INTRODUCTION

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PREGNANCY PRODUCES adaptive modifications in the homeostasis of the maternal immune system in the survival of the fetoplacental graft (4, 12). Natural killer (NK) cells act early in the immune response before specificity can be generated. They mediate first-line defense by direct cytotoxicity against various types of target cells without apparent prior immunization (34). Heat stress during early or mild gestation pregnancy results in a high incidence of embryo mortality (2, 33). Although we have previously demonstrated enhanced immunosuppression produced by heat stress during pregnancy (18), the neuroendocrine mechanisms in the immunosuppression for pregnant mammals, including humans, exposed to heat stress remain to be elucidated.

Since the immunoassay detection of opiate peptide -endorphin (-EP) in placental extracts in 1978 (17), there has been considerable evidence showing the active presence of opiate receptors in human placental villous tissues (1, 35). Genomic and cDNA clones for opioid receptors exist for several animal species, including mouse, rat, guinea pig, and human (14). Human maternal plasma concentration of -EP is elevated during pregnancy (20). Although the endogenous opioid is involved in stress-induced immunosuppression (28), the effect of pregnancy on immunosuppression during stress is unknown. Placental steroids such as estrogens and progesterone (P) exert a positive effect on the -EP content in the pituitary lobes (22). To examine the involvement of the opioid system in enhanced immunosuppression induced by heat stress during pregnancy, we examined the effects of heat exposure and intraperitoneal administration of opioid receptor antagonist naloxone on -EP in blood, pituitary lobes, and placenta as well as splenic NK cell activity (NKCA) and placental steroids in pregnant rats.

	MATERIALS AND METHODS

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Preparation of virgin and pregnant rats for study. Twenty-four Wistar rats at 15-16 days gestation, weighing 270 ± 4.69 g (means ± SE) were studied. For breeding, a male rat was placed in a cage with two females. The environment was controlled in all cases (23 ± 2°C, 50% humidity), with alternating cycles of 12-h light (8 AM-8 PM) and 12-h dark. The onset of pregnancy was determined using vaginal smears. All animals had access to commercial food and tap water ad libitum. The rats were fasted, but given water in the 24 h before the experiment and deprived of food and drink throughout the experiment. This study was approved by the Ethics Committee on Animal Experimentation of Kanazawa University, Takara-machi Campus. In all cases the experimental protocol began at 11 AM. Twenty-four pregnant rats were divided into the following four groups: six rats with intraperitoneal administration of saline, but not exposed to heat; six rats with intraperitoneal administration of saline before heat; six rats with intraperitoneal administration of naloxone, but not exposed to heat; and six rats with intraperitoneal administration of naloxone before heat.

Intraperitoneal administration of naloxone. Naloxone HCl (Sigma, St Louis, MO) was administered intraperitoneally at a dose of 0.2 ml of 10 mg/ml solution in 0.9% saline 30 min before heat exposure. Twelve pregnant rats received naloxone, and 12 received 0.2 ml of the saline alone. The intraperitoneal administration dose of naloxone (2 mg/rat) is known to reverse the effect of the opioid system on immune changes in rats (13, 24).

Exposure to heat stress. The use of a microwave system is ideal for heat exposure because it allows for the administration of an exact quantity of energy (9). The microwave exposure device, described previously (11), was equipped with a magnetron of 2,450 MHz as the source of energy and had an isolator to vary the energy from the magnetron induced by reflection from the applicator (350 × 470 × 455 mm). Twelve pregnant rats (6 rats subjected to saline and 6 rats to naloxone) were put into a semicylindrical acrylic plastic holder (thickness, 5 mm; inside diameter, 60 mm; length, 170 mm) and were exposed to microwaves at 10 mW/cm² incident power density at 2,450 MHz for 90 min. The sham-exposed rats (6 rats subjected to saline and 6 rats to naloxone) were treated in an identical manner, except that the microwave generator was not turned on. During exposure, the environment of the exposure facility was maintained at 21-23°C and 50-60% humidity.

Measurements of blood corticosterone, -EP, estradiol, and P. Blood samples were collected by decapitation of rats immediately after the end of the protocol. Plasma was immediately prepared by transfer of samples to cooled conical centrifuge tubes containing 0.1 mM EDTA followed by centrifugation. The plasma was frozen at 80°C until analysis. Corticosterone (CS) was measured by the fluorometric method of Silber et al. (30). -EP was measured by the radioimmunoassay (RIA) described by Yoshimi et al. (39). In this method, highly purified -EP was labeled with Na ¹²⁵I using chloramine T. The purification of labeled -EP was performed on a carboxymethyl cellulose column. The antiserum against -EP showed negligible cross-reactivity with other fragments of -lipotropin such as -melanocyte-stimulating hormone and ACTH. Estradiol (E₂) and P were analyzed by RIA using the tube solid-phase method of Ratcliffe et al. (25). The intra- and interassay coefficients of variation were 8.0 and 12.5% for CS, 7.0 and 11.0% for -EP, 7.5 and 10.6% for E₂, and 5.4 and 7.6% for P, respectively. The sensitivity of the assays for CS, -EP, E₂, and P were 5 ng/tube and 3, 2.5, and 1.1 pg/tube, respectively.

