BRAIN,
BEHAVIOR,
AND
IMMUNITY
4, 118-128 (1990)
Hypophysectomy and Growth Hormone Replacement on Multiple Immune Responses in Rats
Effects
JERRY H. EXON,JEANINE L. BUSSIERE, ANDJAMES R. WILLIAMS Department
of Veterinary
Science,
University
of Idaho,
Moscow,
Idaho
83843
Male Fischer 344 rats were hypophysectomized at 8 weeks of age. At 10 weeks of age, one group of these animals was treated with 40 log of bovine somatotropin given daily SC for 13 days. A third group was sham-operated and injected daily with saline. All animals were sacrificed on Day 14 and multiple immune responses were assessed in each rat. Immune responses assayed included specific antibody production and delayed-type hypersensitivity (DTH) reactions to antigen, natural killer (NK) cell cytotoxicity, and production of interleukin 2 (IL2). Body and lymphoid organ weights, hematologic parameters, and spleen cell numbers were also recorded. Hypophysectomized (Hx) rats had reduced antibody synthesis, DTH reactions, NK cytotoxicity, IL2 (or IL4) production, body and organ weights, rbc counts, packed cell volumes, and hematocrits compared to sham-operated controls. White blood cell counts were elevated. Treatment of Hx animals with GH restored antibody and IL2 production and thymic weights and partially restored DTH reactions. These data indicate the pituitary is important in maintaining normal immune functions, and part of this effect may be via production of GH. It is postulated that GH may act through stimulation of IL2 (or IL4). o IWO Academic F-MS, IIIC.
INTRODUCTION Animals which have been hypophysectomized generally have reduced thymic and splenic weights accompanied by atrophy and decreased cellularity of these and other lymphoid tissues (Smith, 1930; Feldman, 1951; Lundin, 1958; Kalden, Evans, & Irvine, 1970). These animals also generally have reduced functional immune responses such as antibody production (Lundin, 1960; Enerback, Lundin, & Mellgren, 1961; Nagy & Berczi, 1978; Berczi, Nagy, Kovacs, & Horvath, 1981; Nagy, Berczi, & Frissen, 1983; Pandian & Talwar, 1971; Gisler dz SchenkelHulliger, 1971), graft rejection (Comsa, Leonhardt, & Schwarz, 1975; Nagy & Berczi, 1978), contact sensitivity (Prentice, Lipscomb, Metcalf, & Sharp, 1975; Nagy & Berczi, 1978; Berczi, Nagy, Asa, & Kovacs, 1983), or natural killer cell cytotoxicity (Saxena, Saxena, & Alder, 1982: Cross, Markesbery, Brooks, & Roszman, 1984). Genetically hypopituitary species, such as Snell-Bagg and Ames dwarf mice, certain inbred Weimaraner dogs, and a dwarf strain of White Leghorn chickens, also have reduced lymphoid organs and associated immune dysfunction syndromes, mainly of the cell-mediated arm of the immune system (Bartke, 1964; Baroni, Fabris, & Bertoli, 1969; Baroni, Scelsi, Mingazzini, Cavaliero, & Uncinni, 1972; Baroni, Pesando, & Bertoli, 1971; Fabris, Pierpaoli, & Sorkin, 1971a, b; Duquesnoy, 1972; Wilkinson, Singh, & Sorkin, 1970; Roth, Kaeberle, Greir, Hopper, Spiegel, & McAllister, 1984; Pierpaoli, Baroni, Fabris, & Sorkin, 1969). These data indicate an integral role of pituitary hormones in maintenance of the reticuloendothelial system and regulation of immune responses. Of the various 118
0889-1591/90 $3.00 Copyright 8 1990 by Academic Press, Inc. Au rights of reproduction in any form reserved.
HYPOPHYSECTOMY
GROWTH
HORMONE
IMMUNITY
119
hormones produced by the pituitary, growth hormone in conjunction with thymic hormones appear to be of primary importance for homeostasis of the immune system (reviewed by Kelley, 1989). Treatment of hypophysectomized rats or mice with exogenous growth hormone has been reported to partially or completely restore depressed lymphoid organ weights (Simpson, Evans, & Li, 1949; Feldman, 1951; Maor, Englander, Eylan, & Alexander, 1974) and humoral (Pandian & Talwar, 1971; Nagy et al., 1983; Comsa, Schwarz, & Neu, 1974; Arrenbrechdt & Sorkin, 1973) and cell-mediated immune responses (Comsa et al., 1975; Berczi et al., 1983; Pierpaoli, Fabris, & Sorkin, 1970; Saxena et al., 1982). Growth hormone therapy has also been reported to partially restore immune responsiveness in genetically hypopituitary species (Baroni et al., 1969; Pierpaoli et al., 1969; Fabris et al., 1971b). There is some question at this time whether growth hormone acts alone or in concert with other factors. Receptors for growth hormone are present on human lymphocytes and macrophages (Lesniak, Roth, Gorden, & Gavin, 1983; Kiess & Butenandt, 1985) which have strong cross-reactivity with prolactin (Kover & Moore, 1984). There is also strong evidence that the action of growth hormone on the immune system is mediated via the thymus or enhanced by thymic factors (Comsa et al., 1974, 1975; Arrenbrecht & Sorkin, 1973; Pierpaoli et al., 1970; Pierpaoli, Fabris, & Sorkin, 1971). More recent studies indicate a role of stressrelated hormones (Nagy et al., 1983; Berczi et al., 1983; Berczi, Nagy, Asa, & Kovacs, 1984) or lactogenic hormones in general (Berczi & Nagy, 1986). This study was designed to examine the effects of growth hormone replacement on multiple immune responses of hypophysectomized rats. Humoral, specific cell-mediated and nonspecific cell-mediated immune responses and cytokine production were assessed in each animal on experiment. MATERIALS
AND METHODS
Animals
Eight-week-old male Fischer 344 rats, purchased from Simonsen Laboratories (Gilroy, CA) were randomly assigned to groups of five rats each and caged two or three animals per cage. Rats were housed in stainless steel hanging wire cages in a room with 12-h light/dark cycles and recommended limits of temperature, humidity, and air exchange. Commercial rodent chow and deionized water were provided ad libitum. All hypophysectomized and sham-hypophysectomized rats were maintained on 5% glucose solution. Hypophysectomy
Hypophysectomized and sham-hypophysectomized rats were purchased from Simonsen Labs. The surgeries were performed by the parapharyngeal route 2 weeks prior to initiation of the study. The sella turcica was examined grossly at necropsy to verify completeness of the hypophysectomies. Hormone
Treatment
Two weeks after surgery all rats were injected daily for 13 days, subcutaneously
120
EXON,
BUSSIERE,
AND
WILLIAMS
(SC) in the loose skin over the back, with either 40 pg bovine somatotropin (bGH, Miles Scientific, Naperville, IL) in 0.1 ml sterile physiologic saline or shaminjected with saline alone. Animals were weighed prior to initiation of hormone therapy and twice weekly for the duration of the studies. Gross Pathology
All animals were terminated 14 days after beginning hormone treatment, and complete necropsies were performed on each rat. Whole blood samples were collected by cardiac puncture and hematologic parameters, white and red cell counts, hemoglobin and hematocrit, determined using a Coulter counter, Model ZBI (Coulter Electronics, Hialeah, FL). Body, spleen, thymus, liver, kidney, heart, testis, and adrenal weights were recorded. Assay for Delayed-Type
Hypersensitivity
(DTH)
The delayed-type hypersensitivity response was measured by a modification of the footpad swelling method described by Henningsen, Koller, Exon, Talcott, and Osborne (1984). This method has been shown to induce a typical Type IV DTH reaction. Rats were sensitized by an SCinjection of heat-aggregated keyhole limpet hemocyanin (KLH, Calbiochem, San Diego, CA) at the base of the tail to induce a delayed hyersensitivity reaction (Table 1). Heat-aggregation was achieved by mixing 120 mg of KLH in 6 ml sterile saline (2% w/v) and heating for 1 h at 80°C in a water bath. Rats were injected with 100 ~1 of the aggregate (2 mg KLH). One week later the left footpad was challenged by SCinjection of 2 mg of heat-aggregated KLH suspension in 100 ~1. The right footpad was sham-injected with sterile saline. Twenty-four hours later, footpad swelling in both hind feet was measured with a digital (Model 800) electronic micrometer (Model 4440, EG&G Quality Measurement Systems, Pentield, NY). The DTH reaction was determined by subtracting the thickness of the saline-injected footpad from the KLH-injected footpad. Results are expressed as millimeter difference in swelling. Assay for Antibody
Synthesis
Immediately following the measurement of the DTH reaction, rats were injected SCwith 1 mg of KLH in 0.2 ml of sterile saline to induce a secondary (IgG) TABLE
1
Antigen Injection Schedule
Day 0 I 8 14
Antigen
Dose
loo pl KLH-HA” KLH-HA” loo kl Measure DTH’ response 1 mg KLH (aqueous) Terminate and collect splenocytes for NK and IL2 assays and serum for antibody assay
(2Heat-aggregated keyhole limpet hemocyanin, 2% solution (2 mg/lOO ~1). b Subcutaneous. ’ Delayed-type hypersensitivity.
