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Endocrinology Vol. 140, No. 12 5651-5658
Copyright © 1999 by The Endocrine Society

ARTICLES

Urocortin Messenger Ribonucleic Acid: Tissue Distribution in the Rat and Regulation in Thymus by Lipopolysaccharide and Glucocorticoids1

Kazunori Kageyama, Margaret J. Bradbury, Lingyun Zhao, Amy L. Blount and Wylie W. Vale2

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Wylie W. Vale, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: vale{at}salk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Urocortin (Ucn), a new mammalian member of the CRF family, is a candidate endogenous ligand for type 2 CRF receptors. In a survey of peripheral tissues from adult male rats, we found that Ucn messenger RNA (mRNA) was abundant in the gastrointestinal tract and immune tissues such as thymus and spleen. We next tested the hypothesis that levels of Ucn mRNA levels in thymus and spleen would be altered after immune activation. As measured by ribonculease protection assay, lipopolysaccharide (LPS) induced a 2-fold time-dependent increase in thymic Ucn mRNA levels within 6 h. By contrast, splenic Ucn mRNA levels decreased after LPS. Because LPS activates the hypothalamus-pituitary-adrenal (HPA) axis, we examined whether the effects of LPS on Ucn mRNA might be mediated through changes in HPA axis hormones. Ucn mRNA in thymus, but not spleen, was significantly increased after ACTH injection; however, LPS did not increase Ucn expression in the thymus of adrenalectomized rats with corticosterone replacement, despite substantial increases in ACTH. Finally, sc injection of corticosterone stimulated Ucn mRNA comparably to that of LPS. Together, these results suggest that Ucn mRNA expression can increase after immune activation in a corticosterone-dependent manner, and that such changes in Ucn mRNA may be an additional consequence of HPA axis activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECENTLY, we reported the cloning and characterization of rat urocortin (Ucn), a 40-amino acid novel mammalian CRF family peptide (1). Ucn has also been found in humans (2) and sheep (3). Ucn has 45% sequence identity to rat/human CRF and 63% identity to the fish peptide, urotensin (1). In rat or mouse periphery, Ucn-like immunoreactivity and messenger RNA (mRNA) are detected in the pituitary (4, 5), gastrointestinal tract (5), testis (5), and cardiac myocytes (6), while CRF mRNA has been detected in heart (7), gut (7), placenta (7), ovary (7), testis (7, 8), and fetal lung (7).

There are two receptors for CRF and Ucn, the CRF receptor type 1 (CRF R1) and CRF receptor type 2 (CRF R2) (9). CRF has low affinity for CRF R2 and high affinity for CRF R1 (10), whereas Ucn has high affinity for both (1). Ucn is proposed to function as a ligand for CRF R2 (9). In the brain, the distribution of Ucn or urotensin-like immunoreactive fibers is different from that of some CRF and correlates with the distribution of CRF R2{alpha}, but not CRF R1 (1).

Both CRF and Ucn are posited to influence immune functions. Ucn mRNA and peptide were found in human lymphocytes (11), while CRF mRNA was reported in rat thymus and rat/mouse spleen (7, 12). The functions of CRF and Ucn within the context of immune responses are not completely known. Hypothalamic-produced CRF in response to stressors acts through CRF R1 to stimulate the synthesis and release of ACTH by pituitary corticotropic cells (13, 14). ACTH in turn stimulates production of adrenocorticosteroids (13). In addition to the effect on the hypothalamus-pituitary-adrenal (HPA) axis, CRF modulates immune responses in the periphery both directly and indirectly (15). In in vitro studies, CRF has been shown to stimulate B and T lymphocyte proliferation (16, 17). In addition to well established inhibitory effects on POMC (18, 19) and CRF gene expression (18, 20), glucocorticoids are known to be potent antiinflammatory agents (21) and to modulate production of inflammatory factors such as cytokines (22, 23). Although the role of endogenous Ucn in immune function is not yet clear, endogenous Ucn is able to mimic some of the actions of CRF, such as appetite suppression (24). In addition, both peptides modulate immune and inflammatory responses (9). For example, the administration of Ucn suppresses inflammation (25) and cytokine release more effectively than CRF (26), working independently of endogenous corticosteroids (26). Exogenous Ucn limited the clinical course of autoimmune encephalomyelitis more robustly than did CRF (27). The significance of endogenous Ucn in immune system functions, however, remains an open question.

