Immunomodulation by Bromocriptine

Eva Nagy, I. Berczi, Graham E. Wren, Sylvia L. Asa, and K. Kovacs

Abstract: Treatment of rats with the dopaminergic ergot alkaloid bromocriptine (BRC) inhibited the following immune reactions: contact sensitivity skin reaction to dinitrochlorobenzene (DNCB); antibody formation to sheep red blood cells and to bacterial lipopolysaccharide; adjuvant arthritis; and experimental allergic encephalitis. Immunosuppressive doses of BRC (5 mg/kg) decreased the serum prolactin (PRL) levels from 84.8 +_ 15.9 ng/ml to 4.9 -4- 1.6 ng/ml. Further studies on DNCB contact sensitivity and on antibody formation revealed that the immunocompe- tence of BRC-suppressed animals could be restored by additional treatment with either prolactin (PRL) or growth hormone (GH). Treatment with adrenocorticotropic hormone antagonized the restoring effect of PRL and GH. These results suggest that BRC suppressed immunity by its inhibition of PRL, and possibly also by inhibition of GH secretion.

Key Words: ACTH; Autoimmunity; Bromocriptine; Cellular immunity; Growth hormone; Humoral immu- nity; Prolactin; Rat

INTRODUCTION

Numerous observations indicate that hormones can influence immune reactions (Ahlqvist, 1976). The immunosuppressive action of corticosteroids is well established (Claman, 1975). Adrenocor- ticotropic hormone (ACTH) has a similar effect, although this has been studied to a lesser extent (Comsa and Leonhardt, 1975). Furthermore, it has been shown that growth hormone is needed for the maturation and function of the immmune system (Baroni et al., 1969; Comsa et al., 1974; Fabris et al.; Gisler and Schenkel-Hullinger, 1971). The effect of other pituitary hormones on immune reactions has not been studied in detail.

We have previously demonstrated that humoral, cell-mediated, and autoimmune reactions are impaired in hypophysectomized (Hypox) rats (Nagy and Berczi, 1978), and that the immunocom- petence of Hypox rats could be restored by syngeneic pituitary grafts or by daily treatment with prolactin (Berczi et al., 1981; Berczi and Nagy, 1982; Nagy and Berczi, 1981). Here, we report the effect of bromocriptine on various immune reactions.

Received April 5, 1983; revised and accepted June 7, 1983. From the Immunology Department, Faculty of Medicine, University of Manitoba, 795 McDermot Avenue,

Winnipeg, Manitoba, Canada (E.N.; I.B.; G.W.) and the Pathology Department, University of Toronto, St. Michael's Hospital, 30 Bond Street, Toronto, Ontario, Canada (S.A.; K.K.).

Address requests for reprints to: Dr. Istvan Berczi, The University of Manitoba, Department of Immunol- ogy, 730 William Avenue, Winnipeg, Manitoba R3E OW3 Canada.

© Elsevier Science Publishing Co., Inc., 1983 231 52 Vanderbilt Ave., New York, N.Y. Immunopharmacology 6, 231-243 ( 1 9 8 3 ) 0162-3109/83/$03.00

232 E. Nagy et al.

MATERIALS AND METHODS

Animals

Male and female Fischer (F) rats were obtained from Canadian Breeding Farm Laboratories, Ltd. (Montreal, Quebec, Canada); female Wistar-Furth (W) and female Lewis (L) rats were purchased from A R S - S p r a g u e - D a w l e y (Madison, WI). All the animals weighed 150 -170 g and were maintained on a standard diet (Wayne's Laboratory Blocks, with 6% fat content, Chicago, IL) and on water supplied ad libitum.

Surgical Procedures

Hypox was performed by the parapharyngeal approach (Lostroh and Jordan, 1955; Tarttelin and Gorski, 1972). Some of the Hypox animals received syngeneic pituitary glands under the kidney capsule one week after Hypox (Lu et al., 1977). Immunization was started 2 - 3 weeks after Hypox, as specified in the text. The completeness of Hypox and graft acceptance was determined for each animal by autopsy at the end of the experiment.

