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Endocrinology Vol. 144, No. 5 2102-2110
Copyright © 2003 by The Endocrine Society

Prolactin Suppresses Glucocorticoid-Induced Thymocyte Apoptosis in Vivo

Nithya Krishnan1, Olivier Thellin1, Donna J. Buckley, Nelson D. Horseman and Arthur R. Buckley

College of Pharmacy (N.K., D.J.B., A.R.B.) and Department of Molecular and Cellular Physiology (N.D.H., A.R.B.), University of Cincinnati Medical Center, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Arthur R. Buckley, College of Pharmacy, 3223 Eden Avenue, Cincinnati, Ohio 45267-0004. E-mail: Arthur.Buckley{at}uc.edu; or Nelson D. Horseman, Department of Molecular and Cellular Physiology, 4260 Medical Sciences Building, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0576. E-mail: Nelson.Horseman{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hypothesis that prolactin (PRL) functions as an immunomodulator was based on studies showing lymphocyte PRL receptors, and its effects on growth, differentiation, and apoptosis in lymphoid cells. However, studies of PRL (PRL-/-) and PRL receptor knockout mice indicated that PRL was not required for immune system development or function under basal conditions. Because PRL maintains survival in glucocorticoid (GC)-treated Nb2-T lymphocytes in vitro, and PRL and GCs are elevated during stress, we investigated whether PRL protected T cells in vivo from GC-induced apoptosis. Adrenalectomized mice [PRL -/-, undetectable PRL; pituitary grafted PRL-/- (PRL-/-Graft), elevated PRL; and PRL+/-, normal PRL] were treated with dexamethasone (DEX) or PBS. Thymocytes and splenocytes were isolated and annexin V labeling of phosphatidylserine, DNA fragmentation, and caspase-3 activation were assessed as indices of apoptosis. Total thymocytes and CD4+ and CD8+ T cells obtained from DEX-treated PRL-/- mice exhibited significantly increased annexin V binding. In contrast, binding was not altered by DEX in PRL-/-Graft thymocytes. In addition, DEX induced classic DNA fragmentation in PRL-/- thymocytes. Elevated serum PRL reduced this effect. Thymocytes from DEX-treated PRL-/- mice exhibited increased caspase-3 activation, which was inhibited in cells from PRL-/-Graft mice. Finally, elevated expression of X-linked inhibitor of apoptosis, XIAP, was observed in thymi from DEX-treated PRL -/-Graft mice. This is the first demonstration that elevated PRL antagonizes apoptosis in thymocytes exposed to GCs in vivo. These observations suggest that, under conditions of increased GCs, such as during stress, elevated PRL functions physiologically to maintain survival and function of T-lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STRESS EVOKES A NEUROENDOCRINE response that facilitates adaptation to the external environment. Physiological and psychological stressors generally initiate a response reflecting stimulation of neuronal and endocrine signals that are widely implemented throughout the body. This response, termed the "General Adaptation Syndrome" is, in part, regulated by stimulation of the hypothalamic-pituitary-adrenocortical axis, and results in increased levels of glucocorticoids (GCs) and catecholamines (1).

It is widely held that excessive or prolonged stress negatively impacts health. Indeed, stress is thought to contribute to human cardiovascular pathology, gastrointestinal diseases, and mental disorders (2, 3, 4) and to exert deleterious effects on the immune system. The immunosuppressive consequences of elevated GCs induced by stress have been extensively studied. Cell-mediated immunity appears to be particularly sensitive to the effects of stress, which can cause thymic atrophy, reflecting a loss of immature T cells by apoptosis (5, 6, 7, 8). For example, increased GCs during restraint stress, produced thymic involution, decreased CD4+/CD8+ thymocytes, and caused DNA fragmentation, indicative of apoptosis (7, 8). Due to the reduced availability of T cells from the thymus, coupled with apoptosis in some mature lymphocytes, the number of peripheral lymphocytes also decreases with stress. Importantly, stress-induced thymic involution was shown to be a direct consequence of elevated GCs because it was blocked by surgical or chemical adrenalectomy and by administration of GC receptor II antagonists (7, 8, 9). In addition, thymocytes undergo apoptosis upon administration of GCs in vivo or subsequent to steroid exposure in culture (10, 11). Finally, the immunosuppressive actions of exogenously administered GCs are widely recognized and form the basis of their use in the treatment of transplant organ recipients and patients with autoimmune or inflammatory diseases. The deleterious effects of GCs on immune cells notwithstanding, these hormones also function physiologically to eliminate unneeded thymocytes, thereby shaping the repertoire of T cells within the thymus (reviewed in Ref. 12).

In addition to increased levels of GCs, stress also elevates other hormones that may affect immune system cells including GH, IGF-1, and prolactin (PRL). Each of these has been reported to counteract certain effects of GCs and other stress-mediators in the immune system (13). However, whether these hormones exert physiologically relevant immunomodulatory actions remains controversial.