In the present study, we examined the tissue distribution of Ucn mRNA in rat using ribonuclease (RNase) protection assays. We then studied the regulation of Ucn mRNA levels in thymus and spleen, a tissue of high relative Ucn gene expression, to verify the hypothesis that regulation of Ucn mRNA levels in thymus and spleen would be altered after immune activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats [body weight (BW) 280–320 g] were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). They were housed in a temperature-controlled room with controlled lighting (light 0600–1800 h) and were given free access to laboratory chow and tap water. All procedures were approved by the Salk Institute Animal Use and Care Committee.

Surgery
Jugular vein cannulation. In all rats, a jugular vein catheter (PE 50, Becton Dickinson and Co., Sparks, MD) was inserted into the right atrium under light halothane anesthesia 2 days before experiments. The catheter was filled with heparinized saline, passed through a subcutaneous tunnel, and exteriorized at the back of the neck. After the cannulation, rats were housed in individual cages.

Subcutaneous cannulation. In animals in which ACTH gel or corticosterone was injected, a subcutaneous catheter (PE 20, Becton Dickinson and Co.) was inserted into the back at the same time as jugular vein cannulation. The catheter was filled with saline, passed through a subcutaneous tunnel, and exteriorized at the back of the neck.

Adrenalectomy (Adx). Halothane-anesthetized rats were adrenalectomized bilaterally via a dorsal approach, and implanted sc with slow-release corticosterone pellet (35 mg, 21 day release; Innovative Research of America, Sarasota, FL; Adx + corticosterone). This regimen was chosen for its ability to retain basal CRF and vasopressin mRNA levels in the paraventricular nucleus of the hypothalamus and POMC mRNA levels in the anterior pituitary after Adx (28). A control group of rats (sham) was anesthetized, received the same dorsal incision, and was implanted with a placebo pellet. After surgery, all rats were provided with water containing 0.9% NaCl. Five days later, sham and Adx + corticosterone rats participated in lipopolysaccharide (LPS) experiments. In LPS-injected Adx + corticosterone rats, Adx was verified by the lack of change in plasma corticosterone. In saline-injected Adx + corticosterone rats, Adx was verified by the lack of circadian elevation in plasma corticosterone 8 h after lights-on.

Reagents
LPS (Escherichia coli serotype O26: B6; code 3755, lot 37H4095) and corticosterone were purchased from Sigma (St. Louis, MO). ACTH gel (H.P. Acthar gel) was purchased from Rhone-Poulenc Rorer Pharmaceuticals Inc. (Collegeville, PA).

Experimental procedure
On the day of the experiment, the rats were housed in opaque sampling cages, and the jugular vein catheter was connected to a sampling tube to allow for remote sequential blood sampling. After a period of 2–3 h, experiments were started at 0800–0900 h. The rats were killed by decapitation after final blood sampling, and the tissues were removed and frozen in liquid nitrogen.

Exp 1: Effects of iv injection of LPS (50 µg/kg BW) on plasma ACTH, corticosterone, and Ucn mRNA levels in the thymus and the spleen. After blood sampling for measurement of basal plasma ACTH and corticosterone levels, vehicle (saline, 100 µl) or LPS at a dose of 50 µg/kg BW was injected iv at 0 min. Blood was drawn 30, 60, 120, 240, and 360 min later, and stored for future measurements of plasma ACTH and corticosterone. After blood was sampled at 6 h, some rats were decapitated, and organs were harvested. To examine time-dependent changes in Ucn mRNA levels, other rats were decapitated 2 h and 24 h after vehicle or LPS injection (50 µg/kg BW).

Exp 2: Effects of sc injection of ACTH gel (0.8 U/animal) on plasma corticosterone and Ucn mRNA levels in the thymus and spleen. After blood sampling for measurement of basal plasma corticosterone levels (0 min), vehicle (saline, 100 µl), LPS (50 µg/kg BW, iv), or ACTH gel (0.8 U/rat, sc) was injected. ACTH was also administered at 30 and 60 min. This dose regimen was chosen to ensure a prolonged endogenous corticosterone release (29). Blood was drawn 30, 60, 120, 180, 240, 300, and 360 min after LPS injection or first ACTH injection for plasma corticosterone measurement. After sampling at 6 h, the rats were decapitated for tissue collection.