Splenic NKCA. To measure splenic NKCA, the spleen was surgically excised and dissociated into a single-cell suspension. The splenocytes were suspended in 40 ml phosphate-buffered saline (PBS) and centrifuged in 50-ml tubes at 400 g at room temperature for 30 min over 12 ml Ficoll-Paque (Pharmacia, Piscataway, NJ) to yield mononuclear cells (26). Splenic lymphocytes were collected at the interface, washed twice in PBS solution, and suspended in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% vol/vol fetal bovine serum (FBS; GIBCO), 2 mM L-glutamine, 100 U/ml penicillin, and 100 ug/ml streptomycin, all from GIBCO.

NKCA was measured in a standard 4-h chromium (Cr) release assay that was performed in 0.2-ml volumes in U-bottom microplates. The YAC-1 mouse lymphoma cell line was used as the target for detecting NK cell cytotoxicity. The cells, suspended in culture in RPMI 1640 medium, were labeled with Na₂⁵¹CrO₄ at 1 mCi/ml (New England Nuclear, Boston, MA) for 1 h at 37°C. The cells were washed four times in a tissue culture medium consisting of RPMI 1640 and resuspended in fresh medium, counted, and aliquotted at 1 × 10⁴ target cells/well into 96-well U-bottom microtiter plates containing lymphocytes as effector cells at predetermined concentrations. The effector-to-target cell ratios (E/T) used were 40:1, 20:1, 10:1, and 5:1. After the plates were incubated in 5% CO₂ in air at 37°C for 4 h, the assays were terminated by centrifuging the plate at 400 g for 5 min, after which the medium was harvested from each well using a supernatant-harvesting apparatus (Flow, McLean, VA). All determinations were done in triplicate. Radioactivity was counted using a gamma counter. Spontaneous ⁵¹Cr release, determined by incubating labeled target cells in the medium alone, did not exceed 10% of the maximum release that was determined by adding 1% Triton X-100. NKCA as percentage specific lysis was determined according to the formula: 100 × [mean experimental counts/min (cpm)  mean spontaneous cpm]/(mean maximal cpm  mean spontaneous release cpm). Percent cytotoxicity was calculated at each E/T, and these values were converted to lytic units at 30% (LU₃₀) according to the method of Pross et al. (23). The intra- and interassay coefficients of variance for LU₃₀ as a measure of NKCA were 7.5 and 18.2%, respectively.

Measurement of pituitary and placental -EP. Immediately after the end of the experiment, brains were removed and the anterior pituitary (AP) and neurointermediate pituitary lobe (NIL) were dissected from the isolated pituitary. The dissected regions were sonicated in 1 ml of 0.1 N acetic acid, boiled for 10 min, and then centrifuged twice at 3,000 revolutions/min (rpm) at 4°C for 20 min.

For the excision of the maternal side of placenta, the placental disk adjacent to the endometrium was separated with blunt forceps and mixed with 10 ml PBS. The mixture was sonicated in 1 ml of 0.1 N acetic acid, boiled for 10 min, and then centrifuged twice at 3,000 rpm at 4°C for 20 min.

The supernatants of pituitary and brain extracts were stored at 80°C until analyses. Aliquots of the supernatants were lyophilized and reconstituted in assay buffer for RIA for the measurement of -EP. The pellets were dissolved in 1 N NaOH for protein estimation. Protein concentration was determined as described by Lowry et al. (15) using bovine serum albumin as a standard.

Statistical analysis. Statistical analysis of the difference in the mean values of blood parameters, splenic NKCA, and pituitary and placental -EP was performed by the completely randomized design using two-way analysis of variance (ANOVA). The factors were heat stress, which was composed of two levels (control or heat), and intraperitoneal administration, which was also composed of two levels (saline or naloxone). All statistical tests were two tailed. P values <0.05 were regarded as statistically significant.