Snell Dwarf (dw/dw) mice characterized by PRL, GH, and TSH deficiencies, have been used to study the immunoregulatory functions of hormones in vivo. Initial reports indicated that reduced bone marrow cellularity, a hypoplastic thymus, and suppressed humoral- and cell-mediated immunity exhibited by these mice could be restored by administration of GH, thyroxine, and/or PRL (14, 15, 16, 17, 18, 19). However, more recent studies reported a normal immune system in the dw/dw mice (20). Importantly, the most pronounced immune defects in dw/dw mice were manifest when the animals were maintained under conditions that, in retrospect, may have subjected them to extreme environmental stress (21, 22). This suggests that, when unopposed by hormones such as PRL, stress-induced elevation of GCs produced immunodeficiency in these animals.

In previous studies, we investigated whether mice with a targeted disruption of the PRL gene (PRL-/-) exhibited an immunological phenotype. PRL deficiency resulted in significant deficits in mammary gland development and reproductive function; however, it did not appear to adversely affect the hematopoietic system (23). Subsequently, it was shown that PRL-/- mice were capable of normal humoral- and cell-mediated immune responses following exposure to T-independent and -dependent antigens, and challenges with Listeria monocytogenes (24). These observations were supported by those reported of Kelly and co-workers (25), who showed that PRL receptor knockout mice exhibited a normal immune response to antigenic challenges. Based on these observations, it was concluded that PRL most likely was not required for development or functioning of the immune response under steady-state conditions in vivo. However, the clear capacity of PRL to affect many functions of lymphocytes in vitro indicated that there were likely to be circumstances under which PRL may be critical such as during a stress response.

Using lactogen-dependent rat Nb2 T-lymphoma cells, a widely employed in vitro model of PRL signaling and actions in cells of the immune system, Witorsch and co-workers (26, 27, 28, 29) showed that GCs induced apoptosis, similar to the effects of the steroids in thymic T cells (10). The addition of PRL blocked GC-induced cell death. Subsequent studies designed to investigate the mechanism of apoptosis inhibition in this paradigm showed that PRL, in the presence or absence of GCs, stimulated a rapid increase in expression of pim-1, bcl-2, bcl-xL, and XIAP (X-linked inhibitor of apoptosis), each a well-characterized suppressor of apoptosis (Refs.29 ,31 , and32 ; and Kochendoerfer, S. K., N. Krishnan, D. J. Buckley, and A. R. Buckley, manuscript submitted). These observations suggested that, in Nb2 T-cells, PRL antagonized the cytolytic actions of GCs by activating a survival program, which resulted in increased expression of antiapoptosis genes. However, in spite of a plethora of evidence demonstrating a survival action of PRL in vitro, whether it suppresses lymphocyte apoptosis in vivo has not been evaluated.

In the present study, we demonstrate for the first time that PRL suppresses GC-induced immune cell apoptosis in vivo. Using control PRL-/- mice and PRL-/- mice in which PRL was restored by pituitary grafting, the effect of PRL on GC (dexamethasone, DEX)-induced thymocyte apoptosis was evaluated. The results show that PRL significantly reduced the effects of DEX on early and late indices of apoptosis in thymic T cells, and suggest that PRL may function as a physiological antistress mediator under conditions of elevated GCs in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and supplies
Anti-CD4 tri-color-conjugated (anti-CD4/TC) and anti-CD8 {alpha}-allophycocyanin-conjugated (anti-CD8/APC) rat antimouse monoclonal antibodies were obtained from Caltag Laboratories, Inc. (Burlingame, CA), rabbit antimouse cleaved caspase-3 from Cell Signaling (Beverly, MA), and rabbit antimouse cleaved caspase-8 and antimouse cleaved caspase-9 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat antirabbit IgG peroxidase conjugate was obtained from Sigma (St. Louis, MO). All other reagents were molecular biology grade and obtained from Sigma

Animals
Twenty-five-day-old heterozygous (PRL +/-) or homozygous C57BL6/J PRL knocked-out (PRL-/-) mice (23) were anesthetized by metofane inhalation. The animals were surgically adrenalectomized and maintained with 0.9% saline in the drinking water. In addition, some of the PRL-/- mice received a pituitary gland graft, from a normal C57BL6/J mouse (PRL +/+), beneath the left kidney capsule (PRL-/-Graft). Following 2 wk for recovery, the mice were injected ip with PBS as a vehicle control or DEX (5 mg/kg, 100 µl/100 g, Sigma, DEX-21-phosphate disodium salt). After 8 h, the mice were killed by cervical dislocation. Thymus glands and spleens were removed and placed in Hanks’ buffered saline solution at 4 C. Thymocytes and splenocytes were isolated and suspended in PBS. Water and food were available ad libitum before and after all treatments. All experimental animal protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee and performed in accordance with guidelines detailed in the NIH guide for the Care and Use of Laboratory Animals from the U.S. Public Health Service.