Exp 3: Effects of Adx with corticosterone replacement and LPS injection (50 µg/kg BW) on plasma ACTH and corticosterone and Ucn mRNA levels in the thymus. After blood sampling for measurement of the basal plasma ACTH and corticosterone levels, vehicle (saline, 100 µl) or LPS (50 µg/kg BW, iv) was injected at 0 min in sham or Adx + corticosterone rats. Blood was drawn at 60 and 240 min for ACTH and corticosterone measurements. After the final blood sampling, the rats were decapitated for tissue collection.

Exp 4: Effects of sc injection of corticosterone or restraint stress on plasma ACTH, corticosterone, and Ucn mRNA levels in the thymus. After blood sampling for measurement of the basal plasma ACTH and corticosterone levels, vehicle (100 µl of saline for iv injection and 200 µl of 11% ethanol-containing saline for sc injection), LPS (50 µg/kg BW, iv), a low dose of corticosterone (37.5 µg/rat, sc), or a high dose of corticosterone (125 µg/rat, sc) was injected. Both low and high doses of corticosterone were administered at 0, 30, 60, 120, and 180 min. Some rats that received both vehicle injections were wrapped in cloth towels and restrained by rubber bands and labeling tape for 1 h. Blood was drawn 30, 60, 180, and 360 min after injection or onset of restraint for plasma ACTH and corticosterone measurements. After sampling at 6 h, the rats were decapitated for tissue collection.

RNase protection
Total RNA was extracted using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH).

Rat Ucn and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured simultaneously by RNase protection, using rat GAPDH as an internal loading control. A 361-nucleotide Ucn antisense riboprobe (Fig. 1Go) specific to the rat Ucn mRNA was synthesized using T7 RNA polymerase. A 165-nucleotide GAPDH antisense riboprobe specific to the rat GAPDH mRNA was synthesized using T3 RNA polymerase. All riboprobes were synthesized in the presence of [{alpha}-32P]UTP (3,000 Ci/mmol) and 20 µM UTP, as described (30). The fragments protected by the Ucn and GAPDH riboprobes are 307 and 135, respectively (Fig. 1Go).



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  		Figure 1. Expression of Ucn mRNA in tissue. A, Map of Ucn probe used to detect expression of rat Ucn mRNA. B, A representative image of RNase protection assay of Ucn mRNA. Total RNA isolated from each tissue listed was hybridized with the above antisense probe specific to rat Ucn (5 x 105 cpm) and rat GAPDH (2 x 104 cpm). The protected fragments were resolved on a 6% polyacrylamide urea gel.

 
RNase protection analyses were carried out as previously described (30). RNA samples (50 µg for peripheral tissues; 15 µg for anterior pituitary and Edinger-Westphal nucleus) were hybridized in 24 µl deionized formamide plus 6 µl hybridization buffer containing 5 x 105 cpm of Ucn and 2 x 104 cpm GAPDH antisense riboprobes. After heating at 90 C for 5 min, the samples were hybridized at 42 C for 15 h and subsequently digested by RNase (200 µg/ml RNase A and 350 U/ml RNase T1) at 24 C for 60 min. The samples were resolved on 6% polyacrylamide urea gels. Quantitative analysis was performed using the PhosphorImager system (Molecular Dynamics, Inc., Sunnyvale, CA) and the ImageQuant 4.0 software package. The intensity of each of the protected fragments was corrected for differences in the amount of RNA loaded according to the intensity of the protected GAPDH fragment of the same sample, and results are expressed as corrected arbitary units. The intensity of Ucn mRNA was in proportion to the intensity of GAPDH mRNA in the same thymic sample when added in a range from 12.5 to 50 µg (not shown).

Corticosterone and ACTH measurement
Plasma corticosterone and ACTH were measured in duplicate from unextracted samples. Plasma corticosterone levels were measured with a commercial immunoradiometric assay kit produced by ICN Biomedicals, Inc. (Costa Mesa, CA). Plasma ACTH levels were measured with a commercial immunoradiometric assay kit produced by Nichols Institute Diagnostics (San Juan Capistrano, CA). Repeated samples from individual rats were analyzed within the same assay.