Route SCb Footpad SC
HYPOPHYSECTOMY
GROWTH
HORMONE
IMMUNITY
121
antibody response (Table 1). Six days later the rats were sacrificed by CO, asphyxiation. The animals were fasted for 18 h prior to sacrifice to lower lipid levels in serum. Blood was collected by cardiac puncture. An aliquot of each blood sample was allowed to clot and the serum was removed and stored at 4°C until analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) described by Exon, Koller, Talcott, O’Reilly, and Henningsen (1986). Endpoint IgG antibody titers were defined as the highest dilution of serum which gave an absorbance greater than the statistical mean (0.250) of a normal rat serum control. The normal serum was diluted 1:50 to simulate sample dilutions. Other controls included three groups of wells in which the anti-IgG conjugate, the substrate and the KLH were singularly eliminated from the reaction mixture. To reduce statistical variability, the endpoint titers were then ranked by the method of Wilcoxon with the lowest titer being given the rank of one (Ott, 1977). The ranked data was analyzed by analysis of variance and least-squares mean comparisons. Assay for Natural
Killer
Cell Cytotoxicity
Rat spleens were removed aseptically and cell suspensions were prepared by forcing the spleens through a stainless steel wire mesh into RPM1 Medium 1640 (RPMI, GIBCO Laboratories, Chagrin Falls, OH) containing 100 U/ml penicillin and 100 kg/ml streptomycin (P/S). Spleen cells were centrifuged at 200g for 10 min, resuspended in RPMI, P/S, and counted on a Coulter counter, Model ZBI (Coulter Electronics) to determine total spleen cell numbers. An aliquot of splenocytes was removed at this point for assay of interleukin 2 (IL2) production (see below). The remaining spleen cell suspensions were incubated on nylon wool columns for 45 min in a humidified, 10% CO, atmosphere at 37°C to remove B lymphocytes and phagocytic cell populations. Nonadherent cells were gently flushed from the columns into polystyrene 75-cm2 tissue culture flasks and incubated as before for 1 h to further remove adherent cells. Natural killer (NK) cell cytotoxicity to YAC-I tumor cells was measured by a 4-h chromium-release assay described by Talcott, Exon, and Koller (1984). The percentage cytotoxicity of each sample was calculated from the specific 51Cr release by the formula: Experimental release - spontaneous release x 100%. Maximum release - spontaneous release An NK-insensitive lymphoma cell line, EL-4, was labeled in the same manner as the YAC target cells and incubated with effector cells to serve as a control for non-NK-mediated lysis. Lysis of EL4 cells was less than 2-3% above spontaneous release. Spontaneous release for the NK assay was less than 10%. Assay for Interleukin
2 Production
Synthesis of IL2 was induced in aseptically isolated rat splenocytes (1 x lo6 cells/ml) in RPMI, P/S, containing 0.4 g/liter bovine serum albumin (Sigma), by adding 1.O kg/ml concanavalin A (Sigma) to the cultures, and incubating for 24 h
122
EXON,
BUSSIERE, AND WILLIAMS
at 37°C in a 5% CO*, humidified atmosphere. Identical, unstimulated samples served as negative controls. The amount of IL2 produced by spleen cells was analyzed by its capacity to maintain the growth of CTLL-2 T cells as previously described (Exon et al., 1986). It should be noted that recent evidence suggests this assay may not be capable of distinguishing between IL2 and IL4. The IL2 assay was performed on two separate days using five animals/group/day. Since the assay has innate day-to-day variability, the units were converted to percentage of the daily control and analyzed by analysis of variance based on percentage change. Statistics All data were analyzed with the Statistical Analysis System (SAS Inc., New York, NY). Significant treatment differences were based on analysis of variance and least-squares mean comparisons with an (Ylevel of 0.05. RESULTS
Hypophysectomized rats, with or without bGH treatment, exhibited significantly (p < 0.05) reduced body weights compared to sham-operated rats (Table 2). Weights of hypophysectomized rats which received bGH tended to be greater (p s 0.07) than those which were only hypophysectomized and injected with saline. Thymus, spleen (Table 2), liver, kidney, heart, testes, and adrenal (Table 3) weights were significantly (p < 0.05) reduced in hypophysectomized rats. Organ weights of animals which were hypophysectomized and treated with bGH remained decreased, except for thymic weights which recovered to about normal values. Red blood cell counts, hemoglobin levels, and hematocrits were significantly (p s 0.05) reduced in hypophysectomized rats with or without bGH treatments (Table 4). White blood cell counts were significantly (p < 0.05) elevated in hypophysectomized rats compared to sham-operated controls. Hypophysectomized rats treated with bGH had significantly (r, d 0.05) increased wbc counts compared to either the hypophysectomized group or the sham-operated control. Serum antibody titers to KLH, DTH reactions (Table 5), NK cytotoxicity, IL2 (IL4) production, and spleen cell number (Table 6) were significantly (p C 0.05) TABLE 2 The Effects of Hypophysectomy and Growth Hormone on Body, Spleen, and Thymus Weights (g)
SHAM-HXb HX HX + bGH=
N
Body”
10 10 10
176.1 2 5.6A 106.8 t- 2.8B 116.0 4 4.8B
Spleen 0.26 2 O.OOA 0.18 + 0.01a 0.18 + O.OIB
Thymus 0.152 f 0.007A 0.115 * o.oO!P 0.145 + o.OO!P
a Values are means f the standard error of the mean determined at the time of sacrifice following daily injections of saline or bGH for 13 days. Means with no common capital letters are statistically different (p c 0.05) by analysis of variance and least-squares mean comparisons. b Hypophysectomy. c Bovine growth hormone.
HYPOPHYSECTOMY
GROWTH
HORMONE
123
IMMUNITY
TABLE 3 Effects of Hypophysectomy and Growth Hormone on Weights of Liver, Kidney, Heart, Testes, and Adrenal Weight (q + SEM)”
SHAM-HXb HX HX + bGH’
Liver
Kidney
Heart
Testes
Adrenal
7.0A f 0.3 3.6B +- 0.1 3.9u f 0.2
ow f 0.03 0.36B 5 0.01 0.40B ? 0.01
0.72* 2 0.04 0.31B 4 0.01 0.3SB f 0.02
1.31* f 0.02 0.14B 2 0.03 0.138 +- 0.02
0.04oA r 0.003 0.01on f 0.001 0.0128 f 0.001
0 Values are means 2 standard error of the mean determined at the time of sacrifice following daily injections of saline or bGH for 13 days. Means with no common capital letter are statistically different 0, < 0.05) by analysis of variance and least-square mean comparisons. N = IO/group. b Hypophysectomy. ’ Bovine growth hormone.
lower in hypophysectomized rats compared to sham-operated controls. Natural killer cell cytotoxicity, spleen cell counts, and DTH reactions were also significantly (r, s 0.05) suppressed in rats which were hypophysectomized and received bGH treatment. Antibody titers and IL2 (IL4) production in these animals, however, were not different than controls. The DTH reactions in hypophysectomized animals which received bGH were also significantly (r, s 0.05) greater than those which received only hypophysectomy. DlSCUSSlON
The results of this study indicate the pituitary has an important role in maintenance of normal immune function in rats. Hypophysectomized rats had decreased thymus and spleen weights and reduced spleen cell numbers. Functional immune responses were also suppressed in hypophysectomized rats as noted by decreased antibody and IL2 (IL4) production, DTH reactions, and NK cytotoxic responses. These data are in agreement with those of others which indicate than an intact pituitary is necessary for normal humor-al (Nagy & Berczi, 1978; Berczi et al., 1981; Nagy et al., 1983) and specific (Nagy & Berczi, 1978; Comsa et al., 1975; TABLE 4 The Effects of Hypophysectomy and Growth Hormone on Hematologic Parameters”
SHAM-HX HX HX + bGH
N
RBC ( 106/mm3)
WBC ( 103/mm3)
Hct m
Hbg WV
10 10 9
9.57 e o.osA 8.33 2 0.13B 8.31 * 0.12B
9.5 + 0.3* 13.9 f 1.4n 17.4 5 1.4c
43.9 * 0.4* 35.5 +- 1.1u 37.9 f 1.ou
16.6 2 0.1* 14.1 + 0.2n 13.8 f 0.2B
n Abbreviations used: bGH, bovine growth hormone; Hbg, hemoglobin; Hct, hematocrit; HX, hypophysectomy; RBC, red blood cells; WBC, white blood cells. Values are means + the standard error of the mean of hematology values determined at the time of sacrifice following daily injection of either physiologic saline or 40 kg bGH for 13 days. Means with no common capital letters are statistically different (p < 0.05) by analysis of variance and least-squares mean comparisons.