Determination of PRL concentration
PRL levels in heart blood samples obtained at the time the animals were killed were evaluated by Nb2 cell bioassay using a modification (33) of the method described by Tanaka et al. (34). PRL-dependent rat Nb2–11 cells were maintained at 37 C in Fischer’s medium supplemented with 10% fetal bovine serum, as a source of lactogens, and 10% horse serum (BioWhittaker, Inc., Walkersville, MD), 2-mercaptoethanol (2-ME, 100 µM), penicillin (50 U/ml), and streptomycin (50 µg/ml). Cells were rendered quiescent by incubation for 18–24 h in lactogen-free medium [Fischer’s medium supplemented with 2-ME, antibiotics and 10% nonmitogenic gelding serum (ICN Biochemicals, Irvine, CA)], then cultured in 96-well plates in the presence of various dilutions of mouse serum. After 48 h, cells were harvested onto glass fiber filters using a PHD cell harvester (Cambridge Technology, Watertown, MA). Radioactivity of trichloroacetic acid insoluble material was determined following a 4-h pulse of 3H-thymidine (44–48 h; Amersham Pharmacia Biotech, Arlington Heights, IL; specific activity 90 Ci/mmol, 0.5 µCi/well). Relative concentrations of PRL bioactivity were determined from a standard curve generated using purified PRL (National Hormone and Pituitary Program, NIDDK).

Determination of serum corticosterone concentration
Concentrations of serum corticosterone were determined by a commercial solid-phase RIA validated for the mice. The analysis was conducted by the Endocrinology Laboratory in the Diagnostic Lab at the College of Veterinary Medicine, Cornell University (Ithaca, NY).

Caspase analysis
Isolated thymocytes were suspended in a lysis buffer containing 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 25 µg/ml each of leupeptin and aprotinin. Total protein of the cell lysates was determined by the method of Lowry. Lysates (75 µg) were fractionated using 12% SDS-PAGE, followed by an electrophoretic transfer to Protran nitrocellulose membranes (Schleicher \|[amp ]\| Schuell, Valley Park, MO). The membranes were blocked for 1 h at room temperature with 5% nonfat dried milk in TBS-T (Tris-buffered saline-Tween: 20 mM pH 7.5; 140 mM NaCl; 0.1% Tween-20), followed by 1 h incubation at room temperature with antiactivated caspase-8 (1:200), caspase-9 (1:400), or by overnight incubation at 4 C with antiactivated caspase-3 (1:500). Immunoreactive proteins were visualized by incubation with a goat antirabbit IgG/HRP-conjugated antibody (1:1000), followed by chemiluminescence detection, performed according to recommendations of the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric analysis of the autoradiographs was conducted using a computer assisted image analysis system (Nucleotech, San Carlos, CA).

3'-OH DNA labeling
Suspensions of thymocytes (3 x 106 cells/animal) were fixed in 1% paraformaldehyde in PBS for 10 min at room temperature. After fixing, 50 µl of the suspension was air dried on poly-L-lysine coated-slides at 4 C overnight. The cells were washed, then stained according to the protocol provided with the Apotag Plus In Situ Fluorescein Apoptosis Detection Kit (terminal dideoxyuridine nick end labeling, TUNEL, Intergen, Purchase, NY). Cells were counterstained with propidium iodide. Fluorescence of the cells was observed using a computer-assisted fluorescence microscope (Olympus Corp., Melville, NY; CK40) and photographed using a Spot Insight digital camera (Diagnostic Instruments, Sterling Heights, MI). The percentage of cells staining positive for free 3'-OH DNA ends was calculated by blind counting of four random fields containing at least 500 cells/field from each thymocyte sample.

Agarose gel electrophoresis of fragmented DNA
Thymocyte suspensions (4 x 106cells/animal) in PBS were fixed in 70% ethanol, centrifuged, and lysed in 0.2 M NaH2PO4 and 0.1 M citric acid. The lysates were centrifuged, the supernatant dried, and then treated with Nonidet P-40 (0.25%), ribonuclease A (1 mg/ml), Proteinase K (1 mg/ml). Samples were resolved on 1.8% agarose gels. The laddering pattern of DNA fragments was visualized subsequent to the ethidium bromide-staining of the gel which was photographed under UV illumination.

Flow cytometric analysis
Thymocytes (1 x 106 cells/animal) were centrifuged (300 x g), then incubated for 30 min at 4 C, avoiding exposure to light, in a total volume of 200 µl of PBS containing 2 mM CaCl2 and 0.5% BSA, with 5 µl of fluorescein isothiocyanate (FITC)-conjugated Annexin V (Sigma), 5 µl of anti-CD4/TC and 5 µl of anti-CD8/APC. Control experiments were performed without the addition of annexin V or antibody. Positive controls were performed for each labeling using annexin-V/FITC, anti-CD4/TC or anti-CD8/APC alone. The cells were centrifuged and washed twice with PBS containing 2 mM CaCl2. Fluorescence was measured using an LSR flow cytometer Becton Dickinson and Co., Franklin Lakes, NJ). FITC and TC fluorochromes were excited by an argon-ion laser set for 20 mW at 488 nm, and APC flurorochrome was excited by a HeNe laser set for 17 mW at 633 nm. FITC, TC, and APC fluorescence were detected using 530 nm, 670 nm long path, and 660 nm filters, respectively. Ten thousand cells per sample were analyzed. The percentages of positive cells for each labeling were determined using the CellQuest software (Becton Dickinson and Co.).

