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Endocrinology, doi:10.1210/en.2007-1456
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Endocrinology Vol. 149, No. 4 1436-1450
Copyright © 2008 by The Endocrine Society

Systemic Immune Challenge Activates an Intrinsically Regulated Local Inflammatory Circuit in the Adrenal Gland

Linda Engström, Khadijah Rosén, Anna Angel, Anna Fyrberg, Ludmila Mackerlova, Jan Pieter Konsman, David Engblom and Anders Blomqvist

Department of Clinical and Experimental Medicine (L.E., K.R., A.A., A.F., L.M., D.E., A.B.), Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden; and Laboratory of Psychoneuroimmunology, Nutrition, and Genetics (J.P.K.), Centre National de Recherche Scientifique, Unité Mixte de Recherche 5226, Institut National de Recherche Agronomique, Unité Mixte de Recherche 1286, Université Bordeaux 2, 33076 Bordeaux, France

Address all correspondence and requests for reprints to: Dr. Anders Blomqvist, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden. E-mail: andbl{at}ibk.liu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is evidence from in vitro studies that inflammatory messengers influence the release of stress hormone via direct effects on the adrenal gland; however, the mechanisms underlying these effects in the intact organism are unknown. Here we demonstrate that systemic inflammation in rats elicited by iv injection of lipopolysaccharide results in dynamic changes in the adrenal immune cell population, implying a rapid depletion of dendritic cells in the inner cortical layer and the recruitment of immature cells to the outer layers. These changes are accompanied by an induced production of IL-1β and IL-1 receptor type 1 as well as cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in these cells, implying local cytokine-mediated prostaglandin E2 production in the adrenals, which also displayed prostaglandin E2 receptors of subtypes 1 and 3 in the cortex and medulla. The IL-1β expression was also induced by systemically administrated IL-1β and was in both cases attenuated by IL-1 receptor antagonist, consistent with an autocrine signaling loop. IL-1β similarly induced expression of cyclooxygenase-2, but the cyclooxygenase-2 expression was, in contrast, further enhanced by IL-1 receptor antagonist. These data demonstrate a mechanism by which systemic inflammatory agents activate an intrinsically regulated local signaling circuit that may influence the adrenals’ response to immune stress and may help explain the dissociation between plasma levels of ACTH and corticosteroids during chronic immune perturbations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ADRENAL GLAND plays a critical role in the organism’s response to immune stress, as being the almost exclusive source of corticosteroid production (1). It has since long been established that the activation of the adrenal by immune challenge occurs via hormone release by the hypothalamus and pituitary gland (2, 3), but there is also evidence that inflammatory messengers like cytokines and prostaglandins may influence the release of stress hormone through direct effects on the adrenal cells. For example, in situ perfusion studies have demonstrated that bacterial wall lipopolysaccharide (LPS) enhance corticosterone release (4, 5), likely via local release of IL-1β, and this is further supported by the findings from adrenal cell cultures that IL-1β exerts a stimulatory effect on corticosterone release (6, 7, 8, 9). There is evidence that the effect of IL-1β is exerted through local production of prostaglandin E2 (PGE2) because prostaglandin synthesis inhibitors have been found to attenuate the IL-1β-induced release of corticosterone, both in vitro and during in situ perfusion (4, 7, 8, 10), and treatment of primary bovine adrenal cells with IL-1β have been shown to result in the production of PGE2 (7).

However, in contrast to the large number of observations on local effects of inflammatory mediators on the adrenal gland, which are supported by clinical findings of dissociation between the serum levels of centrally elicited ACTH and adrenally derived corticosteroids (11, 12), so far little has been known about the mechanisms underlying local inflammatory signaling in the adrenal during immune stress. Whereas it has been reported that mRNA for proinflammatory mediators such as IL-1β and cyclooxygenase (Cox)-2 are produced in the rat adrenal gland during severe endotoxemia (13, 14) and that these mediators affect adrenocortical function in vitro (6, 7), there is no information as to which cells that produce and respond to the proinflammatory mediators and how their local release is regulated. In the present study, these issues were examined using an animal model of systemic inflammation. Hence, freely moving rats were injected iv with moderate doses of either LPS or rat recombinant IL-1β (rIL-1β), with or without simultaneous treatment with IL-1 receptor antagonist (IL-1ra). The adrenals were collected at different time points after immune stimulation and examined by using single- and dual-labeling immunohistochemistry, in situ hybridization, and quantitative real-time RT-PCR. The findings show that adrenal immune cells undergo profound changes in response to systemic immune challenge that are associated with their induced and intrinsically regulated expression of inflammatory messengers in a temporal and cell-specific pattern, hence unraveling a mechanism by which inflammatory agents activate local signaling circuits in the adrenals that may regulate stress hormone release.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats (Scanbur BK, Sollentuna, Sweden) were used. They were housed one to a cage at constant room temperature (20 C) on a 12-h light, 12-h dark cycle (lights on at 0700 h) with food and water freely available. All experiments were performed according to Swedish national guidelines and approved by the local Animal Care and Use Committee.

Intravenous administration of LPS, rat rIL-1β, or LPS and IL-1ra
Rats were implanted with a SILASTIC (Dow Corning, Midland, MI) brand catheter in the external jugular vein (15) during anesthesia with Ketalar (400 mg/kg body weight) and Rompun (40 mg/kg body weight). Two days later, between 0900 and 1100 h, they were given an iv injection of either LPS (25 µg/kg body weight; 055:B5; Sigma, St. Louis, MO; n = 27) or rat rIL-1β (2 µg/kg body weight; R&D Systems, Oxon, UK; n = 24) in 0.3 ml saline, or vehicle (n = 51). IL-1ra (16 mg/kg body weight; Kineret; Amgen, Breda, Holland) or saline was given iv simultaneously with the immune stimulus (either LPS or IL-1β; doses as above; n = 24 and 13, respectively). In animals destined for 3-h survival, an additional dose of IL-1ra was also given after 1.5 h.

