Chicken growth hormone, triiodothyronine and thyrotropin releasing hormone modulation of the levels of chicken natural cell-mediated cytotoxicity

Developmental

and Comparative

Immunology, Vol. 15, pp. 65-71,

1991

Printed in the USA. All rights reserved.

0145.305x/91 $3.00 + .oa Copyright 0 1991 Pergamon Press plc

CHICKEN GROWTH HORMONE, TRIIODOTHYRONINE AND THYROTROPIN RELEASING HORMONE MODULATION OF THE LEVELS OF CHICKEN NATURAL CELL-MEDIATED CYTOTOXICITY Eid E. Haddad and Magdi M. Mashaly Department

of Poultry Science, The Pennsylvania (Received

State University, University Park, PA 16802

May 1989; Accepted

qAbstract-Newly hatched White Leghorn male chicks received dietary supplements of either Triiodothyronine (T,) or Thyrotropin Releasing Hormone (TRH) until 8 weeks of age. Chicken growth hormone (cGH) (10 pg/kg BW) was injected into different chicks twice daily for 1 week starting at 7 weeks of age. Separate groups received both T3 and cGH. Natural cell-mediated cytotoxic (NCMC) activity against different target cells was tested. It was found that cytotoxic activity in cells involved in NCMC against P815 mouse mastocytoma was stimulated by cGH alone or in combination with T:, (1 ppm). These findings indicate that cGH and T, stimulate NCMC activity; and that the cells responsible for this activity may be Natural Killer (NK) cells.

August

1989)

been studied mostly in mammalian species, experimental data also have shown that avian species have an effective NK system (reviewed in 22). Chicken NK (cNK) activity has been associated with nonadherent nonphagocytic lymphomononuclear cells (10,20). Morphologically, cNK cells have been described (16). Chicken NK cells appear to lack surface antigens detectable by antiserum against thymic (T) or bursal (B) cells (21). Cells with NK activity have been reported present in the peripheral blood (6,11,12), spleen (4,20,21) and recently in the gut mucosal lymphoid populations (1) of chickens. In humans and mice, NK activity can be modulated by bacterial, viral, and synthetic agents. Certain hormones have been reported to modulate NK cell activity. Administration of estrogen to mice of either sex caused a substantial reduction in the splenic NK cell activity (18), which could have resulted from either blocking NK cell maturation or reducing the number of precursor cells (19). Modulation of NK activity by estrogen was time dependent, for example, stimulatory within the first month of treatment and inhibitory thereafter (17). Mice treated with hydrocortisone have been reported to have decreased NK cell activity against YAC-1 lymphoma (8). Incubation of lymphocytes from cancer patients, with either leucine- or methionine-enkephalin, resulted in significant increases in NK cell activity (5).

qiKeywords-Growth hormone; Triiodothyronine; Thyrotropin releasing hormone; Chickens; Natural cell-mediated cytotoxicity; Tumor cells; NK cells. Introduction Unlike cytotoxic T lymphocytes, lymphomononuclear cells involved in natural cell-mediated cytotoxicity (NCMC) are MHC-unrestricted and have the ability to lyse tumor cell lines without prior immunization or stimulation. One of the cell types that are involved in NCMC has been called natural killer (NK) cells. Although NK cells have Address correspondence to Magdi M. Mashah, Department of Poultry Science, 202 Henning Building, The Pennsylvania State University, University Park, PA 16802. 65