Analysis of survival gene expression
Total RNA was extracted from thymocytes using RNAzol (Tel-Test, Friendswood, TX) and quantitated spectrophotometrically. mRNA (2 µg of total RNA) was reverse transcribed and amplified by PCR using Taq polymerase (Fisher Scientific, Pittsburgh, PA) and gene-specific primers for pim-1, bcl-2, bcl-xL, XIAP, and serum- and glucocorticoid-induced kinase (SGK). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was included as an internal positive control. All primers were synthesized by the University of Cincinnati DNA Core Facility. The PCR products were resolved on agarose gels and stained with ethidium bromide. Stained gels were photographed under UV illumination and subjected to computer-assisted densitometric analysis. Ratios of survival gene and GAPDH intensities were calculated for each animal.

Statistical evaluation
All experimental groups contained at least 7–10 animals, and where applicable, data are presented as means ± SE. Differences among treatment groups were evaluated by two-way and one-way ANOVA followed by the Student Newman-Keul’s post hoc test for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum corticosterone and PRL concentrations
Mouse serum corticosterone was determined to verify the fidelity of adrenalectomy. Serum corticosterone at 0800 h was less than 10 ng/ml (results not shown, normal range: 30–50 ng/ml) in the adrenalectomized animals, indicating that the surgical procedure was complete.

Concentrations of PRL in serum were determined to verify elevated PRL in the pituitary grafted PRL-/- mice (Fig. 1Go). In heterozygous control (PRL+/-) animals, PRL concentrations were found to be 3.6 ± 0.8 ng/ml by bioassay, which was consistent with previous observations (23). Targeted disruption of the PRL gene reduced hormone levels to below the detection limit (< 0.001 ng/ml) of the assay (23). PRL concentrations in the PRL-/-Graft mice were significantly elevated (P < 0.001) compared with either PRL +/- controls or PRL-/- animals. The PRL concentration range in these animals was 130–220 ng/ml.



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  		Figure 1. Serum PRL bioactivity in PRL+/-, PRL-/-, and PRL-/- Graft mice. Five-week-old mice were adrenalectomized. Half of the PRL-/- mice received pituitary glands implanted below the renal capsule. After recovery, mice were treated with DEX (5 mg/kg, ip) or PBS. Eight hours later, cells were isolated and serum PRL concentrations were assessed by Nb2 bioassay. Stationary Nb2–11 cells were cultured in presence of varying dilutions of serum samples or PRL (0.001–1 ng/ml). Cultures were pulse-labeled with 0.5 mCi/well of 3H-thymidine for 4 h and harvested after 96 h. Data are presented as mean ± SEM of six different animals. Bars: Open, PRL+/-; diagonal, PRL-/-; cross-hatched, PRL-/- Graft animals. *, P < 0.001 vs. PRL-/- and PRL+/-.

 
Inhibition of GC-induced apoptosis by PRL in thymocytes and splenocytes
Considerable in vitro evidence has accumulated, indicating that PRL suppressed DEX-induced apoptosis in cultured T cell lines (26, 27, 28, 29, 35). However, whether the hormone functions in a similar manner in vivo remained to be determined. Toward this end, we assessed whether cell surface expression of phosphatidylserine (PS), an early indicator of apoptosis, was similarly affected by the hormone. The presence of PS residues on the outer membrane leaflet was evaluated by flow cytometric analysis of annexin V binding (Fig. 2Go). Subsequent to DEX administration to either PRL+/- or PRL-/- animals, approximately 30% (P < 0.01 and P < 0.05, respectively) of total thymocytes bound annexin V, indicating activation of apoptosis in these cells (Fig. 2AGo). However, the level of annexin V binding in thymocytes obtained from DEX-treated animals with elevated PRL (PRL-/-Graft) did not differ from controls receiving PBS. Annexin V binding to PS residues was also evaluated in splenocytes obtained from the animals (Fig. 2BGo). In control PRL+/- animals, DEX significantly (P < 0.01) increased annexin V binding compared with PBS-treated mice. Administration of the GC did not significantly alter its binding in splenocytes from either group of PRL-/- mice.



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  		Figure 2. Effect of PRL on DEX-induced annexin V labeling of PS residues in thymocytes and splenocytes. Cells were labeled with FITC-conjugated annexin V. Labeled cells were washed and the fluorescence measured. Ten thousand cells/sample were analyzed. A, Thymocytes. B, Splenocytes. Bars: Open, PRL+/-; diagonal, PRL-/-; cross-hatched, PRL-/- Graft animals. *, P < 0.01, PRL+/- DEX vs. PBS. **, P < 0.05, PRL -/- DEX vs. PBS.