Tissue processing
At 1, 3, 5, and 12 h after injection, animals were killed by iv injection of sodium pentobarbital (100 mg/kg; Apoteksbolaget, Uppsala, Sweden). Animals used for immunohistochemical and in situ hybridization histochemical staining were perfused transcardially with 150 ml of 0.9% saline, followed by 400–500 ml of ice-cold 4% paraformaldehyde in phosphate buffer [0.1 M (pH 7.4)]. The adrenal glands were postfixed in 4% paraformaldehyde in phosphate buffer for 3 h, followed by 0.1 M PBS with 25% sucrose overnight, and then cut at 20 µm (for in situ hybridization) or 30 µm (for immunohistochemistry) on a freezing microtome. The sections were collected in sterile bins containing cold cryoprotectant (0.1 M phosphate buffer, 30% ethylene glycol, 20% glycerol), and stored at –20 C. In animals used for real-time PCR analysis, the adrenals were quickly removed, cleared from surrounding adipose tissue, cut into smaller pieces, submerged in RNA stabilizing reagent (RNAlater; QIAGEN, West Sussex, UK), and stored at 4 C for up to 14 d until further processing.

Antibodies
Primary antibodies were: rabbit anti-Cox-1 (1:1000; Cayman Chemicals, Ann Arbor, MI); goat anti-Cox-2 (1:1000; Santa Cruz Biotechnologies, Santa Cruz, CA); rabbit antimicrosomal prostaglandin E synthase (mPGES-1; 1:6000; antiserum raised against a synthetic peptide corresponding to amino acids 59–74 of human mPGES-1 in which Cys 68 was replaced with Ser; kindly provided by Dr. P.-J. Jakobsson, Karolinska Institute, Stockholm, Sweden); goat anti-IL-1β (1:1000; R&D Systems); sheep anti-IL-1 type 1 receptor (IL-1R1) (1:2000; affinity purified antibody raised against the extracellular portion of the rat IL-1R1; kindly provided by Dr. Adrian Bristow, Potters Bar, UK); mouse antirat CD68 (1:500; labels lysosomal components of tissue macrophages, clone ED1; Serotec, Oxford, UK), mouse antirat RT1B [1:1000; labels major histocompatibility complex (MHC) class II (Ia) antigens, clone OX6; Serotec]. Secondary antibodies used for single-labeling peroxidase-based immunohistochemistry were: biotinylated horse antimouse (for ED1 and OX6), biotinylated rabbit antigoat (for Cox-2, IL-1β, and IL-1R1) and biotinylated goat antirabbit (for Cox-1 and mPGES-1) (Vector Laboratories, Peterborough, UK). Secondary antibodies used for immunofluorescence were: Alexa Flour 488 donkey antimouse (for OX6; Invitrogen), and horseradish peroxidase (HRP)-labeled donkey antirabbit (for mPGES-1; Amersham, Uppsala, Sweden), HRP-labeled chicken antigoat (for Cox-2, IL-1β, and IL-1R1; Santa Cruz), and HRP-labeled donkey antimouse (for ED1), the three latter being used with tyramide signal amplification (TSA) technology (TSA-Plus Fluorescence Palette System; PerkinElmer, Boston, MA) for the fluorescent detection. All secondary antibodies were diluted 1:1000.

Single-labeling immunohistochemistry
Sections were incubated in primary antibody overnight at room temperature, followed by secondary antibody and avidin-biotin complexes (Vectastain; Vector), each for 2 h at room temperature. Color was developed using 3,3'-diaminobenzidine-tetrahydrochloride (Sigma) containing 0.01% H2O2 in 0.1 M NaAc buffer (pH 6.0) for 5–8 min.

Dual-labeling immunohistochemistry
After incubation in primary antibodies, sections were incubated with secondary antibodies [HRP-labeled donkey antirabbit (for mPGES-1; Amersham), HRP-labeled chicken antigoat (for Cox-2, IL-1β, and IL-1R1; Santa Cruz), HRP-labeled donkey antimouse (for ED1), and Alexa Flour 488 donkey antimouse (for OX6; Invitrogen)] for 1 h at room temperature. For detection of the HRP-labeled secondary antibodies in combination with Alexa 488-labeled secondary antibodies, sections were incubated for 10 min in tetramethylrhodamine tyramide, diluted 1:50 in a reaction buffer, and mounted on slides using Vectashield mounting medium for fluorescence (Vector). For other dual-labeling experiments, in which antibodies against Cox-2, IL-1β, IL-1R1, mPGES-1, and ED1 were used in different combinations, a protocol for multicolor detection with TSA technology was developed. In brief, sections were incubated in HRP-labeled secondary antibodies targeting the first primary antibody for 1 h at room temperature, followed by incubation in either Cy3 or fluorescein tyramide for 10 min. After quenching of the peroxidase activity with 1% H2O2 in 100% methanol, sections were incubated for 1 h at RT in the HRP-labeled antibody targeting the second primary antibody, followed by 10 min incubation in Cy3 or fluorescein tyramide solution, depending on what fluorochrome that was used for the detection of the first primary antibody. Specificity of the labeling was checked by omission of the primary antibody, and when the tyramide multicolor detection protocol was used, also by developing only one of the two primary antibodies, to ensure that the remaining HRP activity from the secondary antibody was properly quenched before the second tyramide solution was applied.

In situ hybridization
In situ hybridization for the detection of prostaglandin E2 receptors of subtypes 1 and 3 (EP1 and EP3), and IL-1R1 was done as previously described (15). In brief, plasmids containing cloned cDNA fragments were linearized with BamHI (EP1; kindly provided by Dr. Anders Ericsson-Dahlstrand, AstraZeneca, Södertälje, Sweden), XbaI (EP3) (16), and EcoRI (IL-1R1) (17) and transcribed with T7 (EP1) or T3 (EP3 and IL-1R1) polymerase in presence of 33P-labeled deoxyuridine 5-triphosphates. The slides were developed 21 d after exposure.