Statistical analysis
All values are expressed as the mean ± SEM. Statistical analyses of these data were performed using one-way ANOVA, or two-way ANOVA on repeated measures with time and treatment as the factors (followed by Fisher’s least protected significant difference or Duncan’s test). P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue distribution of Ucn mRNA
RNase protection assays were performed to determine the distribution of rat Ucn mRNA (Fig. 1Go). For a preliminary assessment of tissue distribution of Ucn in rat, a rat Ucn-specific antisense riboprobe was synthesized and hybridized with total RNA isolated from a variety of peripheral tissues. Ucn mRNA was abundantly expressed in thymus, spleen, gastrointestinal tract, and testis; lower levels of mRNA were detected in the kidney, heart, and liver.

Effects of LPS on HPA axis activity and on Ucn mRNA in the thymus and spleen
Rats were injected with saline or LPS (50 µg/kg) iv and plasma ACTH and corticosterone levels were measured. Plasma ACTH and corticosterone levels immediately before treatment in both saline and LPS groups were typical of rats under nonstress conditions. Both plasma ACTH and corticosterone levels in vehicle-injected rats were elevated in the evening, consistent with the known diurnal rhythms of these hormones. As expected, iv administration of LPS elicited time-dependent increases in plasma ACTH and corticosterone levels, with peak concentrations measured 1 h (mean ± SEM, 820.42 ± 115.73 pg/ml) and 2 h (mean ± SEM, 522.61 ± 137.34 ng/ml) after injection, respectively.

Figure 2AGo shows time-dependent changes in Ucn mRNA levels in the thymus after iv injection of LPS, which were statistically significant by using ANOVA. Peak Ucn mRNA levels were 2-fold those of the control values 6 h after LPS injection (P < 0.05). Ucn mRNA levels in thymus returned to basal levels 24 h after treatment. By contrast, in the spleen (Fig. 2BGo), Ucn mRNA levels were decreased to less than half those of the control values 6 h after LPS injection (P < 0.05). The decrease in mean values in Ucn mRNA 24 h after LPS injection was not significant.



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  		Figure 2. Time-dependent changes in Ucn mRNA levels in the thymus (A) or the spleen (B) after iv injection of LPS (50 µg/kg BW) or saline (100 µl) in intact male rats. After the final blood sampling, rats were decapitated 2, 6, and 24 h after vehicle or LPS injection, and organs were harvested to examine Ucn mRNA levels. Relative changes in Ucn mRNA levels were compared with saline controls (mean ± SEM) of six or seven animals per group. Statistical analyses were performed using one-way ANOVA. *,P < 0.05 (compared with control).

 
Regulation of thymic and splenic Ucn mRNA by HPA axis hormones
To determine whether changes in Ucn mRNA by LPS were mediated through changes in ACTH or glucocorticoid production, ACTH gel was injected sc, and its effects on plasma corticosterone and Ucn mRNA levels in thymus and spleen were compared with those of LPS injection. In the saline-injected group, plasma corticosterone remained at basal, diurnally-appropriate concentrations throughout the experiment (Fig. 3AGo). Administration of LPS produced a large elevation in plasma corticosterone concentrations. Similarly, sc injection of ACTH gel also significantly elevated plasma corticosterone compared with the saline-injected group.



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  		Figure 3. Effects of sc injection of ACTH gel on plasma corticosterone and Ucn mRNA levels in the thymus and spleen. Vehicle (saline, 100 µl), LPS (50 µg/kg BW, iv) or ACTH gel (0.8U/rat, sc) was injected at 0 min. ACTH was also administered 30 and 60 min later. Data are the mean ± SEM of seven or eight animals per group. A, Effects of sc injection of ACTH gel on plasma corticosterone levels in intact male rats. Blood was drawn 30, 60, 120, 180, 240, 300, and 360 min after LPS injection or first ACTH injection. Statistical analyses were performed using two-way ANOVA on repeated measures. *, P < 0.05 (compared with control). #, P < 0.05 (compared with LPS). B, Regulation of Ucn mRNA in the thymus by sc injection of ACTH gel in intact male rats. The rats shown in panel A were decapitated for tissue collection 6 h after vehicle, LPS, or ACTH gel. Statistical analyses were performed using one-way ANOVA. *, P < 0.05 (compared with control). C, Regulation of Ucn mRNA in the spleen by sc injection of ACTH gel in intact male rats. The rats shown in panel A were decapitated for collection of tissue 6 h after vehicle, LPS, or ACTH gel. Statistical analyses were performed using one-way ANOVA. *, P < 0.05 (compared with control).