 
Because maturation of T cells occurs within the thymus, we next evaluated whether DEX altered annexin V binding in the various thymic subpopulations. As shown in Fig. 3AGo, DEX increased annexin V binding in CD4+ thymocytes obtained from PRL+/- and PRL-/- mice by more than 2-fold (P < 0.05). In the pituitary grafted PRL-/- mice, annexin V binding did not differ in DEX-treated animals compared with that observed in controls administered PBS. Annexin V binding to CD8+ thymocytes (Fig. 3BGo) from PRL-/- animals was also significantly (P < 0.01) increased by DEX. These results suggest that elevated PRL levels protected CD4+ thymocytes from DEX-activated apoptosis (Fig. 3AGo), whereas the hormone at either steady-state or elevated levels suppressed apoptosis in the CD8+ thymic subset (Fig. 3BGo). Annexin V binding was also evaluated in CD4+/CD8+ and CD4-/CD8- thymocytes. Significant differences in annexin V labeling were not observed in these thymic subsets obtained from any of the treatment groups (results not shown).



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  		Figure 3. Effect of PRL on DEX-induced annexin V labeling of phosphatidylserine residues in thymocyte subsets. Thymocytes and splenocytes were labeled with FITC-conjugated annexin V, {alpha}-CD4/TC, and {alpha}-CD8/APC. Fluorescence was measured at an excitation wavelength of 488 nm for FITC and TC and 633 nm for APC. Ten thousand cells/sample were analyzed. Bars: Open, PRL+/-; diagonal, PRL-/-; cross-hatched, PRL-/- Graft animals. A, CD4+/CD8- thymocytes. *, P < 0.05, PRL+/- DEX vs. PRL+/- PBS, and PRL-/- DEX vs. PBS. B, CD4-/CD8+ thymocytes. *, P < 0.01, PRL-/- DEX vs. PBS. C, CD4+/CD8- splenocytes. *, P < 0.05, PRL+/- DEX vs. PRL+/- PBS and PRL-/- DEX vs. PBS.

 
The sensitivity of splenocyte T cell subpopulations to DEX-activated apoptosis and its suppression by PRL was also investigated. Of the subpopulations assessed, only CD4+ spleen cells exhibited increased annexin V binding following administration of DEX to the animals (Fig. 3CGo). As shown, administration of DEX to PRL+/- and PRL-/- animals resulted in significantly (P < 0.05) increased annexin V binding to PS in CD4+ splenocytes. Animals with elevated serum PRL concentrations (PRL-/-Graft) were resistant to this effect of the GC.

Having demonstrated that elevated PRL inhibited DEX-induced annexin V labeling, we next determined the effect of DEX on DNA fragmentation, a hallmark of apoptosis and a late event in the process. In initial experiments, DNA fragmentation was determined by agarose gel electrophoresis (Fig. 4AGo). Administration of DEX to PRL+/- control and nongrafted PRL-/- mice resulted in the appearance of fragmented thymocyte DNA after 8 h. However, no hydrolysis of DNA was observed in DEX-treated PRL-/-Graft mice.



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  		Figure 4. Effect of PRL on DEX-induced T cell DNA fragmentation. A, Thymocytes were lysed using phosphate-citrate buffer, treated with Nonidet P-40, ribonuclease A, and Proteinase K, then resolved on ethidium bromide stained 1.8% agarose gels. B, Photomicrographs of thymocytes fixed in 1% paraformaldeyde. Presence of fragmented DNA was assessed by TUNEL labeling using a fluorescein-tagged conjugate. Representative microscopic figure: green, apoptotic, red, viable. C and D, Four random microscopic fields of stained thymocytes were counted from each animal preparation. Results presented statistical evaluation of mean ± SE counts from thymocytes and splenocytes, respectively. Bars: Open, PRL+/-; diagonal, PRL-/-; cross-hatched, PRL-/- Graft animals. *, P < 0.001, PRL+/- DEX vs. PBS; PRL-/- DEX vs. PBS. **, P < 0.001, PRL-/- DEX vs. PRL-/- Graft DEX.

 
To evaluate DNA fragmentation quantitatively, 3'-OH labeling in situ (TUNEL assay) was performed on thymocytes obtained from the various treatment groups. Presented in Fig. 4BGo are photomicrographs of thymocytes obtained from PRL-/- [PRL-/- (DEX), upper panel] and PRL-/-Graft [PRL-/- Graft (DEX), lower panel] mice treated with DEX. Administration of the steroid caused substantial TUNEL labeling indicating endonuclease hydrolysis resulting in free 3'-OH DNA ends available for labeling. Elevated PRL levels abrogated this effect of DEX. Shown in Fig. 4CGo is the cumulative evaluation of the TUNEL assay results. In PRL+/- and PRL-/- animals, administration of DEX significantly (P < 0.001) increased the percentage of apoptotic cells present within the thymus. In contrast, thymocytes obtained from animals with elevated PRL failed to respond to the apoptotic effects of DEX. The effect of DEX on TUNEL labeling in splenocytes was also determined (Fig. 4DGo). Similar to the effects of DEX in the thymus, the steroid significantly (P < 0.001) increased TUNEL staining in control (PRL+/-) and PRL-/- splenocytes. In contrast to the thymus, in which DEX-induced DNA fragmentation was approximately 50–60% of cells, only approximately 25–30% of splenocytes were sensitive to this effect of the steroid. In splenocytes obtained from animals with elevated PRL, the level of TUNEL labeling in splenocytes from the DEX-treated animals did not differ from that observed in PBS treated PRL-/-Graft mice. These results indicate that thymocytes and splenocytes exhibit differential sensitivity to the apoptotic actions of DEX. Moreover, they demonstrate that increased levels of PRL, above those seen normally, antagonize apoptosis induced by GCs in thymocytes and splenocytes in vivo.

