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Biology of Reproduction 64, 242-248 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Pregnancy Stimulates Secretion of Adrenocorticotropin and Nitric Oxide from Peripheral Bovine Lymphocytes1

V. Deep Dixita, and Nahid Parvizi2,a

a Institute for Animal Science and Animal Behaviour (FAL), Mariensee, D-31535, Neustadt a. Rbg., Germany

ABSTRACT

The cross-talk between the endocrine and the immune systems mediated by a wide array of hormones, cytokines, and neuromodulators is heightened during disease, stress, and presumably, during pregnancy. Adrenocorticotropin (ACTH) and nitric oxide (NO) are two immunomodulators that are also produced from lymphocytes and contribute to the immunomodulation. Thus, we investigated whether the heightened bidirectional communication between the immune and the endocrine systems observed during pregnancy is reflected in production of ACTH and NO from peripheral bovine lymphocytes and if any temporal correlation exists between them. Adrenocorticotropin was analyzed using a sandwich immunoradiometric assay, and nitrite and nitrate (a measure of NO) were estimated in supernatants of cultured peripheral blood lymphocytes (PBLs) using a colorimetric assay based on the Griess reaction. A significantly high secretion of ACTH and NO was noticed from PBLs in all stages of pregnancy compared to that in cyclic and cystic cows. Increased secretory capacity was noticed as early as 7 days after conception, which reached as much as 600% that of nonpregnant animals between Days 90–120 of gestation. Adrenocorticotropin and NO decline 1 mo before the expected time of parturition. Unlike those from cyclic animals, PBLs from pregnant cows were refractory to stimulation by PHA-M (Phytohemagglutinin) and corticotropin-releasing hormone. A strong correlation was observed between ACTH and NO secretion from PBLs in pregnant, in cyclic, and in cystic cows. To our knowledge, this is the first evidence elucidating the induction of ACTH and NO from PBLs during pregnancy, and it implies a new role for ACTH and NO secreted from PBLs in recognition and, probably, maintenance of pregnancy.

ACTH, nitric oxide, ovulatory cycle, pregnancy, reproductive immunology

INTRODUCTION

Evasion of hazardous immune response by a genetically disparate fetus is a vital prerequisite for successful pregnancy. A number of possible mechanisms for this complex event have been proposed over the years. Nearly 50 yr ago, Medawar [1] considered three mechanisms to explain tolerance of the maternal immune system to antigenically foreign conceptus: 1) anatomic separation of the mother and fetus, 2) antigenic immaturity of the fetus, and 3) immunogenetical inertness of the mother. The latter is thought to be achieved predominantly by the local production of regulatory signals with immunomodulatory properties [2, 3]. Lymphocyte function at both the peripheral and the local level is specifically changed after conception and during gestation in mammals [4, 5]. General alterations of the maternal inflammatory and immune system occur during pregnancy, which is apparent from the clinical observation of marked improvement of autoimmune conditions such as rheumatoid arthritis in humans [6]. A shift in T-helper 1 (TH-1) to T-helper 2 (TH-2) cytokines is regarded to be responsible for this immunomodulation during pregnancy [7–9]. Apart from cytokines, which are well-known regulators of the lymphocytic function, hormones of the pituitary gland can modulate immune response (e.g., stimulate or depress the lymphocytic proliferation) [10–12].

Pleiomorphism of the lymphocytic function includes the production of hormones with direct immunomodulatory effects [10]. Adrenocorticotropin (ACTH) is one of the first hormones found to be de novo synthesized and secreted from lymphocytes [13, 14]. Lymphocytic ACTH is apparently similar to its pituitary counterpart [15]. Locally produced ACTH from lymphocytes has a powerful immune suppressor activity independent of its capacity to stimulate glucocorticoid secretion [16]. Adrenocorticotropin has been implicated in inactivating and immobilizing the ameboid leukocytes [17, 18] and in inhibiting proinflammatory TH-1 cytokines [19–21].

Nitric oxide (NO) is a free-radical gas generated by the enzyme NO synthase [22, 23] using arginine as a precursor, and it is also produced from lymphocytes [24, 25]. It mediates critical processes such as neuromodulation, endocrine signal transduction, vasodilation, and immune defense. It exerts immunosuppressive effects by contributing to TH-2 shift [26, 27] via inactivation of the zinc finger transcription factors [28, 29].