 
The level of Ucn mRNA in the thymus was increased 6 h after either LPS or ACTH treatment, to levels approximately twice that measured in saline-injected rats (Fig. 3BGo). The level of thymic Ucn mRNA after the injection of ACTH was not significantly different from that in LPS group. In the spleen (Fig. 3CGo), there were higher mean values in Ucn mRNA levels in the ACTH-injected group, but the difference was not statistically significant.

Effects of endogenous corticosterone on thymic Ucn mRNA
To determine whether the effects of LPS on Ucn mRNA required an increase in endogenous corticosterone, we repeated the previous experiment using Adx rats in which plasma corticosterone was clamped at approximately 50 ng/ml (Adx + corticosterone), or sham-operated intact rats (sham, as a control). In the saline-injected sham rats, plasma ACTH and corticosterone levels exhibited the expected diurnal variation (Fig. 4AGo). Plasma corticosterone in Adx + corticosterone rats was unchanged throughout the sample period. There were significant differences in plasma ACTH levels after LPS injection between sham and Adx + corticosterone rats, probably due to the lack of negative feedback from glucocorticoids in the latter group. Plasma ACTH and corticosterone levels increased 1 h after LPS injection in sham rats. The Adx + corticosterone rats demonstrated substantial ACTH responses after LPS injection, but no change in corticosterone levels.



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  		Figure 4. Effects of Adx on response of plasma ACTH and corticosterone and Ucn mRNA levels in the thymus to LPS injection. Vehicle (saline, 100 µl) or LPS (50 µg/kg BW, iv) was injected at 0 min in sham or Adx + corticosterone rats. Data are the mean ± SEM of seven or eight animals per group. A, Effects of Adx on response to LPS injection on plasma ACTH or corticosterone levels. Blood was drawn at 60 and 240 min for ACTH and corticosterone measurements. Statistical analyses were performed using two-way ANOVA on repeated measures. *, P < 0.05 (compared with sham/saline control). #, P < 0.05 (compared with Sham/LPS). B, Effects of Adx on response to LPS injection on Ucn mRNA levels in the thymus in rats from panel A. The rats were decapitated for tissue collection 4 h after vehicle or LPS injection. Statistical analyses were performed using one-way ANOVA. *,P < 0.05 (compared with sham/saline control).

 
Ucn mRNA in the thymus of Adx + corticosterone animals after vehicle injection was not significantly different from that in vehicle-injected sham rats (Fig. 4BGo). While Ucn mRNA in the thymus increased 2-fold after LPS injection in sham rats, this effect was abolished in Adx + corticosterone rats.

Induction of thymic Ucn mRNA by corticosterone or restraint stress
To determine whether Ucn expression responds directly to changes in plasma corticosterone in the absence of immune challenge, exogenous corticosterone was administered to intact rats. Administration of low (35 µg/rat) and high doses (125 µg/rat) of corticosterone elevated plasma corticosterone concentrations over those of saline-treated rats 30 and 60 min after injection (Fig. 5AGo). Plasma corticosterone concentrations at 30 and 60 min and the area under the curve (AUC) from 0 to 360 min in high doses of corticosterone- and LPS-injected rats were not different (Fig. 5BGo). Low doses of corticosterone injection resulted in plasma corticosterone concentrations at 30 and 60 min and AUC (0–360 min) that were intermediate to those of saline- and high corticosterone dose-injected rats.