66

Modulation of NK activity by thyroid hormones has been partly investigated. Induced hyperthyroidism, by thyroxine (T4) adminstration, enhanced in vitro splenic and peritoneal as well as in vivo NK cell activity in some strains of mice (23). In contrast, hyperthyroidism, either endogenous (Graves' disease) or exogenous (injections to mice and human), was found to reduce the activity of NK cells (13,24). The hypothalamic-pituitary axis has been postulated to regulate several immune activities; NK activity may be one of these. Hypothalamic damage, hypophysectomy, or both caused a significant reduction in the activity of rat NK cells (2). Similar results were also seen in hypophysectomized female mice (I 5) when their spleen NK activity was tested against YAC tumor cells. These authors (15) reported a marked recovery of NK activity after administration of ovine growth hormone (GH) (100 i~g/day, intraperitoneally for 10 days) into hypophysectomized mice. Patients with a chronic deficiency of GH, GH-Releasing Factor (GHRF), or both, had not only a lower percentage of cells expressing NK surface markers but also, reduced activity of cells with NK surface markers (9). Acute administration (1 p.g/kg body weight, i.v.) of GHRF did not restore NK activity (9). Implanted pituitary adenoma cells (GH3), which secrete GH and prolactin in nude mice, failed to modulate NK activity (3); however, subcutaneous injections of ovine GH (750 o.g, twice daily for 5 weeks) to 24-month-old rats enhanced their NK activity (3). There has been no such work concerning the effect of thyroid hormones or GH on the activity of cells involved in NCMC, such as NK cells, in chickens. Previous work (7), as well as the abovementioned reports, prompted examination of the effect of thyrotropin releasing hormone (TRH), triiodothyronine (T3), and chicken growth hormone (cGH) on the natural cytotoxicity in chickens.

E.E. Haddad and M. M. Mashaly

Materials and Methods Animal and Hormone Treatments Newly hatched White Leghorn male (K strain) chicks were used in this study. Experimental design and hormone treatments were previously described in detail (7). Briefly, two experiments, of two replicates each, were conducted. Chicks in the first experiment were fed a diet supplemented with cither T 3 (0.1 and I ppm) or TRH (1 and 5 ppm) for an 8week period starting at hatch. Chicks in the second experiment were injected intravenously (i.v.) with cGH (10 ixg/kg BW), either alone or with T3 supplementation (0.1 and ! ppm), twice daily for I week starting at 7 weeks of age. Two other groups served either as sham-injected controls receiving 0.2 mL sterile saline (i.v.) injected twice daily for 1 week starting at 7 weeks of age or as uninjected controls fed unsupplemented diet.

Media Preparations Medium RPMI-1640 (Gibco Lab., NY) with L-glutamine and without sodium bicarbonate was supplemented with 2 gm/L sodium bicarbonate, 0.5% penicillin/streptomycin (5000 U.mL-1/ 5000 m e g . m L - b , 0.1% gentamicin, 1% HEPES, and either 10% fetal calf serum (FCS) (complete medium) or 2% FCS (washing medium). The pH was adjusted to 6.9 using 1 N HCI before cold filtration. The same complete medium with the addition of !% nonessential amino acid, 1% sodium pyruvate and 2.5% HEPES instead of I% was used to grow and culture (culture media) the target cells.

Leukocyte Separation At 8 weeks of age, blood samples (10 mL) were drawn aspectically (heart

Hormones ancl natural cytotoxicity

puncture) into sterile syringes rinsed with sterile heparin and samples were kept on ice until tested. Blood (10 mL) was diluted with 14 mL of sterile phosphate buffer saline (PBS), pH 7.0. Diluted blood was then aseptically layered over 8 mL (3:1, diluted blood:Histopaque) of Histopaque (Sigma, St. Louis, MO) using sterile round-bottom, screwcapped 50 mL glass bottles. Blood samples were then centrifuged at 1200 rpm for 30 min at room temperature. The leukocyte layer (interface between Histopaque and diluted plasma) was aspirated, washed once in sterile PBS and two more times in washing media, then diluted with 5 mL of complete media. In an attempt to enrich the lymphocyte population and to remove the adherent cells, separated leukocytes were layered in petri dishes (10a cells/dish) previously rinsed with FCS and were incubated for 1 h in a humidified CO2 (5%) incubator at 37°C. Cells were then collected, washed three times with washing media, resuspended to the desired concentration in complete media, and kept under ice while the next step was performed. Viability (trypan blue exclusion) was always greater than 95%. The procedure for enrichment was not fully successful since percent lymphocytes after enrichment was, in most, more than 80% but less than 87% as determined by differential count.