Microscopy
Sections were analyzed in bright-field and dark-field illumination, using a Nikon Eclipse E800 microscope (Nikon, Badhoevedorp, The Netherlands). Micrographs were captured using a SPOT-2 digital camera (Diagnostic Instruments, Sterling Heights, MI), and images were imported into Adobe PhotoShop 7.0 (San Jose, CA) to adjust brightness and contrast. Quantification of dual-labeled cells was done in a Nikon Eclipse E600 microscope connected to a Nikon C1 confocal unit with argon 488 and HeNe 543 lasers. Using a x40 objective and a field zoom of 318.3 µm, three digital pictures were randomly captured from the outer part of the fasciculate layer and the border between the reticulate layer and the medulla, respectively, in three different animals, using an EZ-C1 digital camera and software version 3.1 for Nikon C1 confocal microscopy. For cell counts, the micrographs were imported into Adobe PhotoShop CS. Cells were defined as structures displaying a signal clearly distinguished from background while exhibiting a cell-like appearance (round, oval, dendritic, or spindle shaped).

RNA extraction and cDNA synthesis
RNA was extracted using RNeasy minikit (QIAGEN). The concentrations and purity of the RNA were measured using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The 260:280 ratio was consistently approximately 2.1, ensuring high-purity RNA. A sample of 1 µg of RNA was reversely transcribed using SuperScript III first-strand synthesis kit with oligo dT primers in a volume of 20 µl (Invitrogen). The efficiency of the reverse transcription reaction was evaluated by running five different reverse transcription reactions containing different amounts of RNA, which rendered a linear standard curve in real-time PCR using a β-actin assay (see below). The cDNA was aliquoted and stored at –80 C until used.

Real-time RT-PCR analysis
Real-time RT-PCR was performed as singleplex reactions in duplicates or triplicates on 10 ng of cDNA in a 96-well format on either the ABI Prism 7700 instrument using TaqMan universal PCR master mix (total reaction volume 25 µl) or the Fast 7500 real-time PCR system using the TaqMan Fast universal PCR master mix (total reaction volume 15 µl; Applied Biosystems; Foster City, CA) and with β-actin as endogenous control. The TaqMan gene expression assays were: β-actin (Rn00667869_m1), Cox-2 (Rn00568225_m1), mPGES-1 (Rn00572047_m1), IL-1β (Rn00580432_m1) and IL-1R1 (Rn00565482_m1). Gene expression was calculated using the {Delta}{Delta} threshold cycle (Ct) method.

Statistics
{Delta}Ct values obtained from real-time RT-PCR were compared between immune stimulated animals and their corresponding controls using Student’s t test. P < 0.05 was considered statistically significant. The SEM was obtained by first calculating the SD for the stimulated group (s1) and the control group (s2) and then applying these values in the following formula: [(sp^2(1/n1 + 1/n2)]^0.5, in which sp^2 = s1^2(n1 – 1) + s2^2(n2 – 1)/(n1 + n 2 – 2).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adrenal immune cells undergo profound changes in response to LPS
Because no earlier data are available on the adrenal immune cell population during inflammatory conditions, we first characterized the labeling pattern of immune cells in the adrenals after iv injection of LPS (25 µg/kg), compared with that in control animals. Sections were stained with a monoclonal antibody recognizing MHC class II (clone OX6), a membrane antigen present on professional antigen-presenting cells (18), and with a monoclonal antibody recognizing the rat homolog to human CD68 that predominantly is expressed on lysosomal membranes of myeloid cells, i.e. monocytes and most tissue macrophages, as well as dendritic cells (clone ED1) (19).

The OX6 labeling showed a dense staining pattern in control animals. In particular, there was a heavy labeling in the zona reticularis, radiating into the zona fasciculata, but labeled cells were also seen in the zona glomerulosa and, sparsely, in the medulla (Fig. 1AGo1). The vast majority of the OX6-labeled cells were dendritic in their appearance, with slender, ramifying protrusions extending from a small cell body (Fig. 1AGo2–3). In addition, fusiform cells oriented vertically along the steroid cell column were present preferentially in the outer layer of the zona fasciculata (Fig. 1AGo1). Immune challenge with LPS resulted in no apparent changes in the OX6 labeling at 1 h after injection (Fig. 1BGo); however, at 3 h OX6-labeled cells with larger cell bodies and shorter and less numerous protrusions started to appear in the zona reticularis and zona fasciculata (Fig. 1CGo), and, in addition, OX6 cell density in the zona reticularis was markedly reduced (Fig. 1CGo1). At 5 h, these changes were further pronounced; the OX6 population in the zona fasciculata at this time point mainly consisted of cells with large cell bodies and few or no protrusions. In addition, the cell bodies of the cells in the zona reticularis appeared somewhat thickened and with fewer and coarser protrusions (Fig. 1DGo). At 12 h after LPS injection, the large/medium-sized cells were still numerous, but most of these now showed abundant and slender protrusions. At all time points, OX6-labeled cells in the zona glomerulosa had the same characteristics as those in the zona fasciculata and reticularis, although the larger cells were few. No consistent change in OX6 expression was detectable in the medulla (data not shown).


Figure 1
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  		FIG. 1. LPS-induces depletion of OX6-labeled dendritic cells and the recruitment of immature cells in the adrenal cortex. LPS (25 µg/kg body weight) or saline was injected iv, the rats were killed after 1–12 h, and the adrenals stained for OX6 immunoreactivity. A1-E1, Overviews. A2-E2 and A3-E3, Details from the fasciculate (ZF) and reticulate (ZR) zones. There was a depletion of the dense labeling in ZR and a gradual replacement, preferentially in the ZF, of the dendritic-type cells (seen in saline injected animals; A) by round medium-sized to large cells with no or few protrusions (arrows in C2 and D2), but at 12 h (E), the large cells displayed abundant thin protrusions (arrowheads). Scale bar, 100 µm (A1-E1) and 20 µm (A2,3-E2,3). ZG, Zona glomerulosa; ZM, zona medullaris.