In numerous cell types, including thymocytes (36), activation of the caspase cascade accompanies fragmentation of DNA during apoptosis. Therefore, the effect of DEX or PRL on activation of caspases-3, -8, and -9 was determined by immunoblotting of thymocyte lysates using antibodies that recognize the active forms of the respective cysteine proteases. Administration of DEX to PRL-/- animals increased the level of activated caspase-3 (Fig. 5AGo). In contrast, activated caspase-3 in thymocytes obtained from DEX-treated, pituitary-grafted PRL-/-Graft mice was significantly (P < 0.05) reduced compared with PRL-/- animals treated with DEX (Fig. 5BGo). Because activation of this protease is generally viewed as a terminal event in apoptosis, the demonstration that elevated serum PRL concentrations inhibited its activation further illustrates the antiapoptotic actions of the PRL in vivo. No activation of caspases-8 or -9 was detected in the thymocytes under the experimental conditions employed (results not shown).



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  		Figure 5. Effect of PRL on DEX-induced caspase-3 activation in thymocytes. Cells were lysed, and 75 µg protein was resolved by 12% SDS-PAGE. Determination of cleaved caspase-3, as a measure of its activation, was performed using {alpha}-cleaved caspase-3. A, Representative immunoblot. B, Densitometric analysis of immunoblots from four animals per treatment group. *, P < 0.05, vs. PRL-/- DEX.

 
Effect of DEX on survival gene expression in thymocytes
PRL stimulation in Nb2 T cells was previously shown to rapidly increase the expression of several genes associated with inhibition of apoptosis in GC-treated cultures (Refs.29 ,31 , and32 ; and Kochendoerfer, S. K., N. Krishnan, D. J. Buckley, and A. R. Buckley, manuscript submitted). To determine whether similar increases in survival gene expression were provoked by PRL or altered by DEX treatment in vivo, bcl-2, XIAP, serum- and glucocorticoid-induced kinase (SGK), and pim-1 survival gene levels were evaluated by RT-PCR and compared with the expression of the housekeeping gene, GAPDH, used as an internal control (Fig. 6Go).



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  		Figure 6. Effect of PRL and DEX on expression of XIAP in thymocytes. Total RNA from thymocytes was extracted and quantitated spectrophotometrically. Two micrograms of total RNA were reverse transcribed and amplified using gene-specific primers for XIAP and GAPDH, as an internal control. PCR products were resolved on ethidium bromide-stained agarose gels followed by densitometric analysis. Results are presented as ratios of XIAP and the respective GAPDH densitometric intensities from at least seven animals/treatment. Bars: Diagonal, PRL-/-; cross-hatched, PRL-/- Graft animals. XIAP mRNA expression. *, P < 0.01, PRL-/-Graft DEX vs. PRL-/- DEX.

 
Due to the capacity of XIAP to directly inhibit caspase-mediated proteolysis, it is central to apoptosis regulation (37). Of the genes evaluated, only XIAP consistently responded to DEX + PRL at 8 h (Fig. 6Go). Whereas no differences in its expression were observed in PBS-control or DEX-treated PRL-/- thymocytes, elevated circulating PRL in PRL-/-Graft mice significantly (P < 0.001) increased the level of XIAP mRNA in the DEX-treated animals. Previously, we demonstrated that increased levels of XIAP mRNA expression correlated with its protein levels, which inhibited apoptosis in T cells in vitro (32). Because XIAP suppresses apoptosis by inhibiting caspases including the initiator (caspase-9) and effector (caspase-3, -7) enzymes (38), these results suggest that XIAP may contribute to the suppression of apoptosis observed in these cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous in vitro studies have demonstrated that PRL can function as a survival factor in cells of the immune system. Using rat Nb2 lymphoma T cells as a model, Witorsch and co-workers (26, 27, 28, 29) previously showed that GCs, such as DEX, induced apoptosis in the T cell line and addition of PRL antagonized this effect. Other studies showed that the survival actions of PRL in this paradigm were most likely the result of hormone-mediated augmentation of survival gene expression (39). Although these observations suggested a potential role for PRL as a mediator of immune cell homeostasis, it remained unclear whether the antiapoptotic actions of PRL demonstrated in this transformed cell line could be recapitulated in normal immune cells in vivo.