Immunization

Antibody Formation

Sheep red blood cells (SRBC) were washed three times with phosphate-buffered saline (PBS) at pH 7.2 and 107 cells were injected i.p. to each animal in I mlof PBS. The rats were bled from the tail veins at the time of injection and on every third day afterwards. The serum samples from each animal were individually stored at -20°C and titrated by hemagglutination (Takatsy, 1955) at the end of the experiments. Thymus independent anti- body response was induced againstE, coil 055:B5 lipopolysaccharide (LPS, Difco) (Treiber and Lapp, 1978). Syngeneic rat red blood cells (RRBC) were collected by cardiac puncture with heparin (25 units per ml), were washed three limes in PBS and then incubated with LPS (1 mg per ml of PBS) at 37°C for 45 min. The LPS solution used for coating RRBC was heated at 100°C for 1 hr prior to use. The coated RRBC were washed again and 8 × 10 s cells were injected i.p. to each animal. The animals were sampled as previously described and sera were titrated with LPS coated RRBC by hemagglutination.

Contact Sensitivity

For the induction of contact dermatitis, dinitrochlorobenzene (DNCB) was dissolved at 200 mg/ml concentration in acetone and 20 p,1 was applied to the shaved skin on the dorsal region to an approximate area of 1 cm 2. The diameters of the inflamed skin lesions were measured daily in two diagonal directions and the mean affected skin areas _+ Standard Errors were calculated for each group. DNCB-treated skin areas were excised from control and treated animals on day 6 and fixed in 10% buffered formalin (pH 7.6).

Abbreviations. AA: adjuvant arthritis; ACTH: adrenocorticotropic hormone; BGH: bovine growth hormone; BPRL: bovine prolactin; BRC: bromocriptine; DNCB: dinitrochlorobenzene; EAE: experimental allergic encephalitis; F: Fischer; GH: growth hormone; HCG: human chorionic gonadotropin; Hypox: hypophysectomy; L: Lew/s; LPS: lipopolysaccharide of E. coil 055: B5; PBS: phosphate buffered saline, pH 7.2; PRL: prolactin; RRBC: rat red blood cells; SPG: syngeneic pituitary graft; SRBC: sheep red blood cells; TSH: thyroid stimulating hormone; W: Wistar-Furth.

Immunomodulation by Bromocriptine 233

Skin Allografts Female F rats were grafted with the tail skin of female W animals on the flank, and vice versa. The gypsona bandages protecting the grafts were removed after 7 days, when most grafts were fully vascularized. Graft rejection was followed by daily observation and was considered complete when the entire graft became necrotic.

Adjuvant Arthritis

AA was induced by injecting the right hind leg with 0.1 ml of Freund's complete adjuvant which contained 5 mg/ml of Mycobacterium tuberculosis. Such treatment caused a severe inflammation of all four footpads, which reached the maximum at about day 12 after treatment. The diameter of swollen footpads was measured with a caliper every third day until day 15.

Induction of Experimental Allergic Encephalitis (EAE) EAE was induced in L rats. Rat spinal cords were stored frozen at -20°C, thawed, homogenized, and frozen at -70°C, which was followed by lyophilization. The lyophilized material was then reconstituted to twice its original weight using distilled water and emulsified at a 1 : I ratio in Freund's complete adjuvant which contained M. tuberculosis strain H37Ra (Difco 3113-59). One week after the commencement of BRC treatment, two intradermal injections of emulsion were made on the back and one on the dorsal aspects of each paw. A total of 0.25 ml emulsion was injected into each rat. The severity of the developing encephalomyelitis was scored twice daily, using the following system: I point was given for partial impairment of responsiveness to the environment (e.g., to noises and finger flicks), as well as for the partial paralysis of each of the tail, hind limbs, and bladder. For complete unresponsiveness and complete paralysis of each of the tail, hind limb, or bladder, 2 points were given. Full points were given for all the above parameters plus 2 points if the death of an animal was attributable to EAE, which brought up the score to a maximum of I0. The experiments were terminated when symptoms were no longer present in any of the animals, usually two weeks after the onset of disease. All the animals were killed and halves of their brains were preserved in 10% buffered formalin (pH 7.6).

