HT, DA or NE) in vitro for 30 min followed by a second wash. Leukocytes were placed into capillary tubes for migration assay. This procedure was repeated in a second trial.
In Vitro Exposure
Statistical Analysis
Two trials were conducted. Leukocytes were collected as described above and s a m p l e s w e r e a l i q u o t e d into 3 groups: control, 5-HT--100 ng and 1 ~g or D A - - 1 Ixg and 10 I~g or N E - - 1 I~g and 10 Ixg. A control and two doses of a single monoamine were tested simultaneously. Leukocytes were exposed for 30 min on ice, washed 3 times, and placed in capillary tubes for cell migration.
The data were evaluated by two-way analysis of variance (21). Differences between monoamine treatments and controls within ages were assessed by Dunnett's test at the 0.05 level of significance. Means in the antagonist study were compared by orthogonal contrasts.
Antagonists Pindolol (Sigma) is a 5-HTI antagonist while ketanserin (Janssen Pharmaceutica Inc., Beerse, Belgium) is a 5-HT2 antagonist (17). Apomorphine (Sigma) and metoclopramide (Sigma) are D1 and D2 receptor antagonists (18), respectively. Propranolol (Sigma) is a 13-receptor antagonist (19) and phenoxybenzamine (Ciba-Giegy, Summit, N J) is an a-receptor antagonist (20).
In Vitro Exposure with Antagonists Peripheral blood leukocytes were collected from ten 7-week-old birds and washed. Leukocytes were aliquoted into 6 groups: c o n t r o l , biogenic amine treated in vitro for 30 min, antagonist 1 (Concentration 1/10 & 1/100 of biogenic
Results Peripheral blood leukocytes of 7week-old chickens of both populations had significantly larger migration areas than those of 4-week-old birds (Figs. 1 and 2). None of the monoamines tested (5-HT, DA and NE) significantly affected migration of leukocytes in line U N H 105 chicks (Fig. I). In contrast, NE increased significantly the migration area in 4-week-old B24/B24 chicks (Fig. 2), over that of the control. 5-HT and DA did not affect migration. DA suppressed significantly the migration area of PBL relative to that of the control (Fig. 2) in 7-week-old B24/B24 chicks but 5-HT and NE had no effect. With in vitro exposure of peripheral blood leukocytes to the biogenic amines, cell migration was significantly enhanced by 100 ng 5-HT and significantly suppressed by 1 ~g 5-HT in U N H 105 chicks (Fig. 3). No significant differences were found with in vitro exposure to 5-HT in BZ4/B24 chicks (Fig. 4). DA
88
F.M. McCorkle et al.
I
CONTROL 5-HT DA NE
12.0 lO.O 8.0
/
4.0 r~
2.0 0.0
4 WEEKS
7 WEEKS AGE
Figure 1. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vivo exposure to monoamines for 30 min. Values are expressed as mean standard error.
s i g n i f i c a n t l y s u p p r e s s e s UNH 105 chicks but enhances significantly cell migration in B24/B 24 (Figs. 3 and 4). Conversely, NE enhances significantly migration area in UNH 105 but is significantly suppressed in B24/B 24 (Figs. 3 and 4). 5-HT suppressed leukocyte migration at a dose of 1 Ixg. Both 100 ng and 10 ng pindolol, a 5-HT Z antagonist, blocked
Ill
12.0
"T ~u ~: 10.o
the suppressive effect of 5-HT as evidenced by the significant differences between migration areas (Fig. 5). Ketanserin, a 5-HT 2 receptor antagonist, arr e s t e d 5-HT s u p p r e s s i o n of P B L migration. Ten ng ketanserin produced significantly higher migration while I00 ng of the antagonist resulted in a migration area between those of the control and the 5-HT treatment (Fig. 5). The en-
CONTROL 5-HT
~DA ~ NE
"T 8.o s.o
i,., 'q:
4,0
r~
2.0 0.0
i
i
4 WEEKS
7 WEEKS
AGE Figure 2. Relative migration area of peripheral blood leukocytes from line UNH 105 B24/B24 chicks 1 hr following in vivo exposure to monoamines for 30 min. Values are expressed as mean standard error. Stars indicate significant differences from the control within an age (p < 0.05).
Monoamines and leukocyte migration
89
CONTROL
I
LOW
HIGH 10.0 1
1
L
l
I
6.0-
I,.,.