Target Cells Three target cell lines include K562 (a human myelogenous leukemia line), YAC-I (lymphoma), and P815 (mastocytoma) were kindly provided by Dr. Frederick G. Ferguson (The Pennsylvania State University). All target cells were maintained at 37°C in a humidified atmosphere of 5% COz in the culture medium. The 72-h culture of all target cells were counted, washed, and each labeled with I00 i.tCi of Cr 51 (NaCr5104, NEN, specific activity 400-1200 Ci/g) and incu-

67

bated for 1 h in humidified CO2 (5%) incubator at 37°C. Cultured target cells were washed again three times with washing medium and incubated for another hour, then washed three times and adjusted in complete medium to a concentration of I × 105 cells/mE

Chromium (Ctal) Release Assay Cytotoxic activity of cNK was first tested against the different target cells; and only P815 gave satisfactory results which included ratio (E:T)-response, intravariation, and %SR. Therefore, P815 was used to test effect of hormone treatments on NK activity. Cytotoxicity tests were performed in 96-well, U-bottom microplates (Flow Lab, Inc., McLean, VA) in a total volume of 200 p.L. Different effector:target (E:T) ratios (100, 200, 400: I) were used. Labeled target (I × 104/well) cells were added to the various concentrations of effector cells, centrifuged at 300 rpm for 3 min at room temperature and incubated for 18 h in a humidified COz (5%) incubator at 37°C. The labeled target cells were always plated after the effector cells had been plated. Wells containing labeled target cells either alone or with Triton X-100 were used to determine spontaneous or total Cr 5~ release, respectively. Plates were then centrifuged at 1500 rpm for 15 min at 4°C, then 100 IxL of each well was counted in a gamma counter for 5 min. Percent cytotoxicity was calculated as [(test release - spontaneous release) (total relase - spontaneous release)] × I00. Percent spontaneous release was calculated as (spontaneous release + total release) × 100. Only assays with percent spontaneous release less than 20% were considered valid and used for statistical analyses.

Statistical Analysis The General Linear Model procedures described in the SAS U s e r ' s

68

E. E Haddad and M. M. Mashaly

Guide (14) were applied here. The main effects for each experiment were hormone treatment and replicates. T h e r e was no significant effect due to replicates or b e t w e e n sham and c o n t r o l o p e r a t e d birds; t h e r e f o r e , data w e r e combined for replicates and for sham and c o n t r o l s . Means were c o m p a r e d using Duncan's Multiple Range Test.

Results The c y t o t o x i c activity of c N K cells against different target cells is shown in Table I. It is clear that the P815 mastocytoma cell line was more susceptible to lysis by c N K and had an a c c e p t a b l e %SR as compared to the other targets (Table I) tested in this study. As a consequence of these results as well as other reports (6), we have used P815 in the subsequent assays. Supplementation with T 3 or TRH at any of the concentrations used, did not augment the activity of N K cells (Fig. I) at any of the E:T ratios. The only significant (P < 0.05) differences that were observed were between that of TRH (45.62 +__ 3.21 and 49.44 ___ 4.99 ( m e a n s +_ SEM) for 1 and 5 ppm T R H , respectively) and 0.1 ppm T 3 (30.27 ~ 6.56; mean _+ SEM) at 200:1 E:T ratio. Results from the second experiment are shown in Fig. 2. Injections of cGH significantly (P < 0.05) enhanced the cytotoxic activity o f N K cells at all E:T ratios, approximately 3 - 4 folds above Table 1. Percentage

their respective controls. Combination of cGH and T 3 (I ppm) caused a significant (P < 0.05) enhancement in percent cytotoxicity (31.97 _+ 5.14) relative to the control group (15.58 -+ 2.94), only at 400:1 E:T ratio (Fig. 2).

Discussion The present study shows that peripheral blood lymphocytes exert cytotoxic activity against several target cells as measured by the standard chromium release assay. The cells effective for target cell destruction appear to have charact e r i s t i c s partially similar to those of mammalian species in that they do not adhere to plastic surfaces. Effector cells having the ability to lyse cultured tumor cells in a short-term assay without prior stimulation or sensitization have been called natural killer (NK) cells. Unlike human and murine N K cells, identification of c N K have been based solely on their ability to lyse cultured target cells without prior stimulation or sensitization. We believe that the cytotoxic activity found in this study against P815 mastocytoma was mediated by the same population of cells; N K cells. However, the involvement of other lymphomononuclear cell lines with NCMC cannot be excluded. Possible involvement of adherent cells, which also include thrombocytes, or granulocytes cannot be ruled out because our lymphocyte population after e n r i c h m e n t was not c o m p l e t e l y free o f t h e s e p o p u l a t i o n s ; h o w e v e r ,

o f c y t o t o x t c l t y b y c h i c k e n N K cells

against different target

cells.