124
EXON,
BUSSIERE,
AND WILLIAMS
TABLE 5 The Effects of Hypophysectomy and Growth Hormone on IgG Antibody Titers, Delayed-Type. Hypersensitivity, and Production of IL2”
SHAM-HX HX HX + bGH
N
Ranked Ab titefl
DTH’ (mm)
IL2d % of control
10 10 10
15.9 2 2.8* 8.4 k 2.6’ 12.8 -t 1.9*
2.79 ” 0.13A 1.58 + 0.11’ 2.25 + 0.18’
85.9 ? 6.1* 68.6 + 5.3B 81.0 + 6.4A
a Abbreviations used: Ab, antibody; bGH, bovine growth hormone; DTH, delayed-type hypersensitivity; HX, hypophysectomy; IL2, interleukin 2. Values are means f the standard error of the mean. Means with no common capital letter are statistically different (p < 0.05) by analysis of variance and least-squares mean comparisons. * Antibody endpoint titers to keyhole limpet hemocyanin were determined from serum taken at the time of sacrifice and ranked with the lowest titer receiving the rank of one. ’ Difference (mm) in footpad swelling between right and left. d Production of IL2 (IL4) by splenocytes is reported as a percentage of the mean units of IL2 for normal control animals on the day on which the animals were sacrificed. Control means were 35.2 and 26.9 U.
Berczi et al., 1983; Prentice et al., 1975) and nonspecific cell-mediated (Cross et al., 1984; Saxena et al., 1982) immune responses. It appears that GH can partially or completely restore some of the immune dysfunction associated with hypophysectomy. Animals which received GH treatment and were hypophysectomized had thymus weights and antibody and IL2 (IL4) production comparable to sham-operated controls. The DTH reactions in hypophysectomized rats which were given GH were significantly greater than in animals which were only hypophysectomized, but were still less than shamoperated controls. In contrast, NK responses were similar in hypophysectomized animals with or without GH treatment. This data indicates the humoral (B cell) and specific cell-mediated (T cell) immune response may be more sensitive to restoration by GH than non-specific cell-mediated (NK cell) responses, although ‘TABLE 6 The Effects of Hypophysectomy and Growth Hormone on Splenic Natural Killer Cell Cytotoxicity and Spleen Cell Numbef % Cytotoxicity Effector:Target cell
SHAM-HX* HX HX + bGH’
N
1OO:l
50: 1
Total spleen cell counts (X 10”)
10 10 10
40.4 f 1.6* 20.2 f l.lB 21.8 f 1.3B
31.7 2 1.1* 14.8 + 1.3B 17.2 + 1.4’
2.95 + 0.18A 0.81 f 0.09’ 1.07 + 0.099
n Values are means f the standard error of the mean determined upon sacrifice following daily injections of either saline or 40 pg bGH for 13 days. Means with no common capital letter are statistically diierent (p c 0.05) by analysis of variance and least-squares mean comparisons. * Hypophysectomy. c Bovine growth hormone.
HYPOPHYSECTOMY
GROWTH
HORMONE
IMMUNITY
125
others have reported partial restoration by GH of NK cell cytotoxic responses in hypophysectomized mice (Saxena et al., 1982). Conflicting data on ability of GH or GH-releasing factor to restore reduced NK cell responses in GH-deficient patients has been reported (Crist, Peak, Mackinnon, Sibbit, & Kraner, 1987; Kiess, Malozowski, Gelato, Buten and, Doerr, Crisp, Eisl, Maluish, and Belohradsky, 1988). Several investigators have reported GH treatment can restore humoral (Pandian & Talwar, 1971; Nagy et al., 1983) and T cell-mediated (Comsa et al., 1975; Berczi et al., 1983) immune responses in hypophysectomized animals. A confounding factor in our study was the GH therapy failed to completely restore body and organ weights of hypophysectomized animals. Therefore, this treatment regimen or purity of GH may not have been adequate to restore all immune responses. It has been postulated that the effects of GH may be mediated through the T helper cell population (Arrenbrecht 8z Sorkin, 1973). In our study, IL2 (IL4) production was significantly suppressed in hypophysectomized rats but this effect was reversed by GH treatment. It is well documented that IL2 is important in induction of optimal B and T cell-mediated responses (reviewed by Robb, 1984). Although not shown conclusively in this study, GH may mediate its effects partially through stimulation of IL2. We have previously observed enhanced IL2 production in normal rats treated with GH in our laboratory (unpublished data). It would be interesting to determine if IL2 (or IL4) alone could restore immune responses in hypophysectomized rats. If this is a mechanism by which GH acts, it is less clear why NK cell responses were not similarly restored since IL2 production had returned to normal. The NK cytotoxic response is also reported to be sensitive to ILZinduced enhancement (Yamada, Ruscetti, Overton, Herberman, Birchenall-Sparks, & Ortaldo, 1987). Perhaps a longer treatment period, higher doses, or a species-specific GH may have been more effective. General hematological parameters were also altered in hypophysectomized rats. Red blood cell counts, hematocrits, and hemoglobin levels were significantly reduced in hypophysectomized animals. These effects were not reversed in animals which also received GH treatment. These results are similar to those of Crafts and Meineke (1959) who showed anemia in hypophysectomized rats could be reversed by a combination of GH, thyroxin, and cortisone acetate. However, treatment with any one of the agents was not sufficient to restore normal blood counts. These authors postulated that the posthypophysectomy anemia was due to effects on general metabolism and oxygen need of erythrocytes. A more recent study, however, by Nagy and Berczi (1989) showed that anemia and leukopenia in hypophysectomized female Fisher 344 rats could be reversed by subrenal pituitary grafts, bGH, or prolactin. Since prolactin and GH can bind the same receptors it is unclear which hormone may be most important in restoring hematologic parameters. Also, the activity of the bGH used in that study was greater than ours. Conversely, white blood cell counts were significantly increased in hypophysectomized rats, and this effect was significantly exacerbated by concomitant GH treatment. Increased wbc counts in hypophysectomized rats was a surprising finding since most evidence indicates the converse (reviewed by Kelley, 1989). In
126
EXON,
BUSSIERE,
AND WILLIAMS
fact, Nagy and Berczi (1989) reported a leukopenia in hypophysectomized female F344 rats. These results remain to be confirmed by additional studies. The preponderance of evidence indicates that GH stimulates proliferation of lymphocytes and reduces leukopenia. Therefore, it is not surprising that administration of GH increased even further the leukocytosis. The source of increased numbers of wbc is unknown since differentials were not performed in this study. It is apparent, however, that perturbation at the bone marrow level may have significant implications to altered immune function via effects on progenitor (stem) cells. REFERENCES Arrenbrecht, S., & Sorkin, E. (1973). Growth hormone-induced T cell differentiation. Eur. J. Zmmuml. 3, 601-604. Baroni, C. D., Fabris, N., & Bertoli, G. (1969). Effects of hormones on development and function of lymphoid tissues: Synergistic action of thyroxin and somatotropic hormone in pituitary dwarf mice. Zmmunology 17, 303-314. Baroni, C. D., Pesando, P. C., & Bertoli, G. (1971). Effects of hormones on development and function of lymphoid tissues. II. Delayed development of immunological capacity in pituitary dwarf mice. Immunology 21, 455461. Baroni, C. D., Scelsi, R., Mingazzini, P. L., Cavallero, A., & Uncinni, S. (1972). Delayed hypersensitivity in the hereditary pituitary dwarf SnelYBagg mouse. Nature New Biol. 237, 21%220. Bartke, A. (1964). Histology of the anterior hypophysis, thyroid and gonads of two types of dwarf mice. Anat. Rec. 149, 225-236. Berczi, I., Nagy, E., Kovacs, K., & Horvath, E. (1981). Regulation of humoral immunity in rats by pituitary hormones. Acta Endocrinol. 98, 506-513. Berczi, I., Nagy, E., Asa, S. L., & Kovacs, K. (1983). Pituitary hormones and contact sensitivity in rats. Allergy 38, 325-330. Berczi, I., Nagy, E., Asa, S. L., & Kovacs, K. (1984). The influence of pituitary hormones on adjuvant arthritis. Arthritis Rheum. 27, 682-688. Berczi, I., & Nagy, E. (1986). Prolactin and other lactogenic hormones. In I. Berczi. (Ed.), Pituitary function and immunity, pp. 161-184. CRC Press: Boca Raton, FL. Comsa, J., Schwarz, J. A., & Neu, H. (1974). Interaction between thymic hormone and hypophyseal growth hormone on production of precipitating antibodies in the rat. Zmmunol. Commun. 