4.o-
r~ 2.00.0
SEROTONIN
DOPAMINE
NOREPINEPHRINE
TREA TMEN T Figure 3. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to m o n o a m i n e s for 30 rain. Values are expressed as mean standard error. (Low = 100 ng for 5-HT, 1 p.g for DA and NE; HIGH = 1 p~g for 5-HT, 10 p.g for DA and NE). Stars indicate significant differences from the control within a m o n o a m i n e treatment (p < 0.05).
hancing concentration 5-HT (I00 ng) was not blocked by pindolol. In fact, 1 ng pindolol further enhanced migration above that of the stimulatory dose of 5-HT (Fig. 6). K e t a n s e r i n (10 ng) blocked 5-HT enhancement of migration since the migration area in the presence of antagonist was between the control and 5-HT treatment (Fig. 6).
I
Antagonists for DA and NE reduced their respective monoamine effects. Apomorphine (a D~ antagonist) at 300 ng and 30 ng and metoclopramide (a D 2 antagonist) at 1 p~g and 100 ng blocked the inhibitory effects of DA (Fig. 7). Both concentrations of the [~-receptor antagonist, propranolol, (I p,g and 100 ng) reduced the NE suppression of leukocyte
CONTROL LOW
HIGH
10.0
~k
¢c 8.0 i
¢b
6.o
I
4.0 ~
2.O 0.0
i
SEROTONIN
DOPAMINE
NOREPINEPHRINE
TREA TMENT Figure 4. Relative migration area of peripheral blood leukocytes from line UNH 105 B24/B24 chicks 1 hr following in vitro exposure to m o n o a m i n e s for 30 min. Values are expressed as mean standard error. (Low = 100 ng for 5-HT, 1 p.g for DA and NE; HIGH = 1 I~g for 5-HT, 10 p.g for DA and NE). Stars indicate significant differences from the control within a m o n o a m i n e treatment (p < 0.05).
F . M . M c C o r k l e et al.
90
ab
ab
b
10.0 a
8.0
;~ 0.o I,~ 4.0 e:
2.0 0.0
CONTROL
5-HT
100 ng 10 ng PINDOLOL
100 ng 10 ng KETANSERIN
TREA TMEN T Figure 5. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to an inhibitory concentration of 5-HT or 5-HT + antagonists for 30 rain. Values are expressed as mean standard error, a = value significantly different from the control (p < 0.05); b = value significantly different from the 5-HT treatment (p < 0.05).
migration (Fig. 8). Only 1 Ixg phenoxybenzamine, an a-receptor antagonist, blocked the suppression induced by NE (Fig. 8).
PBL, although McCorkle and Simmons (14) reported its use in turkeys. Leukocytes achieved maximum migration in 1 hr. Addition of cytocholasin B to the cell mixture almost completely inhibited leukocyte migration demonstrating that the cells leave the capillary tube by innate cellular activity rather than by Brownian movement or diffusion. PBL from 7-week-old chicks migrated
Discussion This is the first report using a rapid in vitro migration technique on chicken
ab
10.0
a
a
~
a
I~ 8.0 'IT
i
6.0
NN
4.0 ~
2.0 0.0
iiiiiii ;-:.:.:.:.:.:;:
CONTROL
5-HT
10 n g
ng
PINDOLOL TREA TMEN T
10 n g
1 ng
KETANSERIN
Figure 6. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to a stimulatory concentration of 5-HT or 5-HT + antagonists for 30 min. Values are expressed as mean standard error, a = value significantly different from the control (/9 < 0.05); b = value significantly different from the 5-HT treatment (p < 0.05).
Monoamines and leukocyte migration
91
qq~ 10.0
ab
b e~ 8.o
b
s.o
~
b
~ii!i!
4.0
i!iiil
2.o.