Histology Sections (5 -t~m thick) were made from the paraffin-imbedded brains and skin biopsies and were stained with hematoxylin-eosin. Coded slides were examined and scored independently by two pathologists; the final results of evaluation are presented here.

Hormone and Drug Treatment Porcine adrenocorticotropic hormone (ACTH) was purchased from Sterivet Laboratories (Bolton, Ontario, Canada); human thyroid stimulating hormone (TSH, Thyrotron) from Nordic Pharmaceuticals, Ltd. (Laval, Quebec, Canada); and human chorionic gonadotropin (HCG, Choriogonin) from Calbiochem. Bovine prolactin (BPRL, NIAMDD-BPRL-6) and bovine growth hormone (BGH, NIH-GH-B18) were the generous gift of Dr. Salvatore Raiti, through the National Pituitary Agency, Baltimore, MD. All hormones were administered s.c. daily in various doses as specified for the various experiments. ACTH and HCG were dissolved in oil and the remaining hormones in saline.

Tablets of bromocriptine (Padodel-Sandoz) were dissolved with a few drops of 70% ethyl alcohol and brought to 10 mg/ml concentration with phosphate buffered saline, pH 7.2. Treat-

234 E. Nagy et al.

ment of normal rats was initiated one week before immunization and 5 mg/kg daily doses were given s.c. until the termination of the experiment, if not indicated otherwise.

Evaluation of Experiments

Mean values + 'S.E. were calculated from data obtained in experimental and control groups on the same day of observation and the results are presented in the Figures and Tables. The Students' t- test was used to determine the significance of differences. In EAE experiments, starting with the day on which symptoms first occurred, mean daily scores were compared for a two-week period in the one-way analysis of variance test.

RESULTS

Inhibition of Contact Sensitivity by Bromocriptine

As illustrated by the results presented in Figure 1, administration of 5 mg/kg of BRC daily to W female animals completely suppressed DNCB contact sensitivity, whereas 2.5 mg/kg induced only partial inhibition. A subsequent experiment (see legends to Figure 5) revealed that this immu- nosuppressive dose of BRC also reduced the serum level of PRL from 84.8 _+ 15.9 to 4.9 + 1.6 ng/ml. Bovine prolactin given in 40 and 60 p,g daily doses failed to restore the reactivity of BRC- suppressed animals. As revealed in subsequent experiments (Figure 2), treatment with 100 t~g of BPRL reversed immunosuppression in BRC-suppressed animals. Additional injections of ACTH significantly inhibited the immunorestoring effect of BPRL. The immunocompetence of BRC- suppressed animals could be restored as well by BGH treatment, which was also antagonized by ACTH (Figure 3).

The histological changes induced by DNCB in the skin of normal rats were found to be identical to those described by Willoughby et al. (1965). The characteristic lesion was a large ulcer covered by an amorphous crust consisting of necrotic debris, fibrin fibers, polymorphonuclear leukocytes, and mononuclear inflammatory cells. The dermis was infiltrated in a predominantly perivascular distribution by lymphocytes and macrophages. In the skins of non-Hypox, BRC-treated rats and of Hypox untreated rats, the lesions were minimal. No ulceration was evident and edema of the dermis and inflammatory cell infiltration were very slight. The histologic changes in the skins of BRC-suppressed and GH- or PRL-treated rats did not differ in intensity and extent from those seen in the non-Hypox, untreated rats. ACTH administration to such animals reduced the severity of the histologic changes. The results of the histological studies are summarized in Table 1 and illustrated in Figure 4.