3, 11-18. Comsa, J., Leonhardt, H., & Schwarz, J. A. (1975). Influence of the thymus-corticotropin-growth hormone interaction on the rejection of skin allografts in the rat. Ann. N. Y. Acad. Sci. 249, 387401. Crafts, R. C., & Meineke, H. A. (1959). The anemiaof hypophysectomized animals. Ann. N. Y. Acad. Sci. 77, 501-517. Crist, D. M., Peake, G. T., Mackinnon, L. T., Sibbit, W. L., & Kraner, J. C. (1987). Exogenous growth hormone treatment alters body composition and increases natural killer cell activity in women with endogenous growth hormone secretion. Metabolism 12, 1115-l 117. Cross, R. J., Markesbery, J. A., Brooks, W. H., & Roszman, T. L. (1984). Hypothalamic-immune interactions: Neuromodulation of natural killer activity by lesioning of the anterior hypothalamus. Immunology 51, 3m5. Duquesnoy, R. J. (1972). Immunodeficiency of the thymus-dependent system of the Ames dwarf mouse. J. Zmmunol. 108, 1578-1590. Enerback, L., Lundin, P. M., & Mellgren, J. (1961). Pituitary hormones elaborated during stress: Action on lymphoid tissues, serum proteins, and antibody titres. Acta Pathol. Microbial. Zmmunol. Stand. (SuppZ) 144, 141-144. Exon, J. H., Koller, L. D., Talcott, P. A., O’Reilly, C. A., & Henningsen, G. M. (1986). Immunotoxicity testing, an economical multiple-assay approach. Fundam. Appl. Toxicol. 7, 387-397. Fabris, N., Pierpaoli, W., & So&in, E. (197la). Hormones and the immunological capacity. III. The
HYPOPHYSECTOMY
GROWTH
HORMONE
IMMUNITY
127
immunodeficiency disease of the hypopituitary Snell-Bagg dwarf mouse. Clin. Exp. Immunol. 9, 209-225. Fabris, N., Pierpaoli, W., & So&in, E. (197lb). Hormones and the immunological capacity. IV. Restorative effects of developmental hormones or of lymphocytes on the immunodeficiency syndrome of the dwarf mouse. Clin. Exp. Immunol. 9, 227-240. Feldman, J. D. (1951). Endocrine control of lymphoid tissue. Anat. Rec. 110, 17-39. Gisler, R. H., & Schenkel-Hulliger, L. (1971). Hormonal regulation of the immune response. II. IntIuence of pituitary and adrenal activity on immune responsiveness in vitro. Cell. Immunol. 2, 646-657.
Henningsen, G. M., Koller, L. D., Exon, J. H., Talcott, P. A., & Osborne, C. A. (1984). A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity. J. Immunol. Methods 70, 53-165. Kalden, J. R., Evans, M. M., & Irvine, W. J. (1970). The effect of hypophysectomy on the immune response. Immunology 18, 671-679. Kelley, K. W. (1989). Growth hormone, lymphocytes and macrophages. Biochem. Pharmacol. 38, 705-713. Kiess, K., & Butenandt, 0. (1985). Specific growth hormone receptors on human peripheral mononuclear cells: Reexpression, identification, and characterization. .I. Clin. Endocrinol. Metab. 60, 740-746.
Kiess, W., Malozowski, S., Gelato, M., Butenand, O., Doerr, H., Crisp, B., Eisl, E., Maluish, A., & Belohradsky, B. H. (1988). Lymphocyte subset distribution and natural killer cell activity in growth hormone deficiency before and during short-term treatment with growth hormone releasing hormone. Clin. Immunol. Immunopathol. 48, 85-94. Kover, K., 62 Moore, W. (1984). Comparison of hGH binding to isolated rat liver macrophages and hepatocytes. Horm. Metab. Res. 16, 193-197. Lesniak, M. A., Roth, J., Gorden, P., & Gavin, J. R. (1973). Human growth hormone radioreceptor assay using cultured human lymphocytes. Nature New Biol. 241, 20-22. Lundin, P. M. (1958). Anterior pituitary gland and lymphoid tissue. Acta Endocrinol. (Suppl.) 40, l-83. Lundin, P. M. (1960). Action of hypophysectomy on antibody formation in the rat. Acta Pathol. Microbial. Immunol. Scand. 48, 351-357. Maor, D., Englander, T., Eylan, E., & Alexander, P. (1974). Participation of hormone in the early stages of the immune response. Acta Endocrinol. 75, 205-208. Nagy, E., & Berczi, I. (1978). Immunodeficiency in hypophysectomized rats. Acta Endocrinol. 89, 530-537.
Nagy, E., & Berczi, I. (1989). Pituitary dependence of bone marrow function. Brit. .I. Huemntol.
71,
457-462.
Nagy, E., Berczi, I., & Friesen, H. G. (1983). Regulation of immunity in rats by lactogenic and growth hormones. Acta Endocrinol. 102, 351-357. Ott, L. (1977). An introduction to statistical methods and data analyses, p. 317. Duxburg Press: Massachusetts. Pandian, M. R., & Talwar, G. P. (1971). Effect of growth hormone on the metabolism of thymus and on the immune response against sheep erythrocytes. J. Exp. Med. 134, 1095-I 113. Pierpaoli, W., Baroni, C. D., Fabris, N., & Sorkin, E. (1969). Hormones and immunological capacity. II. Reconstitution of antibody production in hormonally deficient mice by somatotropic hormone, thyrotropic hormone and thyroxin. Immunology 16, 217-230. Pierpaoli, W., Fabris, N., & Sorkin, E. (1970). Developmental hormones and immunological maturation. In G. E. W. Wolstenholme & J. Knight (Eds.), Hormones and the immune response, pp. 126153. Churchill: London. Pierpaoli, W., Fabris, N., & Sorkin, E. (1971). The effects of hormones on the development of the immune capacity. In S. Cohen, G. Cudkowicz, & R. T. McCluskey (Eds.), Cellular interactions in the immune response, p. 25. Karger: Basel. Prentice, E. D., Lipscomb, H., Metcalf, W. K., & Sharp, J. G. (1975). Effects of hypophysectomy on DNCB-induced contact sensitivity in rats. Stand. I. Immunol. 5, 955-961. Robb, R. J. (1984). Interleukin 2: The molecule and its function. Immunol Today 5, 203-209.
128
EXON,
BUSSIERE,
AND WILLIAMS
Roth, J. A., Kaeberle, M. L., Greir, R. L., Hopper, J. G., Spiegel, H. E., &z McAllister, H. A. (1984). Improvement in clinical condition and thymus morphologic features associated with growth hormone treatment of immunodeficient dwarf dogs. Amer. .I. Vet. Res. 45, 1151-1155. Saxena, Q. B., Saxena, R. K., L Adler, W. H. (1982). Regulation of natural killer cell activity in vivo. III. Effect of hypophysectomy and growth hormone treatment on the natural killer activity of the mouse spleen cell population. Int. Arch. Allergy Appl. Immunol. 67, 169-174. Simpson, M. E., Evans, H. M., & Li, C. H. (1949). The growth of hypophysectomized female rats following chronic treatment with pure pituitary growth hormone. I. General growth and organ changes. Growth 13, 151-170. Smith, P. E. (1930). The effect of hypophysectomy upon the involution of the thymus in the rat. Anat. Rec. 47, 119-129. Talcott, P. A., Exon, J. H., & Koller, L. D. (1984). Alteration of natural killer cell-mediated cytotoxicity in rats treated with selenium, diethylnitrosamine and ethylnitrosourea. Cancer Lett. 23, 313-322. Wilkinson, P. C., Singh, H., & Sorkin, E. (1970). Serum immunoglobulin levels in thymus-deficient pituitary dwarf mice. Immunology 18, 437-441. Yamada, S., Ruscetti, F. W., Overton, W. R., Herberman, R. B., Birchenall-Sparks, M. C., & Ortaldo, J. R. (1987). Regulation of human large granular lymphocyte and T cell growth and function by recombinant interleukin 2: Induction of IL2 receptor and promotion of growth of cells with enhanced cytotoxicity. .I. Leukocyte Biol. 41, 505-517. Received May 15, 1989
BEHAVIOR,
AND
IMMUNITY
4, 118-128 (1990)
Hypophysectomy and Growth Hormone Replacement on Multiple Immune Responses in Rats
Effects
JERRY H. EXON,JEANINE L. BUSSIERE, ANDJAMES R. WILLIAMS Department
of Veterinary
Science,
University
of Idaho,
Moscow,
Idaho
83843
Male Fischer 344 rats were hypophysectomized at 8 weeks of age. At 10 weeks of age, one group of these animals was treated with 40 log of bovine somatotropin given daily SC for 13 days. A third group was sham-operated and injected daily with saline. All animals were sacrificed on Day 14 and multiple immune responses were assessed in each rat. Immune responses assayed included specific antibody production and delayed-type hypersensitivity (DTH) reactions to antigen, natural killer (NK) cell cytotoxicity, and production of interleukin 2 (IL2). Body and lymphoid organ weights, hematologic parameters, and spleen cell numbers were also recorded. Hypophysectomized (Hx) rats had reduced antibody synthesis, DTH reactions, NK cytotoxicity, IL2 (or IL4) production, body and organ weights, rbc counts, packed cell volumes, and hematocrits compared to sham-operated controls. White blood cell counts were elevated. Treatment of Hx animals with GH restored antibody and IL2 production and thymic weights and partially restored DTH reactions. These data indicate the pituitary is important in maintaining normal immune functions, and part of this effect may be via production of GH. It is postulated that GH may act through stimulation of IL2 (or IL4). o IWO Academic F-MS, IIIC.