The present study was conducted to investigate whether the heightened cross-talk between the immune and the endocrine systems observed during pregnancy is reflected in the production of ACTH and NO from lymphocytes, and if the secretion of these two immunomodulators from lymphocytes is changed during different physiological states.

MATERIAL AND METHODS

Animals and Blood Sampling

The study was conducted on 49 pregnant cows (40 from the research farm of the Institute for Animal Science and Animal Behaviour, and 9 from a stock not related to the Institute herd) and 18 nonpregnant Holstein-Friesian cows. Animals (3–7 yr old) were divided into groups according to their reproductive status.

Group 1 was comprised of pregnant animals. Blood samples (300 ml each) were collected in EDTA-coated tubes by jugular venipuncture on Days 7–10 (n = 9), 15–35 (n = 12), 90–100 (n = 15), 130–180 (n = 9, animals not related to the Institute herd), and 240–260 (n = 9) of pregnancy. Five of the animals were used in three stages of pregnancy, on Days 23, 95, and 260 after conception. Pregnancy was confirmed by ultrasonographic examination and by plasma progesterone values on Days 40 and 90 after insemination.

Group 2 was comprised of 10 cyclic cows. Blood samples were drawn on Days 10–12 of estrous cycle (n = 10, midluteal phase).

Group 3 was comprised of eight cystic cows, which were selected as being cystic based on the observation of nonreturn to estrus within 25 days, with twice weekly ultrasonographic examination for 2 wk after the expected date of estrus. Blood samples were drawn from these animals on Days 15 and 22 after the day of expected estrus. To judge the nature of cyst, plasma levels of estradiol and progesterone were measured.

Preparation of Lymphocytes

Blood was centrifuged at 3000 rpm for 20 min at 20°C; the buffy coat was harvested and resuspended in 1:2 v:v Hanks' balanced salt solution (HBSS; Sigma, Munich, Germany). Buffy coats were carefully layered on a lymphocyte separation medium (Lymphodex; Fresenius Diagnostics, Wiesbaden, Germany) at a concentration of 2:3 v:v and centrifuged at 2600 rpm for 30 min at 20°C to separate the lymphocytes according to the method described by Hattori et al. [30] and modified for bovines [31]. Briefly, the ring of mononuclear cells was sucked out, and the cells were resuspended and washed twice with HBSS. Contaminating erythrocytes were then lysed through a short incubation in double-distilled water and again washed with HBSS.

Cell Culture

The cell number was adjusted to 2 x 106 cells/ml in Dulbecco's modified Eagle medium F-12 HAM (Sigma) serum-free medium supplemented with 22 mM Hepes and 75 mM glucose (Serva, Heidelberg, Germany). Cells were seeded in culture plates (with 4 wells each; Nalge Nunc International, Darmstadt, Germany) with 1% antibiotic antimycotic (10 000 IU of penicillin/ml, 10 mg of streptomycin/ml, and 25 µg/ml of amphotericin B; Sigma) and incubated for 72 h at 37°C and 5% CO2. The incubations were performed with or without 10 µg of phytohemagglutinin from Phaseolus vulgaricus (PHA-M; Sigma) and bovine corticotropin-releasing hormone (CRH) (Sigma) at doses of 10 and 100 ng/well. The cell viability as assessed by the trypan blue exclusion test was always greater than 95%.

Medium was collected after 72 h of incubation in tubes containing 1% aprotinin (Trasylol; Bayer AG, Leverkusen, Germany). Contaminating lymphocytes were removed after centrifugation, and supernatants were kept at -20°C until the ACTH analysis.

Hormone Assays

Measurement of ACTH ACTH was analyzed using an immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) that has been validated for the bovine ACTH [32] and tested for the measurements in the medium used [31]. This ACTH assay incorporates both a monoclonal and a polyclonal antibody that specifically bind to N- and C-terminals of ACTH, thereby detecting only the intact ACTH molecule, and has no cross-reactivities with other pro-opiomelanocortin peptides. The sensitivity of the assay as defined at the 95% confidence limit is 1 pg/ml, and the intra-assay coefficient of variation is 3.2%.