Inhibition of Antibody Formation by BRC

Humoral immune reactions to thymus-dependent (SRBC) and thymus-independent antigens were suppressed by BRC as they were by Hypox (Figures 5 and 6). The immunosuppressive effect of BRC could again be reversed by treatment with BPRL (Figure 5). In the experiment illustrated in Figure 5, serum levels of PRL were monitored by radioimmunoassay from day 0 to day 15 of immunization. As is obvious from the results given in the legends of this Figure, 5 mg/kg of BRC suppressed serum PRL levels to less than 10% of normal. Pituitary grafts placed under the kidney capsule were able to reconstitute completely the LPS-response of Hypox rats. Bovine prolactin was also capable of restoration, whereas BGH, given in combination with ACTH, HCG, and TSH was ineffective (Figure 6).

Suppression of Adjuvant Arthritis by BRC Studies performed on adjuvant arthritis and skin allograft rejection are presented in Table 2. Bromocriptine significantly suppressed adjuvant arthritis. However, it is clear that the survival of skin allografts could be influenced only minimally, if at all, by BRC treatment.

Immunomodulation by Bromocriptine 235

04

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Figure 1 The inhibition of DNCB contact sensitivity by bromocriptine is dose-dependent. Groups of 5 female Wistar-Furth rats were treated with bromocriptine (2.5 or 5 mg/kg s.c. daily) for one week and then challenged, along with a group of normal controls, with DNCB. Treatment with bovine prolactin was initiated on the day of DNCB challenge at 40 -60 I~/day and maintained for 5 days. No animal died or had to be omitted from this experiment at the final evaluation. Statistics:Control group was significantly different (p <0.01) from all the other groups included in this experiment on days 3 - 7 . BRC-5 mg compared to BRC-2.5 rng differed on days 5 - 6 ; BRC-2.5 mg compared to BRC-2.5 mg+ 40 I~g BPRL, to BRC- 5 mg+ 40 p.g BPRL, and to BRC 5 mg+ 60 ~ BPRL were different on days 4 -5; BRC-5mg compared to BRC-2.5 mg+ 40 t~g BPRL were different on days 5 -7; BRC- 5 mg compared to BRC-5mg + 40 or 60 t#3 BPRL were not different at any time.

236 E. Nagy et al.

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Figure 2

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A BRC + BPRL

• BRC + BPRL +ACTH

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ACTH antagonizes the restoring effect of pro/actin in brornocriptine-suppressed ani- rnals. Groups of 5 Wistar-Furth female animals were treated with 5 rng/kg of brornocriptine s.c. daily; bovine prolactin was given at 100 p.g/day s.c., and ACT/- /at 40 l~/day/anirnal s.c., according to the schedule described for Figure I. All animals survived and were included in the final evaluation of this experiment. Statistics: Controls were significantly different (p <0.01) from the BRC group on days 4 -6 , from the BRC + BPRL group on day 2 only, and from the BRC + BPRL + ACTH group on days 3 - 7 BRC was different from BRC + BPRL and from BRC + BPRL + ACTH groups on days 4 -6 .

Immunomodulaflon by Bromocriptine 237

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Figure 3

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• BRC

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ACTFI antagonizes restoration by growth hormone in bromocriptine-suppressed ani- reals. Groups of 5 female Wistar-Furth rats were treated with 5 mg/kg bromocriptine s.c. daily with 40 p,g/day/animal of bovine growth hormone s.c., and with 40/.,g/day/animal of ACTH s.c., according to the schedule described for Figure 5. All animals suruived and were included in the final evaluation of this experiment. Statistics: Control was significantly different (p <0.01) from the BRC-treated group on days4-6, and from the BRC + BGH + ACTH group on days2-7, but did not differ significantly at any time from the group treated with BRC + BGH: the BRC-treated group was significantly different from BRC + BGH and from BRC + BGH + ACTH on days 4 -6.