INTRODUCTION Animals which have been hypophysectomized generally have reduced thymic and splenic weights accompanied by atrophy and decreased cellularity of these and other lymphoid tissues (Smith, 1930; Feldman, 1951; Lundin, 1958; Kalden, Evans, & Irvine, 1970). These animals also generally have reduced functional immune responses such as antibody production (Lundin, 1960; Enerback, Lundin, & Mellgren, 1961; Nagy & Berczi, 1978; Berczi, Nagy, Kovacs, & Horvath, 1981; Nagy, Berczi, & Frissen, 1983; Pandian & Talwar, 1971; Gisler dz SchenkelHulliger, 1971), graft rejection (Comsa, Leonhardt, & Schwarz, 1975; Nagy & Berczi, 1978), contact sensitivity (Prentice, Lipscomb, Metcalf, & Sharp, 1975; Nagy & Berczi, 1978; Berczi, Nagy, Asa, & Kovacs, 1983), or natural killer cell cytotoxicity (Saxena, Saxena, & Alder, 1982: Cross, Markesbery, Brooks, & Roszman, 1984). Genetically hypopituitary species, such as Snell-Bagg and Ames dwarf mice, certain inbred Weimaraner dogs, and a dwarf strain of White Leghorn chickens, also have reduced lymphoid organs and associated immune dysfunction syndromes, mainly of the cell-mediated arm of the immune system (Bartke, 1964; Baroni, Fabris, & Bertoli, 1969; Baroni, Scelsi, Mingazzini, Cavaliero, & Uncinni, 1972; Baroni, Pesando, & Bertoli, 1971; Fabris, Pierpaoli, & Sorkin, 1971a, b; Duquesnoy, 1972; Wilkinson, Singh, & Sorkin, 1970; Roth, Kaeberle, Greir, Hopper, Spiegel, & McAllister, 1984; Pierpaoli, Baroni, Fabris, & Sorkin, 1969). These data indicate an integral role of pituitary hormones in maintenance of the reticuloendothelial system and regulation of immune responses. Of the various 118
0889-1591/90 $3.00 Copyright 8 1990 by Academic Press, Inc. Au rights of reproduction in any form reserved.
HYPOPHYSECTOMY
GROWTH
HORMONE
IMMUNITY
119
hormones produced by the pituitary, growth hormone in conjunction with thymic hormones appear to be of primary importance for homeostasis of the immune system (reviewed by Kelley, 1989). Treatment of hypophysectomized rats or mice with exogenous growth hormone has been reported to partially or completely restore depressed lymphoid organ weights (Simpson, Evans, & Li, 1949; Feldman, 1951; Maor, Englander, Eylan, & Alexander, 1974) and humoral (Pandian & Talwar, 1971; Nagy et al., 1983; Comsa, Schwarz, & Neu, 1974; Arrenbrechdt & Sorkin, 1973) and cell-mediated immune responses (Comsa et al., 1975; Berczi et al., 1983; Pierpaoli, Fabris, & Sorkin, 1970; Saxena et al., 1982). Growth hormone therapy has also been reported to partially restore immune responsiveness in genetically hypopituitary species (Baroni et al., 1969; Pierpaoli et al., 1969; Fabris et al., 1971b). There is some question at this time whether growth hormone acts alone or in concert with other factors. Receptors for growth hormone are present on human lymphocytes and macrophages (Lesniak, Roth, Gorden, & Gavin, 1983; Kiess & Butenandt, 1985) which have strong cross-reactivity with prolactin (Kover & Moore, 1984). There is also strong evidence that the action of growth hormone on the immune system is mediated via the thymus or enhanced by thymic factors (Comsa et al., 1974, 1975; Arrenbrecht & Sorkin, 1973; Pierpaoli et al., 1970; Pierpaoli, Fabris, & Sorkin, 1971). More recent studies indicate a role of stressrelated hormones (Nagy et al., 1983; Berczi et al., 1983; Berczi, Nagy, Asa, & Kovacs, 1984) or lactogenic hormones in general (Berczi & Nagy, 1986). This study was designed to examine the effects of growth hormone replacement on multiple immune responses of hypophysectomized rats. Humoral, specific cell-mediated and nonspecific cell-mediated immune responses and cytokine production were assessed in each animal on experiment. MATERIALS
AND METHODS
Animals
Eight-week-old male Fischer 344 rats, purchased from Simonsen Laboratories (Gilroy, CA) were randomly assigned to groups of five rats each and caged two or three animals per cage. Rats were housed in stainless steel hanging wire cages in a room with 12-h light/dark cycles and recommended limits of temperature, humidity, and air exchange. Commercial rodent chow and deionized water were provided ad libitum. All hypophysectomized and sham-hypophysectomized rats were maintained on 5% glucose solution. Hypophysectomy
Hypophysectomized and sham-hypophysectomized rats were purchased from Simonsen Labs. The surgeries were performed by the parapharyngeal route 2 weeks prior to initiation of the study. The sella turcica was examined grossly at necropsy to verify completeness of the hypophysectomies. Hormone
Treatment
Two weeks after surgery all rats were injected daily for 13 days, subcutaneously
120
EXON,
BUSSIERE,
AND
WILLIAMS
(SC) in the loose skin over the back, with either 40 pg bovine somatotropin (bGH, Miles Scientific, Naperville, IL) in 0.1 ml sterile physiologic saline or shaminjected with saline alone. Animals were weighed prior to initiation of hormone therapy and twice weekly for the duration of the studies. Gross Pathology
All animals were terminated 14 days after beginning hormone treatment, and complete necropsies were performed on each rat. Whole blood samples were collected by cardiac puncture and hematologic parameters, white and red cell counts, hemoglobin and hematocrit, determined using a Coulter counter, Model ZBI (Coulter Electronics, Hialeah, FL). Body, spleen, thymus, liver, kidney, heart, testis, and adrenal weights were recorded. Assay for Delayed-Type
Hypersensitivity
(DTH)
The delayed-type hypersensitivity response was measured by a modification of the footpad swelling method described by Henningsen, Koller, Exon, Talcott, and Osborne (1984). This method has been shown to induce a typical Type IV DTH reaction. Rats were sensitized by an SCinjection of heat-aggregated keyhole limpet hemocyanin (KLH, Calbiochem, San Diego, CA) at the base of the tail to induce a delayed hyersensitivity reaction (Table 1). Heat-aggregation was achieved by mixing 120 mg of KLH in 6 ml sterile saline (2% w/v) and heating for 1 h at 80°C in a water bath. Rats were injected with 100 ~1 of the aggregate (2 mg KLH). One week later the left footpad was challenged by SCinjection of 2 mg of heat-aggregated KLH suspension in 100 ~1. The right footpad was sham-injected with sterile saline. Twenty-four hours later, footpad swelling in both hind feet was measured with a digital (Model 800) electronic micrometer (Model 4440, EG&G Quality Measurement Systems, Pentield, NY). The DTH reaction was determined by subtracting the thickness of the saline-injected footpad from the KLH-injected footpad. Results are expressed as millimeter difference in swelling. Assay for Antibody
Synthesis
Immediately following the measurement of the DTH reaction, rats were injected SCwith 1 mg of KLH in 0.2 ml of sterile saline to induce a secondary (IgG) TABLE
1
Antigen Injection Schedule
Day 0 I 8 14
Antigen
Dose
loo pl KLH-HA” KLH-HA” loo kl Measure DTH’ response 1 mg KLH (aqueous) Terminate and collect splenocytes for NK and IL2 assays and serum for antibody assay
(2Heat-aggregated keyhole limpet hemocyanin, 2% solution (2 mg/lOO ~1). b Subcutaneous. ’ Delayed-type hypersensitivity.