	RESULTS

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Blood CS, -EP, E₂, and P in heat-exposed or nonexposed pregnant rats with intraperitoneal administration with saline or naloxone are shown in Table 1. Two-way ANOVA revealed that heat stress significantly increased CS and -EP and decreased E₂, independent of naloxone administration. The ANOVA showed that naloxone administration significantly increased -EP. There were significant interactions between heat and naloxone administration on -EP and P. Effects of heat stress and intraperitoneal administration of naloxone on splenic NKCA in pregnant rats are demonstrated in Fig. 1. Whereas heat reduced NKCA, intraperitoneal administration of naloxone reversed it. There was a significant interactive effect on NKCA. We could observe significant increases in placental -EP induced by both heat and naloxone administration and a significant interaction on it.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effects of heat stress and intraperitoneal administration of naloxone on splenic natural killer cell activity (NKCA) in pregnant rats. Values represent means ± SE. Statistical analysis of difference was performed by 2-way analysis of variance. Significant main effect of heat [F(1,20) = 6.67, P < 0.05] and naloxone [F(1,20) = 6.28, P < 0.05] and interactive effect on NKCA [F(1,20) = 9.46, P < 0.01]. LU30, Lytic units at 30%.

Heat was found to increase -EP in AP, NIL, and placenta significantly. Intraperitoneal administration of naloxone significantly increased -EP in placenta. A significant interaction on -EP in placenta was observed (Table 2).

View this table: [in this window] [in a new window]   Table 2.   Effects of heat stress and intraperitoneal administration of naloxone before stress on -EP concentration in the pituitary and placenta of pregnant rats

	DISCUSSION

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Functional networks among nervous, endocrine, and immune systems are now interpreted as a neuroimmunoendocrine function (29). Corticotropin-releasing hormone (CRH; 3, 36) and opiate -EP (16, 37) play roles in modulating neuroendocrine and immune systems as neurotransmitters. In agreement with a previous study (18), heat reduced NKCA in pregnant rats. Furthermore, we observed a promoting effect of naloxone administration as well as an interactive effect between heat and naloxone on NKCA. These results suggest that naloxone administration antagonizes immunosuppression induced by heat. Therefore, immunosuppression after heat stress during pregnancy seems to be mediated by the opioid system.

Gel filtration chromatography for -EP in placental and pituitary extracts has shown that placental -EP is not involved in analgesia induced by opioid-dependent stress, but plays a paracrine and autocrine role during pregnancy (5). Although the elevation in -EP induced by heat stress was seen in blood as well as pituitary and placenta, the interactive effect between heat and naloxone on -EP was seen only in blood and placenta. This result implies that naloxone administration increases -EP in blood and placenta only in heat-exposed rats, supporting the presence of opioid receptors in the placenta, which was shown by many researchers (1, 35). Simultaneously, our data may provide evidence for the involvement of the placental opioid system in heat stress in a paracrine and autocrine fashion. Falconer et al. (8) have indicated that uteroplacental -EP secretes into the maternal circulation in response to hypoglycemic stress. Blockade of the placental opioid system by naloxone during heat stress appears to increase placental -EP, subsequently resulting in hypersecretion of -EP into blood, which was indicated by increased blood -EP in pregnant rats receiving naloxone before heat. Because naloxone administration increased -EP in the placenta, but not in the pituitary of pregnant rats, this appears to support the assumption of Chan and Smith (5) that placental -EP is not involved in systemic immunosuppression. The physiological role of placental -EP may be different from that of pituitary -EP. Several types of opioid receptors have been implicated in the immunosuppression (6, 10, 21), but the type of receptor involved in stress-induced immunosuppression is not clear. Because naloxone is a nonselective opioid receptor antagonist (10, 21), further studies should be designed for determination of the types of opioid receptors involved in heat stress-induced immunosuppression during pregnancy.

Activation of the stress response inhibits the hypothalamic-pituitary-gonadal axis at multiple levels (27, 38). CRH suppresses secretion of luteinizing hormone-releasing hormone in the hypothalamic arcuate nucleus either directly or indirectly via the stimulation of -EP or corticosteroids (7, 19). In the present study, however, heat stress increased P and naloxone administration reversed it. Because progesterone-induced blocking factor administered in vivo significantly prevented the high rates of resorption in mice treated with antiprogesterone, progesterone-mediated suppression of lymphocyte toxicity plays a significant role in the maintenance of pregnancy (32). Taken together with our results showing that effects of naloxone administration on both P and NKCA were seen only in rats exposed to heat, activated placental hormones including P may be involved in the immunosuppression induced by heat stress during pregnancy. However, the effect of naloxone on E₂ in rats with heat was not different from that without heat. On the basis of the fact that stress suppresses placental functions directly or via the opioid systems (7, 19), clarification of the involvement of increased P during heat during pregnancy should be the focus of future work.

Perspectives