238 E. Nagy et al.

Table I Histological evaluation of skin reactions at DNCB challenge sites

Group no. Treatment Severity of reaction

1 Control + + + + 2 Hypox + 3 BRC + 4 BRC + BGH + + + + 5 BRC + BPRL + + + 6 BRC + BGH + ACTH + 7 BRC + BPRL + ACTH +

Suppress ion of EAE by B R C

A relatively mild form of EAE was induced by our method. In one experiment, where all the six control animals showed symptoms of the disease, BRC treatment significantly inhibited (p~0.05) the development of clinical symptoms (mean score for the control group was 2.07 _+ 0.80, whereas for the BRC-treated group it was 0.26 + 0.03). In another experiment, only three of the five controls showed symptoms. EAE was not identified in the BRC-treated group (mean score for controls 1.11 _+ 1.39, for the BRC-group 0.060 _+ 0.005). Although BRC was clearly inhibitory in this experiment also, the results are not significantly different because of the great variation in

controls.

Figure 4 Histology of skin reactions in normal and in BRC-suppressed animals. ( A) The skin of a nonhypophysectomized control rat shows an intense reaction to DNCB; the surface is covered by necrotic debris, fibrin, and inflammatory cells, and there is dermal edema (hematoxylin-eosin stain, original magnification × 32). (13) In a BRC-suppressed rat, the site of DNCB application shows slight dermal edema and minimal inflammation (hematoxylin-eosin stain, original magni- fication × 32).

Immunomodulation by Bromocriptine 239

)--

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Figure 5 Suppression of the antibody response to SRBC by bromocriptine and its reversal by treatment with bovine prolactin. This experiment was performed on female Fischer rats. Hypox was performed three weeks priorto immunization and bromocriptine treatment (5 mg/kg/day s.c.) was initiated one week prior to immunization with SRBC. Bovine prolactin was administered at 100 i.,.g/anirnal/day s. c. for 10 days, commencing on the day of immunization. Animals included in the final evaluation of this experiment were: 0 - 9 , • - 8 , 0 - 8 , ~ - 9 . In this experiment, rat PRL was measured by radioimmunoassay from pooled serum samples. Mean values +_ standard error derived from six measurements (on days O, 3, 6, 9, 12, and 15) are as follows: 0 -84 .8 +_- 15.9; • -25.5 +_ 6.5; 0 - 4 . 9 +_ 1.6; 0 - 6 . 7 +_ 1.1 ng/ml. Statistics: Controls were significantly different (p <0.01) from Hypox animals and from the BRC-treated animals on days 6-15, but were not significantly different at any time from the group treated with BRC + BPRL. Hypox animals were significantly different from the BRC + BPRL on days 6 -15 but did not differ from the BRC-treated group at any time.

240 E. Nagy et al.

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+HCG+TSH • Normal+Bromocriptine -[- Standard errors

8x108 RRBC coated with LPS

I ,

r T T 0 3 6 9 12 15

Days Figure 6 Suppression of the antibody response to LPS by hypophysectomy or by bromocriptine treatment. Groups of 5 female Wistar-Furth rats were used. Hypox was performed three weeks prior to immunization and some of the Hypox rats were transplanted with syngeneic pituitary grafts (SPG) under the kidney capsule one week after Hypox. Hormone treatment was started on the day of immunization and maintained for 10 days. Bovine prolactin was given at 40 l.Lg/animal/day and ACTH, GH, HCG, and TSH at 20 ixg/animal/day s.c. Bromocriptine treatment of normal animals (5 mg/kg/day s.c.) was initiated one week priorto immunization and maintained until the end of the experiment. All animals survived and were included in the final evaluation of this experiment. Statistics: Controls were significantly different (p<O.01) from Hypox, Hypox + ACTH + BGH + HCG + TSH, and from the BRC-treated group on days 6 - 15. Controls differed from the pituitary grafted group on day 12 only and from the Hypox + BPRL group on days 9 and 12.