E:T Target'* Cells

50:1

100:1

200:1

400:1

% SR

YAC-1 P815 K562

ND ND 0%

32.62 ± 5.19" 13.44 _- 1.27 12.48 = 7.25

41.84 _+ 5.51 29.32 -+ 4.53 22.38 -+ 7.06

39.76 _- 4.60 46.64 = 3.81 38.05 -+ 16.75

24.0 19.0 17.4

• Means ~ SEM. °'n - 4 - 5 , E:T--Effector:Target ratio. ND - not done.

Hormones and natural cytotoxicity

69

70

60

[] .1ppmT3 [] 1~13 ~ lppmT~ 1'7]5ppm~

0

I

0

J

E:T(IO0' 1)

E:T(200:1)

E:T(400'1)

FIgure 1. Percentage of specific lysis of cNK cells as measured by the Crsl release assay using PBL of 8-week-old immature male chickens from Experiment 1. Values are means +_ SEM Data were log-transformed. Bars within each E:T ratio having different letters are significantly (P < 0.05) different, n = 5-7. T3-Triiodothyronine, TRH-Thyrotropin Releasing Hormone, E:T Effector:Target ratio.

DC0~

=b I)

J

K

2o

e:tO00:l)

e:t(2oo:)

Rgure 2. Percentage of specific lysis of cNK cells as measured by the Cr 51 release assay using PBL of 8-week-old immature male chickens from Experiment 2. Growth hormone was injected intravenously (10 v.g/kg BW) at 7 weeks of age twice daily for 1 week. Triiodothyronine was added to the feed as indicated in the legends to Fig. 1. Values are means _+ SEM. Bars within each ratio having different letters are significantly (P < 0.05) different, n = 7-12. T3-Triiodothyronine, cGH-Chicken Growth Hormone, E:T Effector:Target ratio.

70

E.E. Haddad and M. M. Mashaly

others have shown that NK activity were not associated with adherent cells (20). In our previous work we showed that T3-supplementation significantly increased serum T3 concentrations (7). This induced hyperthyroidism did not significantly enhance the cytotoxicity of cNK cells in this study. These results partially confirm those reported by Sharma et al. (23), but not others (13,24) where hyperthyroidism was reported to cause a significant enhancement in NK cell cytotoxicity. These inconsistencies between various reports could be due to species differences. Sharma et al. (23) reported that hyperthyroidism augments the activity of NK cells in some strains of mice but not others. The mechanism of the thyroid hormone effect on NK activity has been proposed (23,24). Although the two reports disagree, both have indicated that the effect of thyroid hormones is at the postconjugate formation state. They also indicated that the effect of thyroid hormones is mediated through IL-2, although the latter report suggests an in-

hibitory effect of T4 on IL-2 release. Our results favor the mechanism suggested by Sharma et al. (23). Injections of cGH significantly increased percent specific lysis at all E:T ratios. These findings support those reported by other investigators (2,3,9,15). The present study is the first to demonstrate that purified cGH can enhance NK activity in chickens. The mechanism of GH action of NK cytolytic activity is unknown. The action could be direct or involve lymphokines. It was hypothesized that either hyperthyroidism or GH would enhance NK cell activity, and that both together would be expected to further potentiate the effect of each other. Our data failed to present strong evidence to support this hypothesis, although a significant enhancement was seen when 1 ppm T 3 was fed with cGH injections. The magnitude of enhancement in NK cytolytic activity due to this treatment (! ppm T 3 and cGH injections) was no greater than cGH alone. Nevertheless, there appears to be a positive effect of cGH on the cytotoxicity of cNK cells.

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