 
Cells labeled with the ED1 antibody were present in both the adrenal medulla and cortex in control animals (Fig. 2AGo1). They were less abundant than the OX6-labeled cells and rather evenly distributed throughout the cortex, although slightly more numerous in the innermost layer. Most cells in the zona fasciculata and zona reticularis were dendritic in their appearance, and although possessing a more granular staining pattern (consistent with the antigen being present on lysosomal membranes), they closely resembled those labeled with OX6 in control animals (Fig. 2AGo2). Similar to the OX6 labeling, fusiform cells were also seen and were present in the outer part of the fasciculate layer as well as in the reticulate zone (Fig. 2AGo3). Many of the fusiform cells were associated with blood vessels, which was particularly evident in the reticular layer (Fig. 2DGo3). After stimulation with LPS, significant changes also occurred in the ED1 staining pattern, with the appearance an array of different cell shapes and sizes in a temporal sequence. Thus, at 1 h after immune challenge, small round and homogenously stained cells started to appear in the fasciculate zone (Fig. 2BGo1–2). At 3 h, such cells were seen also in the reticular layer, but, in addition, larger but more weakly stained cells appeared in the fasciculate zone (Fig. 2CGo) and was a dominant cell type in this zone at 5 h after injection. At the latter time point, the cells of the zona reticularis appeared thickened with coarser protrusions (Fig. 2DGo), similar to what was seen for the OX6- and ED1-positive cells At 12 h, the large/medium-sized cells were again sparse, with preferentially small and round cells being seen in the zona fasciculata and almost only dendritic cells in the zona reticularis (Fig. 2EGo3). At all time points, the zona glomerulosa contained small round and homogenously stained cells, in addition to dendritic cells (data not shown).


Figure 2
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  		FIG. 2. ED1-immunoreactive cells display a series of morphological changes after LPS. Rats were injected with LPS (25 µg/kg body weight) or vehicle, killed after 1–12 h, and the adrenals stained with ED1 antibody. Vehicle-injected rats (A) displayed small dendritic-type cells or fusiform cells with thin protrusions. At 1 h after LPS (B), small, round cells appeared (small arrow), preferentially in the fasciculate zone (ZF), and at later time points medium-sized to large cells with no or few protrusion were present (C and D; large arrows). In the reticulate zone (ZR), the cells became somewhat enlarged (C3 and D3) and were associated with blood vessels (v). At 12 h small to medium-sized cells were again seen. Scale bar, 100 µm (A1-E1) and 20 µm (A2,3-E2,3). ZG, Zona glomerulosa; ZM, zona medullaris.

 
LPS-induced changes in immune cells are associated with induced expression of proinflammatory mediators
Because the findings showed that the immune cell population in the adrenals undergoes profound changes both in morphology and distribution in response to circulating LPS and because immune cells are a common source of proinflammatory mediators such as cytokines and prostaglandins, we next examined the expression of these mediators and their receptors in the adrenals during systemic inflammation, by using immunohistochemistry and/or in situ hybridization. The proteins and/or transcripts studied included the cytokine IL-1β and its receptor type 1 (IL-1R1), the constitutive and inducible cyclooxygenases Cox-1 and Cox-2, the terminal PGE2 synthesizing enzyme mPGES-1, and the PGE2 receptors EP1 and EP3, i.e. the subtypes that have been implicated in the hypothalamic-pituitary adrenal axis response to immune stress (20).

Constitutive expression.
Animals injected with saline and killed 3–5 h later (as a control for any handling effects) showed weak expression of IL-1β in the medulla that involved both chromaffin-like cells and fusiform cells associated with blood vessels, but there was no IL-1β labeling in the cortex (Fig. 3AGo1–2). Similarly, IL-1R1 was weakly expressed in the medulla but absent in the cortex (data not shown). Of the prostaglandin-synthesizing enzymes examined, Cox-1 and mPGES-1 were constitutively expressed in the medulla as well as in the cortex, with labeled dendritic and fusiform cells seen in the reticular layer (Fig. 4Go, A1–2 and B1–2), whereas Cox-2 was constitutively expressed in chromaffin cells of the medulla but not seen in the adrenal cortex (Fig. 5AGo1–2). Finally, EP1 and EP3 receptors, demonstrated by in situ hybridization, were densely expressed in the medulla, whereas weak and inconsistent signal was seen in the zona reticularis and zona fasciculata of the adrenal cortex (Fig. 4Go, E and F).


Figure 3
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  		FIG. 3. IL-1β immunoreactivity is induced by LPS. LPS (25 µg/kg body weight) or saline was injected iv, and the rats were killed after 1–12 h. There was a prominent induction at 1 h (B1) after LPS injection occurring in dendritic type cells (B2–3). Small round cells appeared at 3 h (arrow in C2) and medium-sized to large round cells at 5 h (arrow in D2). Scale bar, 100 µm (A1-E1) and 20 µm (A2,3-E2,3). ZF, Zona fasciculata; ZG, zona glomerulosa; ZM, zona medullaris; ZR, zona reticularis.

 

Figure 4
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  		FIG. 4. Cox-1, mPGES-1, and EP receptors are constitutively expressed, but mPGES-1 is also induced by immune challenge. LPS (25 µg/kg body weight), rIL-1β (2 µg/kg body weight), or vehicle was injected iv, and the rats were killed after 1–12 h. Cox-1 (A) and mPGES-1 (B) were constitutively expressed in small cells in the reticulate zone (ZR), whereas the induced expression of mPGES-1 (C and D) occurred among large round cells (arrows) in the fasciculate zone (ZF). Note the large difference in induction between LPS and rIL-1β (C and D). E and F, Dark-field micrographs showing the expression of EP1 (E) and EP3 (F) receptors in the adrenal medulla and inner cortical layers, as demonstrated by radiolabeled in situ hybridization Scale bar, 100 µm (A1-D1), 20 µm (A2-D2), and 250 µm (E–F). ZG, Zona glomerulosa; ZM, zona medullaris.

 

Figure 5
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  		FIG. 5. Cox-2 immunoreactivity is induced in the adrenal cortex by LPS. In saline-injected rats, Cox-2 immunoreactivity (Cox-2 IR) was seen only in the medulla (A), whereas iv injection of 25 µg/kg body weight LPS (B) resulted in the appearance of preferentially medium-sized to large Cox-2 IR cells throughout the cortex. There was a significant difference in induction in the outer part of the zona fasciculata (ZF) between animals injected with LPS (25 µg/kg body weight) (B) and those injected with rIL-1β (2 µg/kg body weight) (C). Scale bar, 100 µm (A1-C1) and 20 µm (A2-C3). ZG, Zona glomerulosa; ZM, zona medullaris; ZR, zona reticularis.