Route SCb Footpad SC
HYPOPHYSECTOMY
GROWTH
HORMONE
IMMUNITY
121
antibody response (Table 1). Six days later the rats were sacrificed by CO, asphyxiation. The animals were fasted for 18 h prior to sacrifice to lower lipid levels in serum. Blood was collected by cardiac puncture. An aliquot of each blood sample was allowed to clot and the serum was removed and stored at 4°C until analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) described by Exon, Koller, Talcott, O’Reilly, and Henningsen (1986). Endpoint IgG antibody titers were defined as the highest dilution of serum which gave an absorbance greater than the statistical mean (0.250) of a normal rat serum control. The normal serum was diluted 1:50 to simulate sample dilutions. Other controls included three groups of wells in which the anti-IgG conjugate, the substrate and the KLH were singularly eliminated from the reaction mixture. To reduce statistical variability, the endpoint titers were then ranked by the method of Wilcoxon with the lowest titer being given the rank of one (Ott, 1977). The ranked data was analyzed by analysis of variance and least-squares mean comparisons. Assay for Natural
Killer
Cell Cytotoxicity
Rat spleens were removed aseptically and cell suspensions were prepared by forcing the spleens through a stainless steel wire mesh into RPM1 Medium 1640 (RPMI, GIBCO Laboratories, Chagrin Falls, OH) containing 100 U/ml penicillin and 100 kg/ml streptomycin (P/S). Spleen cells were centrifuged at 200g for 10 min, resuspended in RPMI, P/S, and counted on a Coulter counter, Model ZBI (Coulter Electronics) to determine total spleen cell numbers. An aliquot of splenocytes was removed at this point for assay of interleukin 2 (IL2) production (see below). The remaining spleen cell suspensions were incubated on nylon wool columns for 45 min in a humidified, 10% CO, atmosphere at 37°C to remove B lymphocytes and phagocytic cell populations. Nonadherent cells were gently flushed from the columns into polystyrene 75-cm2 tissue culture flasks and incubated as before for 1 h to further remove adherent cells. Natural killer (NK) cell cytotoxicity to YAC-I tumor cells was measured by a 4-h chromium-release assay described by Talcott, Exon, and Koller (1984). The percentage cytotoxicity of each sample was calculated from the specific 51Cr release by the formula: Experimental release - spontaneous release x 100%. Maximum release - spontaneous release An NK-insensitive lymphoma cell line, EL-4, was labeled in the same manner as the YAC target cells and incubated with effector cells to serve as a control for non-NK-mediated lysis. Lysis of EL4 cells was less than 2-3% above spontaneous release. Spontaneous release for the NK assay was less than 10%. Assay for Interleukin
2 Production
Synthesis of IL2 was induced in aseptically isolated rat splenocytes (1 x lo6 cells/ml) in RPMI, P/S, containing 0.4 g/liter bovine serum albumin (Sigma), by adding 1.O kg/ml concanavalin A (Sigma) to the cultures, and incubating for 24 h
122
EXON,
BUSSIERE, AND WILLIAMS
at 37°C in a 5% CO*, humidified atmosphere. Identical, unstimulated samples served as negative controls. The amount of IL2 produced by spleen cells was analyzed by its capacity to maintain the growth of CTLL-2 T cells as previously described (Exon et al., 1986). It should be noted that recent evidence suggests this assay may not be capable of distinguishing between IL2 and IL4. The IL2 assay was performed on two separate days using five animals/group/day. Since the assay has innate day-to-day variability, the units were converted to percentage of the daily control and analyzed by analysis of variance based on percentage change. Statistics All data were analyzed with the Statistical Analysis System (SAS Inc., New York, NY). Significant treatment differences were based on analysis of variance and least-squares mean comparisons with an (Ylevel of 0.05. RESULTS
Hypophysectomized rats, with or without bGH treatment, exhibited significantly (p < 0.05) reduced body weights compared to sham-operated rats (Table 2). Weights of hypophysectomized rats which received bGH tended to be greater (p s 0.07) than those which were only hypophysectomized and injected with saline. Thymus, spleen (Table 2), liver, kidney, heart, testes, and adrenal (Table 3) weights were significantly (p < 0.05) reduced in hypophysectomized rats. Organ weights of animals which were hypophysectomized and treated with bGH remained decreased, except for thymic weights which recovered to about normal values. Red blood cell counts, hemoglobin levels, and hematocrits were significantly (p s 0.05) reduced in hypophysectomized rats with or without bGH treatments (Table 4). White blood cell counts were significantly (p < 0.05) elevated in hypophysectomized rats compared to sham-operated controls. Hypophysectomized rats treated with bGH had significantly (r, d 0.05) increased wbc counts compared to either the hypophysectomized group or the sham-operated control. Serum antibody titers to KLH, DTH reactions (Table 5), NK cytotoxicity, IL2 (IL4) production, and spleen cell number (Table 6) were significantly (p C 0.05) TABLE 2 The Effects of Hypophysectomy and Growth Hormone on Body, Spleen, and Thymus Weights (g)
SHAM-HXb HX HX + bGH=
N
Body”
10 10 10
176.1 2 5.6A 106.8 t- 2.8B 116.0 4 4.8B
Spleen 0.26 2 O.OOA 0.18 + 0.01a 0.18 + O.OIB
Thymus 0.152 f 0.007A 0.115 * o.oO!P 0.145 + o.OO!P
a Values are means f the standard error of the mean determined at the time of sacrifice following daily injections of saline or bGH for 13 days. Means with no common capital letters are statistically different (p c 0.05) by analysis of variance and least-squares mean comparisons. b Hypophysectomy. c Bovine growth hormone.
HYPOPHYSECTOMY
GROWTH
HORMONE
123
IMMUNITY
TABLE 3 Effects of Hypophysectomy and Growth Hormone on Weights of Liver, Kidney, Heart, Testes, and Adrenal Weight (q + SEM)”
SHAM-HXb HX HX + bGH’
Liver
Kidney
Heart
Testes
Adrenal
7.0A f 0.3 3.6B +- 0.1 3.9u f 0.2
ow f 0.03 0.36B 5 0.01 0.40B ? 0.01
0.72* 2 0.04 0.31B 4 0.01 0.3SB f 0.02
1.31* f 0.02 0.14B 2 0.03 0.138 +- 0.02
0.04oA r 0.003 0.01on f 0.001 0.0128 f 0.001
0 Values are means 2 standard error of the mean determined at the time of sacrifice following daily injections of saline or bGH for 13 days. Means with no common capital letter are statistically different 0, < 0.05) by analysis of variance and least-square mean comparisons. N = IO/group. b Hypophysectomy. ’ Bovine growth hormone.
lower in hypophysectomized rats compared to sham-operated controls. Natural killer cell cytotoxicity, spleen cell counts, and DTH reactions were also significantly (r, s 0.05) suppressed in rats which were hypophysectomized and received bGH treatment. Antibody titers and IL2 (IL4) production in these animals, however, were not different than controls. The DTH reactions in hypophysectomized animals which received bGH were also significantly (r, s 0.05) greater than those which received only hypophysectomy. DlSCUSSlON
The results of this study indicate the pituitary has an important role in maintenance of normal immune function in rats. Hypophysectomized rats had decreased thymus and spleen weights and reduced spleen cell numbers. Functional immune responses were also suppressed in hypophysectomized rats as noted by decreased antibody and IL2 (IL4) production, DTH reactions, and NK cytotoxic responses. These data are in agreement with those of others which indicate than an intact pituitary is necessary for normal humor-al (Nagy & Berczi, 1978; Berczi et al., 1981; Nagy et al., 1983) and specific (Nagy & Berczi, 1978; Comsa et al., 1975; TABLE 4 The Effects of Hypophysectomy and Growth Hormone on Hematologic Parameters”
SHAM-HX HX HX + bGH
N
RBC ( 106/mm3)
WBC ( 103/mm3)
Hct m
Hbg WV
10 10 9
9.57 e o.osA 8.33 2 0.13B 8.31 * 0.12B
9.5 + 0.3* 13.9 f 1.4n 17.4 5 1.4c
43.9 * 0.4* 35.5 +- 1.1u 37.9 f 1.ou
16.6 2 0.1* 14.1 + 0.2n 13.8 f 0.2B
n Abbreviations used: bGH, bovine growth hormone; Hbg, hemoglobin; Hct, hematocrit; HX, hypophysectomy; RBC, red blood cells; WBC, white blood cells. Values are means + the standard error of the mean of hematology values determined at the time of sacrifice following daily injection of either physiologic saline or 40 kg bGH for 13 days. Means with no common capital letters are statistically different (p < 0.05) by analysis of variance and least-squares mean comparisons.