In this paper, we show that administration of DEX in mice induced apoptosis in thymic and splenic lymphocytes, consistent with previous observations (40). In addition, we demonstrate for the first time that the apoptotic effects of GCs were significantly inhibited in animals with elevated serum PRL concentrations. Here we report that elevated PRL levels, induced by pituitary grafting in PRL-/- mice, significantly inhibited exposure of PS residues on the T cell surface, blocked caspase-3 activation, and suppressed DNA fragmentation, each induced by DEX in PRL-/- mice, with undetectable PRL levels, and in animals with normal levels of the hormone. Because activation of the hypothalamic-pituitary-adrenocortical axis by stress rapidly increases the circulating levels of GCs coupled to release of PRL by the pituitary gland, these observations suggest that elevated serum PRL in this setting may serve a survival function, particularly in thymus-derived T cells. It is important to note that the PRL levels observed in the PRL-/-Graft animals are similar to those observed in lactating mice (41) or in mice subjected to 1 h of restraint stress (42). Thus, under various physiological conditions in vivo, significantly elevated PRL occurs presumably to maintain homeostasis. Therefore, inhibition of GC-induced cell death by PRL could block a deleterious reduction in the functional capacity of cell-mediated immunity.

The hypothesis that PRL plays a physiological role as a modulator of immune cells has been extensively investigated for more than two decades. During this period, numerous investigators have demonstrated stimulatory effects of PRL alone or in combination with various cytokines and lectins on T and B lymphocytes and natural killer cells in culture (43, 44, 45, 46, 47, 48). In addition to the reported comitogenic effects of PRL in vitro, others showed that administration of PRL to hypophysectomized or bromocryptine-treated rats abrogated the immunological defects observed in these animals in the absence of the hormone (49, 50, 51). Despite the reported immunomodulatory effects of PRL in vitro and following its administration to hypoprolactinemic rats, it was recently shown that the immune response in mice developed and functioned normally in PRL-/- and PRL receptor knockout animals (25). However, the clear capacity of PRL to affect many functions in immune system cells, including suppression of GC-mediated T cell apoptosis in vitro, led us to investigate whether, under conditions that mimic those provoked by stress, e.g. elevated serum concentrations of GCs and PRL, elevated PRL altered the response of thymic or splenic lymphocytes to adrenal steroids.

Previously, Mann et al. (36) demonstrated in rats that administration of DEX induced apoptosis in thymocytes by a mechanism requiring de novo gene expression that was distinct from that activated by serum deprivation. To evaluate the effect of PRL on GC-induced lymphocyte apoptosis, which occurs during stress PRL+/-, PRL-/-, and PRL-/- Graft mice received a single administration of DEX; various biochemical indices of apoptosis were assessed in thymocytes and splenocytes 8 h later.

A relatively early marker of apoptosis is movement of PS residues to the external leaflet of the plasma membrane (52). This is thought to facilitate macrophage recognition, engulfment, and removal of dying cells (53). The results of the present study indicate that in unfractionated thymocytes (Fig. 2Go) and in CD4+ cells from thymus and spleen (Fig. 3Go), DEX increased the proportion of PS exposure-positive cells in PRL+/- and PRL-/- mice, whereas elevated PRL blocked this effect. DEX also increased external PS staining in CD8+ cells from PRL-/- animals but not in the PRL+/- animals or those with elevated PRL suggesting that the absence of PRL renders this subpopulation more sensitive to the apoptotic actions of GCs. In unfractionated splenocytes, only those obtained from PRL+/- mice exhibited increased external exposure of the phospholipid suggesting that PRL levels, altered from normal, may sensitize or suppress responsiveness of specific subpopulations to DEX. For example, splenic CD4+ cells from PRL-/- animals are sensitive, whereas those from animals with elevated PRL resisted the effects of DEX (Fig. 3CGo).

Because activation of caspase-3 represents a step in apoptosis that is upstream of DNA fragmentation (54), we evaluated whether PRL, similar to its effect on PS exposure on the cell surface, antagonized activation of this effector protease. Activated caspase-3 is present as a heterodimer composed of 12- and 17-kDa fragments that are derived from a 32-kDa proenzyme (55). Treatment of PRL-/- mice with DEX increased activated caspase-3 in thymocytes; this effect was significantly inhibited in cells from the animals with elevated PRL levels (Fig. 5Go).

The recently described survival protein, XIAP, suppresses apoptosis by inhibiting activation of caspases (56). During T lymphocyte apoptosis in culture, XIAP has been shown to be cleaved by caspases resulting in two fragments that retain specificities for caspases-3 and -7 (57). Moreover, the role of XIAP in the immune system has also been evaluated in vivo. Thymocytes from XIAP-transgenic mice resist several apoptotic stimuli and show altered maturation (58). Our observation of elevated XIAP expression in thymocytes from DEX-treated PRL-/-Graft mice suggests that PRL may suppress apoptosis by increasing XIAP, which in turn, may reduce levels of activated caspase-3. This is consistent with our previous observation that PRL increased XIAP expression in DEX-treated Nb2 T cells and that overexpression of XIAP inhibited DEX-induced apoptosis in this model (32). Moreover, we have recently observed that PRL also augments XIAP levels by antagonizing DEX-induced ubiquitination and proteolysis of XIAP (59). Thus, our results indicate that XIAP is most likely an important survival protein regulated by PRL that may mediate the antiapoptotic effect of the hormone in vitro and in vivo.