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  		Figure 5. Effects of sc injection of corticosterone (low corticosterone or high corticosterone) or restraint stress on plasma ACTH, corticosterone, and Ucn mRNA levels in the thymus. Vehicle (100 µl of saline for iv injection and 200 µl of 11% ethanol-containing saline for sc injection), LPS (50 µg/kg BW, iv), a low dose of corticosterone (37.5 µg/rat, sc), or a high dose of corticosterone (125 µg/rat, sc) was injected. Both low and high doses of corticosterone were administered at 0, 30, 60, 120, and 180 min. A separate set of rats was restrained for 1 h. Data are the mean ± SEM of seven or eight animals per group. A, Effects of sc injection of corticosterone or restraint stress on plasma ACTH or corticosterone levels. Blood was drawn 30, 60, 180, and 360 min after injection or onset of restraint. Statistical analyses were performed using two-way ANOVA on repeated measures. *,P < 0.05 (compared with control). #,P < 0.05 (compared with LPS). B, AUC of plasma corticosterone levels, calculated from the data of panel A was also determined. *, P < 0.05 (compared with control). #, P < 0.05 (compare with a low dose of corticosterone). C, Regulation of Ucn mRNA in the thymus by sc injection of corticosterone or restraint stress. The rats were decapitated for collection of tissue 6 h after vehicle, LPS, corticosterone injection, or onset of restraint. Statistical analyses were performed using one-way ANOVA. *, P < 0.05 (compared with control).

 
Figure 5CGo shows effects of corticosterone injections on Ucn mRNA in the thymus. Ucn mRNA levels in the thymus doubled 6 h after injection of the high dose of corticosterone compared with saline-injected rats. This increase was of the same magnitude as that seen in LPS-injected rats. Injections of the low dose-corticosterone did not elevate Ucn mRNA expression.

To determine whether Ucn expression could be stimulated by a nonimmune stress, some saline pretreatment/saline treatment rats were physically restrained for 1 h. The restraint stress produced marked elevations of plasma corticosterone levels that were similar in magnitude and AUC to those of high doses of corticosterone injection and LPS injection (Fig. 5BGo). Ucn mRNA in the thymus was stimulated by restraint stress 6 h after the initiation of restraint to a level similar to that seen in LPS- or high doses of corticosterone-injected rats (Fig. 5CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As presented here, Ucn is broadly expressed in the periphery. The levels of the mRNA in the peripheral tissues were varied, with the greatest abundance in components of the immune system and in the gastrointestinal tract. We have reported previously the presence of Ucn-like peptide in the duodenum (1). We also found that immune system activation by LPS injection in conscious rats increased Ucn mRNA levels in the thymus. The increase in thymic Ucn mRNA after corticosterone or ACTH injection, or restraint stress in the absence of immune stimulation, indicates that the increased Ucn mRNA by endotoxin is induced by HPA axis activation. It is likely that, as an additional consequence of activation of the HPA axis, plasma corticosterone elevations are largely responsible for Ucn gene stimulation; LPS injection did not stimulate thymic Ucn gene expression in the Adx + corticosterone rats, despite huge increases in plasma ACTH. The direct injection of high doses of corticosterone, but not low doses of corticosterone, elevated the thymic Ucn mRNA levels in intact rats. To our knowledge, our results show for the first time that the Ucn mRNA expression can be positively regulated by endogenous glucocorticoid stimulation in any tissue; by contrast, in the spleen, the mRNA levels were reduced after LPS injection, but not by ACTH or corticosterone stimulation. There is no obvious explanation for the discrepancy between these two tissues from our data. This differential Ucn mRNA expression, however, may be attributable to tissue-specific glucocorticoid-sensitive factors, or others such as cytokines, and cell types found exclusively in thymus and spleen (31, 32).

The role of Ucn in the immune system has not been determined. On the other hand, CRF has previously been demonstrated to modulate the immune or inflammatory responses of various tissues, although the significance of such findings has yet to be put in physiological and pathological context. Exogenous administration of CRF has been shown to reduce inflammation and suppress some immune functions (33, 34). In contrast to these reports, it has been demonstrated that administration of CRF antiserum or receptor antagonist decreased acute and subacute inflammations in vivo in several models of inflammation (15, 35, 36). The recent identification of Ucn, however, suggests that Ucn, instead of CRF, may represent an endogenous mediator of some of these effects, because both Ucn and CRF can act through CRF R1 and R2 (1). Further, some antisera previously used were found to detect not only CRF but also Ucn (11). For instance, although the production of CRF-like immunoreactivity had been demonstrated in human T lymphocytes (37, 38), Bamberger et al. (11) clearly demonstrated that Ucn mRNA, but not CRF, was expressed in the human lymphocytes through the use of CRF- and Ucn-specific probes (11).