124
EXON,
BUSSIERE,
AND WILLIAMS
TABLE 5 The Effects of Hypophysectomy and Growth Hormone on IgG Antibody Titers, Delayed-Type. Hypersensitivity, and Production of IL2”
SHAM-HX HX HX + bGH
N
Ranked Ab titefl
DTH’ (mm)
IL2d % of control
10 10 10
15.9 2 2.8* 8.4 k 2.6’ 12.8 -t 1.9*
2.79 ” 0.13A 1.58 + 0.11’ 2.25 + 0.18’
85.9 ? 6.1* 68.6 + 5.3B 81.0 + 6.4A
a Abbreviations used: Ab, antibody; bGH, bovine growth hormone; DTH, delayed-type hypersensitivity; HX, hypophysectomy; IL2, interleukin 2. Values are means f the standard error of the mean. Means with no common capital letter are statistically different (p < 0.05) by analysis of variance and least-squares mean comparisons. * Antibody endpoint titers to keyhole limpet hemocyanin were determined from serum taken at the time of sacrifice and ranked with the lowest titer receiving the rank of one. ’ Difference (mm) in footpad swelling between right and left. d Production of IL2 (IL4) by splenocytes is reported as a percentage of the mean units of IL2 for normal control animals on the day on which the animals were sacrificed. Control means were 35.2 and 26.9 U.
Berczi et al., 1983; Prentice et al., 1975) and nonspecific cell-mediated (Cross et al., 1984; Saxena et al., 1982) immune responses. It appears that GH can partially or completely restore some of the immune dysfunction associated with hypophysectomy. Animals which received GH treatment and were hypophysectomized had thymus weights and antibody and IL2 (IL4) production comparable to sham-operated controls. The DTH reactions in hypophysectomized rats which were given GH were significantly greater than in animals which were only hypophysectomized, but were still less than shamoperated controls. In contrast, NK responses were similar in hypophysectomized animals with or without GH treatment. This data indicates the humoral (B cell) and specific cell-mediated (T cell) immune response may be more sensitive to restoration by GH than non-specific cell-mediated (NK cell) responses, although ‘TABLE 6 The Effects of Hypophysectomy and Growth Hormone on Splenic Natural Killer Cell Cytotoxicity and Spleen Cell Numbef % Cytotoxicity Effector:Target cell
SHAM-HX* HX HX + bGH’
N
1OO:l
50: 1
Total spleen cell counts (X 10”)
10 10 10
40.4 f 1.6* 20.2 f l.lB 21.8 f 1.3B
31.7 2 1.1* 14.8 + 1.3B 17.2 + 1.4’
2.95 + 0.18A 0.81 f 0.09’ 1.07 + 0.099
n Values are means f the standard error of the mean determined upon sacrifice following daily injections of either saline or 40 pg bGH for 13 days. Means with no common capital letter are statistically diierent (p c 0.05) by analysis of variance and least-squares mean comparisons. * Hypophysectomy. c Bovine growth hormone.
HYPOPHYSECTOMY
GROWTH
HORMONE
IMMUNITY
125
others have reported partial restoration by GH of NK cell cytotoxic responses in hypophysectomized mice (Saxena et al., 1982). Conflicting data on ability of GH or GH-releasing factor to restore reduced NK cell responses in GH-deficient patients has been reported (Crist, Peak, Mackinnon, Sibbit, & Kraner, 1987; Kiess, Malozowski, Gelato, Buten and, Doerr, Crisp, Eisl, Maluish, and Belohradsky, 1988). Several investigators have reported GH treatment can restore humoral (Pandian & Talwar, 1971; Nagy et al., 1983) and T cell-mediated (Comsa et al., 1975; Berczi et al., 1983) immune responses in hypophysectomized animals. A confounding factor in our study was the GH therapy failed to completely restore body and organ weights of hypophysectomized animals. Therefore, this treatment regimen or purity of GH may not have been adequate to restore all immune responses. It has been postulated that the effects of GH may be mediated through the T helper cell population (Arrenbrecht 8z Sorkin, 1973). In our study, IL2 (IL4) production was significantly suppressed in hypophysectomized rats but this effect was reversed by GH treatment. It is well documented that IL2 is important in induction of optimal B and T cell-mediated responses (reviewed by Robb, 1984). Although not shown conclusively in this study, GH may mediate its effects partially through stimulation of IL2. We have previously observed enhanced IL2 production in normal rats treated with GH in our laboratory (unpublished data). It would be interesting to determine if IL2 (or IL4) alone could restore immune responses in hypophysectomized rats. If this is a mechanism by which GH acts, it is less clear why NK cell responses were not similarly restored since IL2 production had returned to normal. The NK cytotoxic response is also reported to be sensitive to ILZinduced enhancement (Yamada, Ruscetti, Overton, Herberman, Birchenall-Sparks, & Ortaldo, 1987). Perhaps a longer treatment period, higher doses, or a species-specific GH may have been more effective. General hematological parameters were also altered in hypophysectomized rats. Red blood cell counts, hematocrits, and hemoglobin levels were significantly reduced in hypophysectomized animals. These effects were not reversed in animals which also received GH treatment. These results are similar to those of Crafts and Meineke (1959) who showed anemia in hypophysectomized rats could be reversed by a combination of GH, thyroxin, and cortisone acetate. However, treatment with any one of the agents was not sufficient to restore normal blood counts. These authors postulated that the posthypophysectomy anemia was due to effects on general metabolism and oxygen need of erythrocytes. A more recent study, however, by Nagy and Berczi (1989) showed that anemia and leukopenia in hypophysectomized female Fisher 344 rats could be reversed by subrenal pituitary grafts, bGH, or prolactin. Since prolactin and GH can bind the same receptors it is unclear which hormone may be most important in restoring hematologic parameters. Also, the activity of the bGH used in that study was greater than ours. Conversely, white blood cell counts were significantly increased in hypophysectomized rats, and this effect was significantly exacerbated by concomitant GH treatment. Increased wbc counts in hypophysectomized rats was a surprising finding since most evidence indicates the converse (reviewed by Kelley, 1989). In
126
EXON,
BUSSIERE,
AND WILLIAMS
fact, Nagy and Berczi (1989) reported a leukopenia in hypophysectomized female F344 rats. These results remain to be confirmed by additional studies. The preponderance of evidence indicates that GH stimulates proliferation of lymphocytes and reduces leukopenia. Therefore, it is not surprising that administration of GH increased even further the leukocytosis. The source of increased numbers of wbc is unknown since differentials were not performed in this study. It is apparent, however, that perturbation at the bone marrow level may have significant implications to altered immune function via effects on progenitor (stem) cells. REFERENCES Arrenbrecht, S., & Sorkin, E. (1973). Growth hormone-induced T cell differentiation. Eur. J. Zmmuml. 3, 601-604. Baroni, C. D., Fabris, N., & Bertoli, G. (1969). Effects of hormones on development and function of lymphoid tissues: Synergistic action of thyroxin and somatotropic hormone in pituitary dwarf mice. Zmmunology 17, 303-314. Baroni, C. D., Pesando, P. C., & Bertoli, G. (1971). Effects of hormones on development and function of lymphoid tissues. II. Delayed development of immunological capacity in pituitary dwarf mice. Immunology 21, 455461. Baroni, C. D., Scelsi, R., Mingazzini, P. L., Cavallero, A., & Uncinni, S. (1972). Delayed hypersensitivity in the hereditary pituitary dwarf SnelYBagg mouse. Nature New Biol. 237, 21%220. Bartke, A. (1964). Histology of the anterior hypophysis, thyroid and gonads of two types of dwarf mice. Anat. Rec. 149, 225-236. Berczi, I., Nagy, E., Kovacs, K., & Horvath, E. (1981). Regulation of humoral immunity in rats by pituitary hormones. Acta Endocrinol. 