 
Immune challenge with LPS.
Intravenous injection of LPS (25 µg/kg) resulted in induced expression of IL-1β, IL-1R1, Cox-2, and mPGES-1 in the adrenal cortex (Figs. 3–7GoGoGoGoGo) along a time course that differed between these substances. Hence, prominent induced labeling was seen for IL-1β already at 1 h, whereas IL-1R1, Cox-2, and mPGES-1 started to show strongly induced labeling at somewhat later time points (3 h for the Cox-2 protein, 3 and 5 h for the IL-1R1 transcript and protein, respectively, and 5 h for the mPGES-1 protein).


Figure 6
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  		FIG. 6. Cells expressing IL-1β/IL-1R1 are immune cells. Confocal micrographs showing dual labeling of IL-1β/IL-1R1 and the immune cell markers ED1 and OX6 5 h after iv injection of LPS (25 µg/kg body weight). Arrows point at dual-labeled and arrowheads indicate single-labeled cells. Scale bar, 25 µm. ZF, Zona fasciculata; ZR, zona reticularis.

 

Figure 7
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  		FIG. 7. Cells expressing mPGES-1 coexpress Cox-2, ED1, OX6, IL-1β, and IL-1R1. Confocal micrograph showing dual-labeling for mPGES-1 and Cox-2, ED1, OX6, IL-1, and IL-1R1 5 h after iv injection of LPS (25 µg/kg body weight). Arrows point at dual-labeled and arrowheads indicate single-labeled cells. In E2 note the membrane localization of the MHC type II antigen (OX6 labeling) in dendritic like cells (dual arrows), and the intracellular location in the immature-type cells (dual arrowheads). Scale bar, 25 µm. ZF, Zona fasciculata; ZR, zona reticularis.

 
The appearance of the cells labeled for IL-1β along the time course studied (1, 3, 5, and 12 h; Fig. 3Go, B–E) followed that demonstrated for the cells labeled with OX6 and ED1 (see above). Thus, at 1 h the IL-1β expressing cells were dendritic or fusiform (Fig. 3BGo); at 3 h small, round IL-1β-labeled cells appeared (Fig. 3CGo) and at 5 h large, round cells, both types with few or no protrusions (Fig. 3DGo). At this time point, the IL-1β-labeled dendritic-like cells in the inner part of the reticular layer displayed a thickened soma and coarser protrusions than seen at the earlier time points (Fig. 3DGo3). At the last time point examined, 12 h, the density of the IL-1β-labeled cells was considerable lower, but both dendritic-like cells and large round cells were still seen (Fig. 3EGo1–3).

The cells expressing IL-1R1 (Figs. 6Go, E1-H1, and 7Go, I2-J2) and Cox-2 (Fig. 5BGo) and those that appeared at 3–5 h after injections consisted mainly of the cells of the small and large round cell type, with the former being most abundant in the zona reticularis and inner part of the zona fasciculata, and the latter, which were preferentially seen at 5 h, in the outer part of the zona fasciculata. In addition, IL-1R1 and Cox-2 labeling was also displayed by the thickened dendritic-type cells in the inner part of the reticular zone. In contrast, induced mPGES-1 expression occurred almost exclusively in large round cells in the fasciculate zone (Fig. 4CGo), in addition to being displayed by the thickened fusiform cells in the inner part of the reticular zone (a location that displayed constitutive mPGES-1 expression; see above).

LPS-induced expression of IL-1β, IL-1R1, Cox-2, and mPGES-1 occurs in immune cells in a cell-specific pattern
Because these data clearly suggested that the cells displaying immune-induced expression of IL-1β, IL-1R1, Cox-2, and mPGES-1 were immune cells, we performed dual labeling immunohistochemical staining to determine whether these cells also expressed ED1 and OX6 and whether the same cells expressed all proteins. The findings, obtained by confocal microscopy, are illustrated in Figs. 6Go and 7Go, and quantitative data are given in Table 1Go. The results confirmed that the expression of IL-1β, IL-1R1, and mPGES-1 occurred in ED1- (<90%) and OX6 (65–84%)-positive cells (Figs. 6Go and 7Go, C–F), indicating that it exclusively occurred in immune cells. Almost all mPGES-1-expressing cells coexpressed Cox-2, IL-1β, and IL-1R1 (Fig. 7Go, A, B, and G–J), although the latter populations were larger and as evident from their expression pattern (seen above) also contained cells that were negative for mPGES-1. However, as shown in Table 1Go, there was a particularly high degree of coexpression between mPGES-1 and Cox-2, IL-1β, and IL-1R1 in the inner part of the reticular layer, suggesting that the immune activated cells at this location or at least a considerable proportion of them, similar to the large round cells in the fasciculate zone coexpress all these proteins.


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  		TABLE 1. Dual labeling for proinflammatory mediators and immune cell markers

 
Additionally, confocal microscopy showed that OX6 labeling was localized to the outer cell membrane in the dendritic cells, whereas it had an intracellular localization in the larger cells that displayed no or few protrusions (Fig. 7EGo2), being consistent with a different localization of the MHC class II antigen between immune cells at different maturation stages (see Discussion).

LPS induces expression of IL-1β, Cox-2, IL-1R1, and mPGES-1 mRNA in a temporal sequence
To obtain a quantitative estimate of the immune-induced induction of IL-1β, IL-1R1, Cox-2, and mPGES-1 in the adrenals, we performed real-time RT-PCR analysis of the mRNA expression at 1, 3, 5, and 12 h after immune stimulation with LPS (25 µg/kg iv, i.e. same dose as used for the immuno- and hybridization histochemical analyses). The results, shown in Fig. 8Go, A–D, demonstrated a strong and rapid induction (180-fold) of IL-1β (Fig. 8AGo) after LPS, consistent with the immunohistochemical findings. There was also a strong and rapid induction of Cox-2 mRNA (190-fold; Fig. 8CGo), whereas the induction of IL-1R1 (Fig. 8BGo) and mPGES-1 (Fig. 8DGo), which in consistence with morphological data appeared at 3–5 h after injection, was much weaker (~4-fold for IL-1R1 and 6- to 7-fold for mPGES-1). The small increase in IL-1R1 and mPGES-1, compared with IL-1β and Cox-2, probably reflects a more prominent constitutive expression of IL-1R1 and, in particular, mPGES-1 (as seen from a higher basal level of these transcripts in control animals in real-time RT-PCR; data not shown) and, for the latter, also the smaller size of the population in which induced expression occurred. Notably, at 12 h the level of mRNA expression was not significantly different from that of control animals injected with saline, except for a small (~3-fold) increase of IL-1β, which is consistent with the close to normalized histological picture seen at this time point.