Antiinflammatory actions of exogenous Ucn have recently been reported (25). Exogenous Ucn was shown to be a potent antiinflammatory factor after thermal injury of skin (25), and an antinecrotic factor following hypoxia (6). Recently, it was shown that exogenous Ucn directly inhibited LPS-induced tumor necrosis factor (TNF) production both in vivo and in vitro more potently than did CRF, supporting the hypothesis postulating an antiinflammatory role for Ucn (26). The role of endogenous Ucn in immune tissues, however, is still unclear. Endogenous Ucn in the immune tissues might act on lymphocytes, in an autocrine or paracrine manner, as lymphocytes have been shown to possess CRF binding sites (39). Ucn secreted locally in a glucocorticoid-dependent manner after immune challenge could possibly modulate cellular immunity or the differentiation and/or proliferation of T lymphocytes in the thymus.

The increase in Ucn mRNA in the thymus is dependent on glucocorticoid production. The thymus has been found to exhibit high concentrations of glucocorticoid receptor (31). In the thymus, glucocorticoids take a major part in the growth of the epithelial cells, differentiation of thymocytes, and regulation of T cell development (32, 40). The molecular mechanisms underlying the regulation of Ucn are currently under investigation. In both the mouse and human Ucn promoters, there is a consensus cAMP response element (CRE) site, which has been shown to mediate the regulation of Ucn expression by cAMP (41). Four base pairs upstream of the CRE site, there is a consensus half-site for the glucocorticoid response element (GRE) with sequence AGAACA in the antisense orientation. It exists in the same position in both mouse and human Ucn promoters. The glucocorticoid receptor has been shown to activate gene expression through both simple GREs or composite GREs, sites that have both GRE and other transcription factor binding sites (42, 43). It is possible that Ucn regulation by corticosterone is mediated through this potential composite GRE in the thymus.

Although Ucn mRNA in the spleen was decreased after LPS challenge, the decrease was independent of HPA changes. The mechanism of modulation of Ucn mRNA in the spleen after LPS was not determined in this study. During activation of the immune system or exposure to nonimmune stresses, proinflammatory mediators such as interleukin (IL)-1ß, IL-6, and TNF{alpha}, are known to be elevated in the systemic circulation (15, 44), and it has been shown that CRF is regulated by such cytokines in vivo and in vitro (15). The Ucn promoter has a CCAAT/enhancer-binding protein site, sequences associated with cytokine signaling in CRF promoter (40, 45); therefore, it is most likely that cytokines, such as IL-1ß, IL-6, and TNF{alpha}, produced after LPS challenge, might modulate Ucn mRNA. Such cytokine effects would be expected to modify the humoral immunity and the cellular immunity in the spleen and thymus, respectively (32).

In summary, our present data demonstrate 1) that the immune-related tissues relatively express high levels of Ucn mRNA, 2) that endotoxin stimulates Ucn mRNA in thymus, whereas it inhibits Ucn mRNA in spleen, 3) that endotoxin-induced stimulation of Ucn mRNA in thymus requires elevations in plasma corticosterone above basal levels, and 4) that high levels of plasma corticosterone can stimulate Ucn gene expression in the absence of immune challenge. Together, these results suggest that Ucn gene expression in the thymus is dependent on corticosterone stimulation. The significance of increased Ucn expression after immune challenge is currently under investigation.


   Acknowledgments
 
We thank Sandra Guerra and Dave Dalton for assistance with manuscript preparation, and Dr. Susan Akana for helpful advice on corticosterone and ACTH doses. We also thank Catherine Rivier’s laboratory for technical assistance.


   Footnotes
 
1 This work was supported by NIH Program Project DK-26741, the Foundation for Research, The Third Department of Internal Medicine, Hirosaki University School of Medicine (K.K.), The Kleberg Foundation (K.K.), and the Adler Foundation (M.B.). Back

2 Foundation for Research Senior Investigator. Back

Received May 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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