Immunomodulat ion by Bromocriptine 241

Table 2 The effect of bromocriptine on adjuvant arthritis and on skin allograft re)ection a

Adjuvant arthritis (percent Skin allograft Group no. Treatment increase in paw diameter) survival (days)

I Control 56 ± 9.2 (5) 9.2 ± 1.6 (9) 2 Hypox 6 ± 3.3 (4) a 16.8 __ 2.0 (7) ~ 3 Hypox + SPG 13 ___ 3.0 (5) a 11.4 _ 3.7 (8) 4 Hypox + BPRL 26 + 6.1 (5) c 9.2 ___ 1.6 (7) 5 Normal + BRC 5 ± 2.8 (5) ~ 11.2 ± 1.9 (9) b

aGroups of 5 male Fischer rats were used for the adjuvant arthritis experiment. Hypophysectomy, grafting with syngeneic pituitaries, treatment with bovine prolactin and with bromocriptine of the various groups were performed as described for Figure 6. The number of animals alive at the end of the various reactions studied are given in brackets after the results for each group. Skin grafting was performed from Wistar-Furth donors to female Fischer recipients. Approximately I cm 2 of tail skin was transplanted in each case. Hypox, pituitary grafting, hormone, and bromocriptine treatment were performed as described for Figure 6. The reactivity of control and treated groups was compared with the Student's t-test and the results are shown in the Table as follows: b = p < 0.05; c = p < 0.01; d = p < 0.001.

Histologic examination of the brains of control rats with EAE revealed perivascular cuffing by lymphocytes and plasma cells with occasional neutrophils (Figure 7). BRC-treated animals show- ed no lesion.

DISCUSSION

Our results indicate that BRC is capable of suppressing cell-mediated, humoral, and autoimmune reactions. This immunosuppressive effect is most likely due to inhibition of prolactin (Meites and Clemens, 1972; Parkes, 1979) and possibly of growth hormone secretion by the pituitary gland (Bansal et al., 1981; Bazan et al., 1981), since the immunocompetence of BRC-treated animals is readily restored by injections of prolactin or growth hormone. However, further work is required to study the effect of BRC on other targets, such as lymphocytes (LeFur et al., 1981) or blood vessels (Cavero et al., 1982), which may contribute to its immunosuppressive effect.

We have previously established that prolactin and growth hormone are necessary for the maintenance of humoral and cell-mediated immune reactions (Nagy et al., 1983). The present experiments further support the immunoregulatory role of these two pituitary hormones. It is noteworthy that ACTH antagonized immunorestoration by PRL or GH in BRC-treated animals as it did in earlier studies in Hypox rats (Berczi et al., 1983). Bromocriptine-suppressed animals needed up to 5 × more PRL or GH for immune restoration than did Hypox rats. The reason for this difference is not known. However, unlike Hypox rats, ACTH is secreted in BRC-treated animals and thus could exert an immunosuppressive effect, resulting in higher hormone require- ment (Figure 6) (Berczi and Nagy, 1981; Nagy and Berczi, 1981).

In man, the effects of PRL and GH on immunity have not been elucidated. Leukocytes obtained from three hyperprolactinemic patients exhibited altered (i.e., one was enhanced and two suppressed) chemotactic activity. The chemotaxis of leukocytes from some (but not all) normal donors was also suppressed at high PRL concentrations ( 1 - 2 p~g/ml) (Harris et al., 1979). It is a long-standing observation that acromegaly is associated with thymic and lymphatic hyper- plasia (reviewed by Ahlqvist, 1976). X-linked hypogammaglobulinemia associated with isolated growth hormone deficiency has also been observed in a family study (Fleisher et al., 1980). Because of the widespread and ever increasing use of bromocriptine and of other dopaminergic drugs in medicine, the possible immunomodu la to ry effect of these agents needs urgent investigation.

The authors are indebted to Drs. A.H. Sehon and H.G. Friesen for their continued interest, advice, and critical evaluation of this work. We also thank Dr. Friesen and his associates for the radioimmunoassay of prolaciin in

242 E. Nagy et al.

Figure 7 Histology of EAE. Photomicrograph of the brain of a control rat with EAE shows perivascular cuffing by mononuclear inflammatory ceils. BRC-treated animals showed no lesion (hematoxylin-eosin stain; original magnification × 80).

some of these experiments. The donation of BGH and BPRL by Dr. Salvatore Raiti, through the National Pituitary Agency, Baltimore, Maryland, is gratefully acknowledged. This work was supported by the MRC of Canada and the Arthritis Society of Canada; Dr. S.L. Asa is an MRC Fellow.

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