Figure 8
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  		FIG. 8. IL-1β and Cox-2 are strongly and rapidly induced by both LPS and rIL-1β but differently regulated by IL-1ra. LPS (25 µg/kg body weight) or rIL-1β (2 µg/kg body weight) was injected iv, and adrenal expression of IL-1β (A), IL-1R1 (B), Cox-2 (C), and mPGES-1 (D) was examined using real-time RT-PCR after 1, 3, 5, and 12 h. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. saline injected controls. E, IL-1ra (16 mg/kg body weight) was injected iv at the same time point as LPS or rIL-1β or at two time points (0 and 1.5 h), and IL-1β and Cox-2 expression in the adrenal was examined after 1 and 3 h, respectively. F, Cox-2 expression in the hypothalamus in the same experiment as in E. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 between animals given LPS/rIL-1β and IL-1ra and animals injected with LPS/rIL-1β and vehicle (n = 3–6).

 
The expression of IL-1β and Cox-2 is intrinsically regulated
Injection of rat rIL-1β (2 µg/kg) resulted in an induced expression of IL-1β, IL-1R1, Cox-2, and mPGES-1 mRNA with a similar time course as that elicited by LPS (Fig. 8Go, A–D). However, whereas IL-1β thus induced its own expression, this was less prominent than that seen after LPS with the peak value (at 1 h), at least in the present dose, which elicits a systemic response (21, 22), corresponding to a 19-fold increased in IL-1β mRNA, i.e. approximately a tenth of that elicited by LPS (Fig. 8AGo). The induced expression of Cox-2 after rIL-1β was also weaker (51-fold increase after rIL-1β, compared with 190-fold after LPS; Fig. 8CGo), whereas the differences for IL-1R1 was in the 1- to 2-fold range (Fig. 8BGo) and that for mPGES-1 in the 2- to 4-fold range (Fig. 8DGo). The differences in magnitude between LPS and rIL-1β in the induction of Cox-2 and mPGES-1 were corroborated in immunohistochemical staining, which showed that rIL-1β largely failed to induce expression of the prostaglandin synthesizing enzymes in the fasciculate zone (cf. Figs. 4Go, C and D, and 5Go, B and C).

To further examine the role of the endogenously produced IL-1β, we next injected IL-1ra [16 mg/kg iv, a dose that has been shown to attenuate LPS-induced fever (23)]. The injection was given together with LPS or rIL-1β, and the animals were killed 1 or 3 h later; in the latter case, a second dose of IL-1ra was given after 1.5 h. This resulted in an attenuation of the LPS-induced IL-1β mRNA production at 3 h (Fig. 8EGo, right columns) but not at 1 h (data not shown); in contrast, there was a significant increase in the induced Cox-2 expression at 3 h (Fig. 8EGo, middle columns), whereas the mPGES-1 induction was not changed (not shown). As a control for the efficiency of the IL-1ra, we also injected the same dose in conjunction with iv injection of rIL-1β, which resulted in a complete extinction of the induced Cox-2 mRNA expression, in both the adrenal (Fig. 8EGo, left columns) and the hypothalamus (Fig. 8FGo, left columns). Furthermore, when combined with iv injection of LPS (25 µg/kg), the IL-1ra also attenuated the Cox-2 induction in the hypothalamus (Fig. 8FGo, right columns), being consistent with its ability to attenuate the febrile response.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the interpretation of the above data, two prerequisites that relate to the experimental design should be pointed out. First, the effects of injection of LPS or rIL-1β were determined by comparison with animals injected with vehicle (saline). Whereas such injection controls for the effects of vehicle load and animal handling, the possibility remains that it by itself may produce a minor increase in inflammatory factors above background. Accordingly, the constitutive expression pattern reported in the present study includes any such influences. Second, our data on the effects of LPS are based on iv injection of 25 µg/kg. Whereas this dose of LPS is well within the range of doses that give rise to a number of acute phase responses, including a robust stress hormone release (24), the mechanisms of immune activation may vary as a function of infectious load (25). Accordingly, the data reported here reflect the conditions during moderate endotoxemia; during more severe infections, alternative inflammatory mechanisms may be initiated.

Given these qualifications, the present work demonstrates that the immune cells in the cortex of the adrenal glands display profound morphological changes in response to acute immune challenge with LPS and that these morphological changes are associated with the induced production of IL-1β, its receptor IL-1R1, and the prostaglandin synthesizing enzymes Cox-2 and mPGES-1. We also show that IL-1β induces its own expression and regulates, in a complex pattern, the expression of Cox-2. In addition, we demonstrate a constitutive expression of mPGES-1, associated with Cox-1 in immune cells localized at the border to the medulla, and we show that PGE2 receptors are present in the adrenal cortex and medulla. Taken together, these findings, schematically illustrated in Fig. 9Go, demonstrate a mechanism by which systemic inflammatory agents activate an intrinsically regulated local signaling circuit that may influence the adrenals’ response to immune stress.


Figure 9
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  		FIG. 9. Schematic drawing summarizing the findings of the present study. The adrenals contain large populations of mature immune cells, labeled with ED1 and OX6 antibodies, with the morphological characteristics of dendritic cells and, in the innermost part of the reticular zone (ZR), resident macrophages. The latter display a constitutive expression of Cox-1 and mPGES-1, and Cox-1 is also expressed by dendritic cells in the inner part of the zona fasciculata (ZF). The changes in the immune cell population after systemic immune challenge with LPS includes the depletion of dendritic cells from ZR and the recruitment of immature cells, characterized by their rounded cell bodies and the absence of visible protrusions, into the outer cortical layers, as well as reactive changes, such as swelling of the cell body and processes, of the macrophage-like population in the innermost part of the cortex. These changes are associated with strong and rapid induction of IL-1β synthesis, followed by expression of IL-1R1, Cox-2, and, in a restricted population of immature cells, mPGES-1. As the cells mature, they again assume dendritic cell characteristics, and they cease to produce IL-1β, its receptor, and the prostaglandin synthesizing enzymes. Note expression of EP receptors in the cortex and medulla consistent with a role for PGE2 in the release of corticosteroids as well as catecholamines and medullary neuropeptides.