Estimation of estradiol and progesterone Plasma estradiol concentrations were measured using a RIA as described previously [33]. The antibody (E2-6 #3) was kindly provided by N.R. Mason (Eli Lilly, Indianapolis, IN), and 125I-labeled estradiol (Amersham, Braunschweig, Germany) was used as a tracer. The sensitivity of the assay at 95% confidence limit was 1.5 pg ml-1. The intra-assay coefficient of variation was 9%. The cross-reactions were 1.5% with estradiol benzoate, 0.6% with estrone, 0.1% with estriol, and less than 0.1% with progesterone, testosterone, and hydrocortisone.

Plasma progesterone concentrations were analyzed using an enzyme immunoassay as described previously [34]. The sensitivity of the assay was 1 ng ml-1, with a 12% intra-assay coefficient of variation. The cross-reactions with 17{alpha}-hydroxyprogesterone, estradiol, and cortisol were 0.005, less than 0.002, and 0.1%, respectively.

Measurement of NO Nitrite and nitrate are stable end products of NO metabolism and, thus, serve as indirect markers for the presence of NO [35]. Nitrite levels were measured in cell-culture supernatants after previous conversion of nitrate in samples to nitrite using NADH-dependent nitrate reductase enzyme by an colorimetric assay based on the Griess reaction [36]. All reagents were freshly prepared before assay. Briefly, samples were centrifuged at 13 000 x g for 6 min and incubated with freshly aliquoted B-NADH and nitrate reductase (both reagents from Sigma) from Aspergillus sp. in 96-well plates for 16 h. Total nitrite in the sample was assayed using equal amounts of sample and Griess reagent (1% sulfanilamide and 0.1% N-[1-naphthylene] ethylenediamine in 5% concentrated phosphoric acid). Amounts of nitrite were estimated from a standard curve of sodium nitrite, and absorbance was measured spectrophotometrically at 540 nm.

Statistical Analysis

Results are expressed as the mean ± SEM. Differences between mean ACTH levels during different stages of gestation and effects of treatments were analyzed by two-way ANOVA using SAS software [37]. Significant differences were determined using Tukey's test, which protects the significance tests of all combinations of pairs [38]. A correlation between ACTH and NO values was tested using Pearson's method, followed by a linear regression analysis (Sigma Stat software package) at a 95% confidence limit with NO as a dependent variable.

RESULTS

Secretion of ACTH from Lymphocytes

Lymphocytic ACTH secretion in cyclic cows was 1.5 ± 0.2 pg ml-1 (Fig. 1). This basal secretion was significantly enhanced (P <= 0.05) under treatments with CRH at doses of 10 and 100 ng/well and with PHA-M at a dose of 10 µg/well to 5.2 ± 1.71, 4.58 ± 1.74, and 3.97 ± 1.05 pg ml-1, respectively (Fig. 1). During all stages of gestation tested, the lymphocytic ACTH secretion was significantly higher (P <= 0.05) than that during estrous cycle. On Days 7–10 of pregnancy, lymphocytes released 6.67 ± 0.56 pg ml-1, which increased to 7.95 ± 0.71 pg ml-1 on Days 12–35 of pregnancy and peaked at 9.43 ± 0.63 pg/ml on Days 90–100 of gestation.



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  		FIG. 1. Secretion of ACTH from peripheral bovine lymphocytes of pregnant and cyclic cows expressed as mean ± SEM; {ddagger}, below the detection limit of the assay; for significant differences, see the text

The lymphocytic ACTH release activity attenuated after Days 130–180 of gestation. At this stage, ACTH values decreased to 4.71 ± 0.48 pg ml-1. A further decline of ACTH levels to 3.12 ± 0.5 pg ml-1 was observed on Days 240–260 of pregnancy. The levels on Days 130–180 and 260 were still significantly elevated (P <= 0.05) compared to ACTH values during the luteal phase of the cycle (Fig. 1). We noticed no significant differences between plasma ACTH levels of cyclic (16.4 ± 4.5 pg ml-1) and pregnant cows (11.1 ± 2.07 pg ml-1).

Lymphocytes separated from cows having ovarian cysts secreted very low amounts of ACTH. These values were less than 1 pg ml-1, which was the lower limit for reliable measurement in our assay system. As indicated in Table 1, mean estradiol-17ß levels in cows with cysts were higher than those in luteal-phase cows. The difference, however, was due to large individual variations that were not significant (cystic animals: 3.97 ± 1.63 pg ml-1; cyclic animals: 1.72 ± 0.71 pg ml-1). Plasma progesterone levels were comparable to those in pregnant animals in two out of eight cystic cows (Table 2). Mean progesterone values in cystic animals were, however, significantly lower (P <= 0.05) than those in pregnant animals. Interestingly, no relation was observed between lymphocytic ACTH secretion and plasma progesterone or estradiol levels in cystic animals (Table 2).