98, 506-513. Berczi, I., Nagy, E., Asa, S. L., & Kovacs, K. (1983). Pituitary hormones and contact sensitivity in rats. Allergy 38, 325-330. Berczi, I., Nagy, E., Asa, S. L., & Kovacs, K. (1984). The influence of pituitary hormones on adjuvant arthritis. Arthritis Rheum. 27, 682-688. Berczi, I., & Nagy, E. (1986). Prolactin and other lactogenic hormones. In I. Berczi. (Ed.), Pituitary function and immunity, pp. 161-184. CRC Press: Boca Raton, FL. Comsa, J., Schwarz, J. A., & Neu, H. (1974). Interaction between thymic hormone and hypophyseal growth hormone on production of precipitating antibodies in the rat. Zmmunol. Commun. 3, 11-18. Comsa, J., Leonhardt, H., & Schwarz, J. A. (1975). Influence of the thymus-corticotropin-growth hormone interaction on the rejection of skin allografts in the rat. Ann. N. Y. Acad. Sci. 249, 387401. Crafts, R. C., & Meineke, H. A. (1959). The anemiaof hypophysectomized animals. Ann. N. Y. Acad. Sci. 77, 501-517. Crist, D. M., Peake, G. T., Mackinnon, L. T., Sibbit, W. L., & Kraner, J. C. (1987). Exogenous growth hormone treatment alters body composition and increases natural killer cell activity in women with endogenous growth hormone secretion. Metabolism 12, 1115-l 117. Cross, R. J., Markesbery, J. A., Brooks, W. H., & Roszman, T. L. (1984). Hypothalamic-immune interactions: Neuromodulation of natural killer activity by lesioning of the anterior hypothalamus. Immunology 51, 3m5. Duquesnoy, R. J. (1972). Immunodeficiency of the thymus-dependent system of the Ames dwarf mouse. J. Zmmunol. 108, 1578-1590. Enerback, L., Lundin, P. M., & Mellgren, J. (1961). Pituitary hormones elaborated during stress: Action on lymphoid tissues, serum proteins, and antibody titres. Acta Pathol. Microbial. Zmmunol. Stand. (SuppZ) 144, 141-144. Exon, J. H., Koller, L. D., Talcott, P. A., O’Reilly, C. A., & Henningsen, G. M. (1986). Immunotoxicity testing, an economical multiple-assay approach. Fundam. Appl. Toxicol. 7, 387-397. Fabris, N., Pierpaoli, W., & So&in, E. (197la). Hormones and the immunological capacity. III. The
HYPOPHYSECTOMY
GROWTH
HORMONE
IMMUNITY
127
immunodeficiency disease of the hypopituitary Snell-Bagg dwarf mouse. Clin. Exp. Immunol. 9, 209-225. Fabris, N., Pierpaoli, W., & So&in, E. (197lb). Hormones and the immunological capacity. IV. Restorative effects of developmental hormones or of lymphocytes on the immunodeficiency syndrome of the dwarf mouse. Clin. Exp. Immunol. 9, 227-240. Feldman, J. D. (1951). Endocrine control of lymphoid tissue. Anat. Rec. 110, 17-39. Gisler, R. H., & Schenkel-Hulliger, L. (1971). Hormonal regulation of the immune response. II. IntIuence of pituitary and adrenal activity on immune responsiveness in vitro. Cell. Immunol. 2, 646-657.
Henningsen, G. M., Koller, L. D., Exon, J. H., Talcott, P. A., & Osborne, C. A. (1984). A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity. J. Immunol. Methods 70, 53-165. Kalden, J. R., Evans, M. M., & Irvine, W. J. (1970). The effect of hypophysectomy on the immune response. Immunology 18, 671-679. Kelley, K. W. (1989). Growth hormone, lymphocytes and macrophages. Biochem. Pharmacol. 38, 705-713. Kiess, K., & Butenandt, 0. (1985). Specific growth hormone receptors on human peripheral mononuclear cells: Reexpression, identification, and characterization. .I. Clin. Endocrinol. Metab. 60, 740-746.
Kiess, W., Malozowski, S., Gelato, M., Butenand, O., Doerr, H., Crisp, B., Eisl, E., Maluish, A., & Belohradsky, B. H. (1988). Lymphocyte subset distribution and natural killer cell activity in growth hormone deficiency before and during short-term treatment with growth hormone releasing hormone. Clin. Immunol. Immunopathol. 48, 85-94. Kover, K., 62 Moore, W. (1984). Comparison of hGH binding to isolated rat liver macrophages and hepatocytes. Horm. Metab. Res. 16, 193-197. Lesniak, M. A., Roth, J., Gorden, P., & Gavin, J. R. (1973). Human growth hormone radioreceptor assay using cultured human lymphocytes. Nature New Biol. 241, 20-22. Lundin, P. M. (1958). Anterior pituitary gland and lymphoid tissue. Acta Endocrinol. (Suppl.) 40, l-83. Lundin, P. M. (1960). Action of hypophysectomy on antibody formation in the rat. Acta Pathol. Microbial. Immunol. Scand. 48, 351-357. Maor, D., Englander, T., Eylan, E., & Alexander, P. (1974). Participation of hormone in the early stages of the immune response. Acta Endocrinol. 75, 205-208. Nagy, E., & Berczi, I. (1978). Immunodeficiency in hypophysectomized rats. Acta Endocrinol. 89, 530-537.
Nagy, E., & Berczi, I. (1989). Pituitary dependence of bone marrow function. Brit. .I. Huemntol.
71,
457-462.
Nagy, E., Berczi, I., & Friesen, H. G. (1983). Regulation of immunity in rats by lactogenic and growth hormones. Acta Endocrinol. 102, 351-357. Ott, L. (1977). An introduction to statistical methods and data analyses, p. 317. Duxburg Press: Massachusetts. Pandian, M. R., & Talwar, G. P. (1971). Effect of growth hormone on the metabolism of thymus and on the immune response against sheep erythrocytes. J. Exp. Med. 134, 1095-I 113. Pierpaoli, W., Baroni, C. D., Fabris, N., & Sorkin, E. (1969). Hormones and immunological capacity. II. Reconstitution of antibody production in hormonally deficient mice by somatotropic hormone, thyrotropic hormone and thyroxin. Immunology 16, 217-230. Pierpaoli, W., Fabris, N., & Sorkin, E. (1970). Developmental hormones and immunological maturation. In G. E. W. Wolstenholme & J. Knight (Eds.), Hormones and the immune response, pp. 126153. Churchill: London. Pierpaoli, W., Fabris, N., & Sorkin, E. (1971). The effects of hormones on the development of the immune capacity. In S. Cohen, G. Cudkowicz, & R. T. McCluskey (Eds.), Cellular interactions in the immune response, p. 25. Karger: Basel. Prentice, E. D., Lipscomb, H., Metcalf, W. K., & Sharp, J. G. (1975). Effects of hypophysectomy on DNCB-induced contact sensitivity in rats. Stand. I. Immunol. 5, 955-961. Robb, R. J. (1984). Interleukin 2: The molecule and its function. Immunol Today 5, 203-209.
128
EXON,
BUSSIERE,
AND WILLIAMS
Roth, J. A., Kaeberle, M. L., Greir, R. L., Hopper, J. G., Spiegel, H. E., &z McAllister, H. A. (1984). Improvement in clinical condition and thymus morphologic features associated with growth hormone treatment of immunodeficient dwarf dogs. Amer. .I. Vet. Res. 45, 1151-1155. Saxena, Q. B., Saxena, R. K., L Adler, W. H. (1982). Regulation of natural killer cell activity in vivo. III. Effect of hypophysectomy and growth hormone treatment on the natural killer activity of the mouse spleen cell population. Int. Arch. Allergy Appl. Immunol. 67, 169-174. Simpson, M. E., Evans, H. M., & Li, C. H. (1949). The growth of hypophysectomized female rats following chronic treatment with pure pituitary growth hormone. I. General growth and organ changes. Growth 13, 151-170. Smith, P. E. (1930). The effect of hypophysectomy upon the involution of the thymus in the rat. Anat. Rec. 47, 119-129. Talcott, P. A., Exon, J. H., & Koller, L. D. (1984). Alteration of natural killer cell-mediated cytotoxicity in rats treated with selenium, diethylnitrosamine and ethylnitrosourea. Cancer Lett. 23, 313-322. Wilkinson, P. C., Singh, H., & Sorkin, E. (1970). Serum immunoglobulin levels in thymus-deficient pituitary dwarf mice. Immunology 18, 437-441. Yamada, S., Ruscetti, F. W., Overton, W. R., Herberman, R. B., Birchenall-Sparks, M. C., & Ortaldo, J. R. (1987). Regulation of human large granular lymphocyte and T cell growth and function by recombinant interleukin 2: Induction of IL2 receptor and promotion of growth of cells with enhanced cytotoxicity. .I. Leukocyte Biol. 41, 505-517. Received May 15, 1989