Translational up-regulation of XIAP, thought to be mediated by an important internal ribosome entry site element located within an XIAP 5'-untranslated region (60), correlates with increased resistance to apoptosis (32, 61). Because this mechanism has been shown to be active under conditions of cellular stress and elevated PRL increases XIAP mRNA expression in the presence of DEX (Fig. 6Go), it is possible that the antiapoptotic action of PRL during stress might involve activation of internal ribosome entry site-mediated XIAP translation.

The results from this study suggest that subpopulations of T cells are differentially sensitive to the antiapoptotic actions of PRL. In thymic and splenic CD4+ cells obtained from DEX-treated PRL+/- mice, basal PRL levels were insufficient to inhibit GC-induced apoptosis (Fig. 3Go, A and C). In these cells, significantly elevated PRL concentrations were required to attenuate DEX-induced apoptosis. However, basal levels of PRL blocked the effect of DEX to increase PS labeling in thymic CD8+ cells (Fig. 3BGo).

One possible explanation for the observed differences in sensitivity of CD4+ and CD8+ T cells to PRL may reflect variation in the ratios of long and short forms of the PRL receptor (PRLR) expressed in these cells. Both isoforms, which are derived from differential mRNA splicing of the intracellular signal transducing domain of the receptor, are expressed in murine immune cells (62). The long form is coupled to signal transduction, whereas the short form may serve as a decoy (63). The latter does not appear to activate signaling mechanisms and its coexpression with the long form reduces PRL sensitivity due to competition for the hormone (64). Moreover, heterodimers of long and short PRLR isoforms are inactive (65). Therefore, in T cell subpopulations, in which the ratio of long to short PRLR isoform favors the long form, PRL could regulate survival at lower hormone concentrations. This may be the case for CD8+ T cells. However, if the ratio were to favor the short PRLR, higher PRL concentrations would be required to activate signaling. This may underlie the observed effects of PRL in CD4+ T cells.

The previous observations by us and others indicate that PRL blocked GC-induced apoptosis in vitro most likely by activating specific intracellular mechanisms leading to expression of survival genes. Results from the present study show that elevated PRL inhibited apoptosis in thymocytes and splenocytes and that certain T cell subsets exhibited differential sensitivity to the pro-survival effects of PRL in animals treated with DEX. A role for PRL in maintenance of immune system integrity is further supported by our recent observation that burn stress enhanced myelopoiesis and attenuated the splenic T cell proliferative response to mitogens in PRL-/- mice (66). Thus, in both cell culture and in vivo, PRL functions to maintain cell-mediated immunity.

In this study, elevated circulating PRL was achieved by pituitary transplantation in PRL-/- mice. This approach for increasing serum PRL has been thoroughly investigated (67, 68). Specificity for PRL secretion from the grafted glands stems from tonic inhibition of PRL release by hypothalamic dopamine, which is absent in the transplanted tissue (69, 70). In contrast, secretion of each of the other anterior pituitary hormones, such as GH, is primarily regulated by hypothalamic releasing hormones, which are also absent. However, we cannot rule out a possible contribution of an unidentified factor that may be released from the transplants in combination with PRL. Future studies will use exogenous PRL administration in combination with various inducers of physiological and psychological stress to further investigate the relationships between PRL and its antagonistic effects on GC actions in the immune system.

The observations reported in this paper are consistent with the work of Lilly et al. (71), who showed that in rhesus macaques exposed to severe acute stress, serum GCs were significantly elevated, PRL levels were modestly increased, and several parameters of cell mediated immunity were suppressed. However, when challenged with a one year period of controlled, chronic stress, GCs levels fell to below basal levels and PRL concentrations were significantly increased to levels exceeding those observed with acute stress. In this setting, CD4+, CD8+, and total lymphocytes recovered to unstressed levels. In toto, the observations made in rodent T cells in culture and in vivo suggest that elevated PRL functions physiologically to antagonize GC-mediated immunosuppression during stress.


   Acknowledgments
 
We thank Kathryn M. Nieport (Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH) for expert technical assistance with the animal experiments.


   Footnotes
 
This work was supported in part by grants from the NIH (DK-52134 and DK-53452), The Shriners Hospital for Children, and the American Institute for Cancer Research.

1 N.K. and O.T. each contributed equally to this study and should be considered co-first authors. Back

Abbreviations: APC, {alpha}-Allophycocyanin; DEX, dexamethasone; dw/dw, Snell Dwarf mice; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, glucocorticoid; PRL, prolactin; PRLR, PRL receptor; PS, phosphatidylserine; TC, tri-color conjugate; TUNEL, terminal dideoxyuridine nick end labeling; XIAP, X-linked inhibitor of apoptosis.

Received January 10, 2003.

Accepted for publication January 27, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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