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  		TABLE 1. Plasma estradiol-17{ß} and progesterone levels in pregnant, midluteal, and cystic cows.a


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  		TABLE 2. Plasma estradiol-17{ß} and progesterone levels and lymphocytic ACTH release in individual cows with ovarian cysts

Production of NO by Lymphocytes

Measurement of nitrite from lymphocyte-conditioned medium showed a significant increase (P <= 0.05) during pregnancy when compared to that of cyclic cows (Fig. 2). Lymphocytes from cyclic cows had a nitrite production of 3.8 ± 0.39 µM, which increased to 11.8 ± 3.1 µM on Day 7, 14.9 ± 2.4 µM on Days 12–35, and 16.2 ± 1.8 µM on Days 90–100 of pregnancy (Fig. 2). Around Day 260 of gestation, nitrite production fell to 4.22 ± 0.48 µM, which was not significantly different from levels in cyclic animals. Lymphocytes of cystic cows produced significantly lower (P <= 0.05) amounts of nitrite than pregnant cows.



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  		FIG. 2. Secretion of NO from peripheral bovine lymphocytes during pregnancy and in cyclic and cystic cows expressed as mean ± SEM. a and a1 vs. b, c, and d = P <= 0.05 (one-way ANOVA followed by Tukey's test)

A strong correlation (r = 0.70, P <= 0.001) was observed between ACTH and NO (Fig. 3) at the 95% confidence limit.



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  		FIG. 3. Multiple regression curve between ACTH and NO at the 95% confidence limit for secretion from peripheral bovine lymphocytes. The dependent variable is NO, r = 0.70, and P <= 0.001

DISCUSSION

The cross-talk between the immune and the endocrine systems is heightened during pregnancy, when a variety of cytokines, growth factors, and hormones are engaged in a complex communication for successful maintenance of pregnancy [2, 7]. Results of the present study indicate a high production of lymphocytic ACTH and NO in pregnant cows. We have not come across any comparable study on ACTH and NO secretion from lymphocytes during pregnancy in any other species.

We noticed a marked, early increase in secretion of ACTH from lymphocytes, which was maintained throughout the pregnancy. The increased lymphocytic ACTH secretion beginning around Day 7 of gestation correlates well to the time at which bovine embryo expresses the paternal major histocompatibility complex (MHC) molecules and is regarded as the time of immunological recognition of pregnancy [39]. Another factor that is reportedly important for fetal survival is interferon-tau (IFN-{tau}), an antiluteolytic factor produced from trophoblast and that is strongly immunosuppressive [40] in ruminants. In maternal plasma, IFN-{tau} is observed from Days 12–35 of pregnancy. The peak levels can be measured around Days 15–19. In ruminants, IFN-{tau} is regarded as vital for conceptus survival [40]. In the present study, lymphocytic ACTH production enhanced around Day 7 of pregnancy, and maximum levels, which reached as much as 600% of those in nonpregnant animals, were measured between Days 90–100 after conception. This may indicate that the initiation of lymphocytic ACTH release is not dependent on IFN-{tau} secretion.

Progesterone apparently plays little or no role in elevation of ACTH secretion from lymphocytes during pregnancy. Support for this assumption comes from findings that lymphocytes harvested from animals during the luteal phase or from cows having ovarian cysts with low or high plasma progesterone levels produce very low amounts of ACTH. Cysts have been defined as structures larger than 20 mm in diameter that persist for at least 10 days in the absence of corpus luteum [41]. Plasma progesterone concentration is not a reliable parameter for determining the nature of cysts [42–44]. Plasma estradiol-17ß concentrations, together with progesterone levels and ultrasonographic observations, revealed that two out of eight animals had luteal cysts and two other follicular cysts; however, both kind of cysts produced progesterone as well as estradiol-17ß.