 
The rat adrenals have been shown to contain resident dendritic cells and macrophages (26, 27). However, the present study is the first to examine these cells in the adrenal during inflammatory conditions. Dendritic cells, which are of the myeloid lineage, are present in nonlymphoid tissues of the body and constitute an important link between the innate and adaptive branches of the immune system. The precursors of dendritic cells are derived from the bone marrow and enter the tissues via the blood stream to become resident cells (28). They are highly sensitive to LPS; systemic administration of LPS has been shown to drain organs such as kidneys and heart of resident cells (29), whereas at the same time immature cells are recruited. In contrast to the resident cells, the immature cells have few processes; they possess the ability to process antigens, and they display high levels of CD68 (ED1) and intracellular MHC class II (OX6). After immune challenge they undergo several steps of maturation that include loss of antigen processing capacity, redirection of MHC class II molecules from the lysosomes to the cell membrane, rearrangement of the cytoskeleton resulting in slender processes, and the ability to migrate from the tissue to the regional lymph nodes in which they present antigens to lymphocytes (28, 30). The present observations on the immune activated adrenals are consistent with such a process. Thus, at 1 h after iv injection of LPS, small round cells resembling dendritic precursor cells were seen in the ED1-stained material, and at 3–5 h, cells of an immature cell type, encompassing medium-sized to large cells with few or no protrusions and that displayed the MHC class II antigen (OX6 labeling) at an intracellular location, became abundant. Notably, these cells were seen preferentially in the outer cortical layers, i.e. the part of the cortex that is first reached by the arterial blood supply. At the same time period, cell density in the reticular layer (as seen by OX6 labeling) became greatly reduced, consistent with a depletion of the dendritic cells from this location to the regional lymph nodes. At 12 h after iv injection of LPS, the medium-sized to large cells displayed several thin protrusions, consistent with a maturation process in these cells (28).

In addition to the depletion of cells in the zona reticularis, the cells in this part of the cortex displayed more subtle changes in response to iv injection of LPS. Hence, the remaining cells, particularly those located in the inner part of the zona reticularis, became somewhat thickened with coarser protrusions after immune challenge, and many of these cells appeared to be associated with blood vessels. In control animals, cells at the same location displayed a constitutive expression of mPGES-1. Their perivascular location and morphological characteristics suggest that they are macrophages, being in line with the demonstration of the macrophage marker ED2 in the inner part of the reticular zone (26). In contrast to what has been suggested from previous work, performed on human adrenals (31), the present data do not provide any evidence for a significant constitutive expression of IL-1β in the adrenal cortex and strongly indicate that it is induced exclusively in immune cells because virtually all IL-1β-expressing cells were also labeled with OX6 or ED1. The response was very rapid, being most pronounced at 1 h after iv injection of LPS. This implies that it is a direct effect of LPS on the immune cells, via Toll-like receptor 4, which is known to be expressed in the adrenal (10, 32, 33). However, injection of rIL-1β also resulted in induced expression of IL-1β, and furthermore, we demonstrated that IL-1R1 was present on the same cells as those that produced IL-1β and that it was up-regulated in response to LPS. This implies an autocrine mechanism by which immune-induced IL-1β may enhance its own expression, similar to what has been seen in fibroblasts (34). In support of this idea, we found that the administration of IL-1ra attenuated the IL-1β expression after LPS at the time point when the expression of IL-1R1 was significantly induced (3 h), whereas it had no effect at 1 h. IL-1ra is present in the adrenal and has been shown to be up-regulated upon immune challenge with LPS (35), suggesting the presence of autoregulatory mechanisms for the control of the IL-1β response.

Similar to what was found for IL-1β, Cox-2 expression was also strongly and rapidly induced after LPS administration. Whereas we did not perform dual labeling for Cox-2 and immune cell markers, the morphological characteristics of the Cox-2-expressing cells indicated that they were immune cells. Furthermore, almost all cells that expressed mPGES-1 also expressed Cox-2 as well as immune markers and IL-1β, demonstrating the immune cell identity of that subpopulation of Cox-2-expressing cells. Accordingly, it is likely that the Cox-2 expression, similar to the IL-1β expression, occurred in immune cells and that it largely occurred in cells that coexpressed IL-1β. Also, administration of rIL-1β resulted in a strong and rapid induction of Cox-2 (50-fold increase after rIL-1β vs. 190-fold after LPS), suggesting that at least part of the Cox-2 expression after LPS may have been induced indirectly, via locally produced IL-1β. A similar Cox-2 induction in response to IL-1β has been demonstrated in other tissues, such as the endothelial cells of the blood-brain barrier (36). However, it is not clear whether the Cox-2 expression that was induced by rIL-1β is a physiological response because administration of IL-1ra increased the LPS induced Cox-2 expression and decreased the IL-1β expression, pointing to the possibility that endogenous IL-1β may inhibit Cox-2 synthesis. An enhanced PGE2 production by IL-1ra has been shown for placental membranes and decidual cells (37, 38, 39), and whereas those observations suggest that IL-1ra may act in synergy with IL-1β (37, 38, 39), it is also possible that IL-1β triggers negative feedback circuits that control the Cox-2 response, such as those involving antiinflammatory cytokines, e.g. IL-10 (40, 41).

In contrast to Cox-2, the expression of mPGES-1, the terminal PGE2-synthesizing enzyme, seemed to be induced exclusively in immature cells located in the fasciculate zone, as determined by their morphology and intracellular location of the MHC class II antigen (OX6 labeling). As mentioned, almost all mPGES-1-expressing cells coexpressed IL-1β, IL-1R1, and Cox-2, and at least in the fasciculate zone, these events appeared to occur in a temporal sequence after the transformation of dendritic precursor cells into large immature cells. Real-time RT-PCR analysis showed that mPGES-1 mRNA was induced at 3–5 h after LPS, and induced mPGES-1 protein expression, which occurred in the fasciculate layer, was most evident at 5 h time point. Accordingly, mPGES-1 induction occurred later than the Cox-2 induction, consistent with its downstream location in the prostaglandin synthesizing cascade.