~i~ i~!i
0.0
CONTROL
DA
300 ng 30 ng APOMORPHINE
iii!iiii
.... ili!iil
1 ug 100 ng METOCLOPRAMIDE
TREA TMEN T Figure 7. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to DA or DA + antagonists for 30 min. Values are expressed as mean standard error, a = value significantly different from the control (p < 0.05); b = value significantly different from the DA treatment (p < 0.05).
into significantly larger areas than those of 4-week-old chicks in both populations tested (Figs. 1 and 2), indicating that migration ability has matured. In vivo exposure to monoamines did not affect PBL migration in line U N H 105 chicks. The genetic heterogeneity of this line which segregates for many genes including the B complex may have masked the influence of monoamines. NE treatment resulted in a significant enhancement of PBL migration in 4week-old B24/B ~ chicks after a 30 min in vivo exposure (Fig. 2). 5-HT and DA had no significant effect on PBL migration in 4-week-old B24/B 24 chicks. In
contrast, DA significantly suppressed the leukocyte migration in 7-week-old B24/B 24 chicks. Direct action of NE and DA on leukocytes in vivo seems plausible since none of the monoamines tested cross the blood-brain barrier (26,27) to exert effects through the hypothalamic-pituitary-adrenal axis. This possibility does not exclude direct action of the monoamines on the pituitary or the adrenal since they lie outside the blood-brain barrier. Capillary tube migration represents a method of evaluating an early leukocyte response to compounds which may utlimately alter important immune functions
10.0
kl '¢
8.0
a
b
b ......~......
a
iiiiiiiiiiilil
s.o C) 4.o
~
2.o 0.0 CONTROL
NE
1 ug
100 ng
PROPANOLOL
1 ug 100 ng PHENOXYBENZAMINE
TREA TMEN T Figure 8. Relative migration area of peripheral blood leukocytes from line UNH 105 chicks 1 hr following in vitro exposure to NE or NE + antagonists for 30 min. Values are expressed as mean standard error, a = value significantly different from the control (p < 0.05); b = value significantly different from the NE treatment (p < 0.05).
92
F.M. McCorkle et al.
such as antigen recognition or phagocytosis of foreign material. The study suggests that modulated monoamine levels affect immune responses of birds in agreement with reports from the mammalian literature (4-7). Age and genetic composition of the bird also influence these monoamine effects. No evidence of sex effects have been found. The increased migration area in older birds indicates a maturation of the immune system. Differential effects between line UNH 105 chicks and B24/B 24 homozygotes suggest that the latter birds may have a higher concentration of monoamine receptors, or a greater receptor sensitivity due to their different genetic composition. Additional studies are in progress to assess whether this effect is associated with the B complex or with other background genes. While the in vivo tests demonstrated an effect of the biogenic amines on leukocyte migration, the result could be due to the activation of a second messenger such as cAMP, IL-1, IL-2, or release of a hormone. In vitro exposure of leukocytes to the biogenic amines demonstrated significant alterations of leukocyte migration in both UNH 105 and B24/B24 chicks which is suggestive evidence for receptors on the leukocytes. Additional evidence for the presence of amine receptors comes from the antagonist studies. Pindolol (a mammalian 5-HT 1 receptor antagonist) blocked the suppressive but not the enhancing effects of 5-HT. Ketanserin (a mammalian 5-HT2 receptor antagonist), blocked both enhancing and suppressing effects of 5-HT. Therefore, these results suggest that 5-HTI and 5-HT2 receptors are present on chicken leukocytes. Similar consequences were found with DA an-
tagonists. The in vitro DA response, suppressed leukocyte migration, was blocked by apomorphine (a mammalian D I receptor antagonist and'by metoclopramide (a mammalian D2 receptor antagonist), suggesting that chicken leukocytes possess D 1 and D z dopamine receptors. The presence of ot and 13 NE receptors on chicken leukocytes is demonstrated by the blockage of NE suppression in the presence of phenoxybenzamine and propanolol respectively. Monoamine receptors have been found on mammalian lymphocyte and monocyte surface membranes (22-25). The present study suggests that there may be avian receptors similar to both types of known mammalian receptors for each amine (5-HT, DA, and NE) on avian leukocytes. Monoamine effects may be mediated by these leukocyte cell surface receptors to influence the synthesis of rate-limiting enzymes as suggested in mice by Besedovsky and coworkers (7). The present study does not explain if the biogenic amines are altering leukocyte migration by direct action following attachment to the receptors or by indirect action through cAMP, IL-1, IL-2, or other intercellular messengers. These modes of action could be the basis for genetic differences observed between the line UNH 105 and B24/B 24 populations.
Acknowledgements--The
authors
thank
Jackie Packard for clerical assistance and Tom Thorpe for animal care. Critical reviews of the manuscript by P. J. Battista, W. M. Collins, and D. G. Simmons are gratefuly acknowledged. Scientific Contribution No. 1457 of the New Hampshire Agricultural Experiment Station.
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Monoamines and leukocyte migration
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