Production of ACTH from lymphocytes is reported to be stimulated by CRF [13]; in our study, a stimulatory effect of CRF on ACTH secretion from lymphocytes was noticed only in cyclic animals. Likewise, PHA-M, which stimulates proliferation of lymphocytes, was effective in enhancing the ACTH release in cyclic, but not in pregnant, animals. This indicates that the lymphocytes are refractory to secretagogues such as PHA-M and CRF during pregnancy, that the mechanisms regulating ACTH secretion from lymphocytes differ during pregnancy from those during the estrus cycle, or both.

Multiple mechanisms have been proposed to explain the tolerance of lymphocytes to fetal allograft [4, 5]. Downregulation of MHC class I and expression of Fas ligand on placenta are not the sole requisites for successful allogenic pregnancy. Thus, other factors must prevent a harmful maternal immune response [3, 45–47]. Ability of lymphocytes to secrete immunomodulators could be one of the possible mechanisms. Peripheral mononuclear cells produce hCG as early as Days 7–11 after embryo transfer. Production of hCG remains high during human pregnancy and is presumed to be vital for immunotolerance [48]. Secretagogues like prostaglandin E2 from the embryonic or endometrial compartment has been also suggested to specifically alter the functional properties of peripheral blood lymphocytes in cows [49].

During pregnancy, a strong shift toward TH-2 cytokines occurs, resulting in less production of TH-1 cytokines [7]. This is proposed as an important mechanism of fetal survival. An increase in proinflammatory TH-1 cytokines is associated with abortions [8, 50, 51]. Both ACTH and NO are reported to cause a shift toward TH-2 cytokines by increasing levels of TH-2 cytokines and decreasing levels of TH-1 cytokines [19–21, 26, 27]. We observed a strong, positive correlation between production of ACTH and NO from lymphocytes during different reproductive states. The net result of cytokine-mediated self-amplification and -regulation is that once a T-cell immune response begins to develop along one pathway, namely TH-1 or TH-2, it tends to become progressively polarized in that direction [52]. The TH-2 shift can be induced by the presence of fertilized ovum [53] that progresses as the gestation proceeds. During infection with the human immunodeficiency virus (HIV), a shift toward TH-2 cytokines is also noticed [54], and interestingly, HIV infection also leads to increased production of ACTH from lymphoid cells [55]. Increased NO production from lymphocytes together with ACTH, as observed in the present study, can act synergistically to skew the balance of T-helper cytokines toward TH-2. To our knowledge, no comparable data are in the literature that indicate a strong, positive correlation between ACTH and NO production from lymphocytes during pregnancy. However, stress-induced pituitary ACTH release is NO dependent, and a positive correlation exists between lipopolysaccharide-induced ACTH secretion and NO levels in paraventricular nucleus [56].

The observed increase in secretion of ACTH from peripheral lymphocytes was not reflected in the circulation, because plasma ACTH levels did not differ between nonpregnant and midpregnant cows. An increase in circulating ACTH levels could be obstructive by stimulating secretion of glucocorticoids and causing a strong, general immunosuppression that could possibly predispose the maternal immune system to infections. However, ACTH produced from lymphocytes could have significant modulatory effects by autocrine and paracrine mechanisms. Adrenocorticotropin is a central part of the hypothalamic-pituitary-adrenal axis. This system is crucial for the development of several other hormonal systems in the fetus [57], and it determines the onset of parturition [58]. Recently, it was shown that lymphocytes cocultured with adrenocortical cells can stimulate adrenal androgen secretion via cell contact [59]. Elucidation of the significance of lymphocytic ACTH and NO secretion during pregnancy and clarification of trigger mechanisms need further investigation. Nevertheless, early appearance of increased ACTH and NO secretion from lymphocytes, sustained till the very end of pregnancy, sheds new light on the area of fetomaternal immune interactions and the mechanisms governing the fetal survivability.

ACKNOWLEDGMENTS

We would like to thank Mrs. Iris Stelter, Mr. Ronald Wittig, Mr. P. Aldag, Mr. K.G. Hadeler, and Mr. H.G. Sander for technical support.

FOOTNOTES

First decision: 30 June 2000.

1 V.D.D. carried out the work under a German Academic Exchange Service (DAAD) fellowship. Back

2 Correspondence: N. Parvizi, Institute For Animal Science and Animal Behaviour (FAL), Höltystrasse 10, 31535, Neustadt, Germany. FAX: 49 5034 871143; parvizi{at}tzv.fal.de Back

Accepted: August 16, 2000.

Received: May 17, 2000.

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