Taken together, these data indicate that immune challenge with LPS elicits a series of events in the adrenal glands that results in prostaglandin synthesis, including the synthesis of PGE2, and that may influence stress hormone release. Whereas the bulk of in vivo experiments suggests that the effect of immune challenge on the adrenals is exerted at the level of the hypothalamus (2, 42), in situ perfusion studies show that both LPS and IL-1β enhance corticosterone release by acting directly on the adrenal gland (4, 5). This effect is likely to be exerted through the action of IL-1β because it can be blocked by an IL-1ra (4, 5). A direct action of proinflammatory agents on the adrenals is further supported by the finding that hypophysectomized rats, despite the absence of circulating ACTH, still respond with corticosterone release after systemic injections of IL-1β or LPS (6, 43). There is also substantial evidence from in vitro experiments on primary cell cultures that IL-1β exerts a stimulatory effect on corticosterone release (6, 7, 8, 9). However, it has been suggested that the dose of IL-1β needed to elicit corticosterone release in vitro by far exceeds the levels of IL-1β in plasma that occur during endotoxemia (43). Whereas this suggests that circulating IL-1β may not be important for initiating corticosterone release in the intact organism, it does not exclude that IL-1β produced in the adrenals plays a role, particularly considering that an autocrine/paracrine mechanism most likely involves a high local concentration of the intra-adrenally produced IL-1β.

Our finding that IL-1R1 seems to be almost exclusively expressed on immune cells and hence not on steroid producing cells, suggests that any IL-1β derived effect on hormone release during immune challenge should be exerted indirectly via other mediators, derived from either the immune cells or cells of the medulla. Hence, whereas isolated cortical steroid-producing cells from the fasciculate and reticulate zones were found to display signs of hypertrophy and secretory activity when the rat previously had been exposed to high levels of circulating IL-1β, these cells did not respond to IL-1β stimulation when dispersed in vitro (44), indicating that the effect of IL-1β is exerted via intraadrenal paracrine mechanisms. Several lines of evidence suggest that such mechanisms involve prostaglandins, which has long been known to stimulate corticosteroidogenesis (4, 7, 45). Thus, treatment of bovine primary cell cultures with IL-1β results in the production of PGE2 (7), and prostaglandin synthesis inhibitors have been shown to attenuate the IL-1β-induced release of corticosterone both in vitro and during in situ perfusion (4, 7, 8, 10). Taken together with the present data, these observations suggest that corticosterone release may be regulated locally in the adrenals through IL-1β-induced synthesis of prostaglandins, similar to what has been demonstrated for the PGE2 production in blood-brain barrier (36). In particular, it has been suggested that PGE2 mediates the corticosterone release induced by ACTH (46, 47), pointing to how central and local mechanisms may interact in the stress hormone response to inflammatory challenge. It is well established from in vivo studies that cyclooxygenase inhibitors attenuate the immune-induced corticosterone response (48, 49, 50, 51), but it remains to be clarified whether this effect is due to central or peripheral prostaglandin inhibition. In either case, there is evidence that PGE2 is involved, shown by an attenuated corticosterone response to acute immune challenge in animals with a deletion of the mPGES-1 gene (Elander, L., L. Engström, J. Ruud, L. Mackerlova, C. Nilsberth, and A. Blomqvist, manuscript in preparation).

In addition to the induced expression of prostaglandin synthesizing enzymes, we also demonstrated constitutive expression of mPGES-1, associated with Cox-1, in cells localized to the innermost part of the adrenal cortex. Immune cells in the same location that were positive for mPGES-1 showed reactive changes after LPS administration, with induced expression of Cox-2, as well as IL-1β and IL-1R1, indicating that these cells produce PGE2 both during constitutive conditions and as a response to immune challenge. Their juxtamedullary position and frequent association with blood vessels suggest that the PGE2 released by these cells may influence medullary cells, being consistent with the strong EP1 and EP3 receptor expression seen in the adrenal medulla. These observations are in line with the extensive functional and anatomical connections that have been demonstrated between the cortex and the medulla (52, 53, 54, 55) and that have been shown to be important for the IL-1β-induced stress hormone response (6, 44) and hence indicate a potential role for PGE2 in the release of catecholamines (56) and costored neuropeptides such as ACTH, CRH, and enkephalins (57) that in turn may influence corticosterone release (58, 59, 60).

In conclusion, we here show that systemic inflammation induces the expression of inflammatory messengers within the adrenal gland. This represents a mechanism that in addition to the HPA axis regulates the adrenal response to immune challenge and that may have implication for the release of stress hormone during such conditions. These observations may help explain the findings of dissociation between plasma levels of pituitary released ACTH and adrenally derived corticosteroids during late phases of sepsis and other chronic immune perturbations (11, 12).


   Footnotes
 
This work was supported by Grant 7879 from the Swedish Research Council-Medicine, Grant 4095 from the Swedish Cancer Foundation, the County Council of Östergötland, and Konung Gustaf V:s 80-årsfond.

Disclosure Statement: L.E., K.R., A.A., A.F., L.M., and J.P.K. have nothing to declare. D.E. and A.B. are inventors on international patent application PCT/EP2004/009734. Author contributions: L.E, D.E., and A.B. designed research; L.E. collected tissue and performed real-time RT-PCR and single- and dual-labeling immunohistochemistry; K.R. and A.A. contributed with single-labeling immunohistochemistry; A.F. and D.E. performed in situ hybridization; L.M. performed surgery and collected tissue; J.P.K. characterized the IL-1R1 antibody; L.E. and A.B. analyzed data; and L.E. and A.B. wrote the paper.

First Published Online January 3, 2008

See editorial p. 1433.

Abbreviations: Cox, Cyclooxygenase; HPA, hypothalamic-pituitary-adrenal; HRP, horseradish peroxidase; IL-1ra, IL-1 receptor antagonist; IL-1R1, IL-1 type 1 receptor; LPS, lipopolysaccharide; MHC, major histocompatibility complex; mPGES-1, microsomal prostaglandin E synthase-1; PGE2, prostaglandin E2; TSA, tyramide signal amplification.

Received October 23, 2007.

Accepted for publication December 27, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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