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Molecular Cardioprotection and Inflammation Group (R.B., O.B., K.Z.), Department of Anaesthesia and Department of Endocrinology, Diabetes, Rheumatology (M.S.), University Hospital Dusseldorf, 40225 Dusseldorf, Germany; Department of Anaesthesia (N.T., A.K., P.A.Z., K.Z.), Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom; Department of Anaesthesia (G.B., P.K.), University Hospital Bonn, 53105 Bonn, Germany; Department of Medicine (W.K., S.R.B.), University of Dresden, 01307 Dresden, Germany; and Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (S.L.L.), Bristol BS1 3NY, United Kingdom
Address all correspondence and requests for reprints to: Professor Kai Zacharowski, M.D., Ph.D., Molecular Cardioprotection and Inflammation Group, Department of Anesthesia, Bristol Heart Institute, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom. E-mail: kai.zacharowski{at}bristol.ac.uk.
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Abstract |
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Objective: We investigated whether TLR-9 is expressed in human and murine adrenal glands and whether its activation is associated with an adrenal response.
Design: Human fetal and adult adrenal glands; wild-type, C57BL/6 and TLR-9 deficient (TLR-9–/–) mice; and in vitro cell line models were used in the study.
Setting: The study took place at a university hospital.
Results: TLR-9 is expressed in human and murine adrenal glands, as well as in in vitro cell lines (Y-1 and NCI-H295R cells). CpG-oligodeoxynucleotide challenge caused a 3-fold increase in plasma levels of corticosterone in wild-type mice. This effect was not observed in TLR-9–/– mice. Furthermore, CpG-oligodeoxynucleotide challenge resulted in a strong release of several inflammatory cytokines, such as TNF-
, and IL-1ß, -6, -10, and -12 in vivo as well as in vitro. Again, this effect was not present in TLR-9–/– mice.
Conclusions: TLR-9 is present in both murine and human adrenal glands. TLR-9 stimulation led to a corticosterone and inflammatory cytokine response. TLR-9 may play a role in the regulation of the hypothalamic-pituitary-adrenal axis during conditions in which bacterial DNA is present.
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Introduction |
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, and TNF-, which eventually lead to the synthesis and release of glucocorticoids by activating the HPA axis (6, 7). The increased glucocorticoid levels, in turn, feed back and suppress the excessive immune response. During inflammation, these cytokines are capable of maintaining high glucocorticoid output, suggesting a shift from neuroendocrine to immune-endocrine regulation of the adrenal (8). Therefore, a coordinated response of the adrenal and immune system is of vital importance for survival during severe inflammation (9, 10). Bacterial DNA containing cytosine-phosphate-guanine (CpG) motifs can initiate an innate immune response via Toll-like receptor 9 (TLR-9), potentially leading to septic shock (11, 12). Bacterial CpG motifs are unmethylated CpG dinucleotides and are predominantly prevalent in bacterial DNA, but not in mammalian DNA (13). The effects of bacterial DNA can be mimicked by synthetic oligonucleotides via the same receptor system (14, 15).
The identification of TLRs has been a major advance in the understanding of the pathogenesis of septic shock, and a role for TLR-2 and 4 in the immune-adrenal stress response has been shown (16, 17).
However, to our knowledge, nothing is known about the role of TLR-9 in the adrenal gland, but constitutive expression of mRNA has been detected in human adrenal gland tissue (18). Therefore, we performed an analysis of adrenal function in relation to TLR-9.
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Materials and Methods |
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TLR-9 deficient (TLR-9–/–) mice, backcrossed onto a C57BL/6 background, were kindly provided by S. Akira (Osaka University, Osaka, Japan) (15). Animals were handled according to the principles of laboratory animal care (National Institutes of Health publication no. 86-23, revised 1985). Experimental procedures were approved by the German government ethical and research boards. C57BL/6 [wild-type (WT)] and TLR-9–/– mice were kept at 24 C, 55% humidity, 12-h d/night rhythm, standard chow and water ad libitum, and used at 10–12 wk of age (20–25 g body weight). For numbers (n), please refer to the figure legends.
Fetal (12-wk) and adult human adrenal tissue were obtained from the adrenal tissue bank approved by the ethical committee of the University of Dresden, Germany.
Treatment
Mice were ip sensitized with 1 mg/kg D-galactosamine (D-GalN) (Roth, Karlsruhe, Germany). Thirty minutes later, mice received ip either 1 ml/kg saline or 1 nmol/g CpG-oligodeoxynucleotide (ODN) (Thioat 1668; containing a "CG-motif": 5'-TCC ATG ACG TTC CTG ATG CT; TibMolBiol, Berlin, Germany). Animals were killed by an overdose of pentobarbital (Sigma-Aldrich, Munich, Germany) 15 min up to 6 h after the second challenge. Adrenal glands, spleen, and plasma samples were taken and stored at –80 C until assayed.
Cell culture
Mouse adrenal cortex tumor cell line Y-1 was purchased from the European Collection of Cell Cultures and was cultured at 37 C in a 5% CO2 atmosphere. For experiments, cells were plated and grown on six-well plates for 24 or 48 h until 80–90% confluence. CpG-ODN 1668 (2 µmol) was added with fresh medium, and at indicated time points, cell and supernatant samples were collected. The human adrenal cells (NCI-H295R; adherent) were plated for the experiments into 24-well culture plates (Falcon; Becton Dickinson, Heidelberg, Germany) at a density of 70,000 cells/cm2 and treated as previously described (19). CpG-ODN 2006-G5 (5'-TCG TCG TTT TGT CGT TTT GTC GTT GGG GG-3', TibMolBiol) or CpG-ODN 2216 (5'-GGG GGA CGA TCG TCG GGG GG-3', TibMolBiol) (1–6 µM) alone or in combination with forskolin (Sigma-Aldrich) were added with fresh medium to the cells. After 24, 48, and 72 h of stimulation, supernatant samples were collected.
Isolation of RNA
Total RNA from mice tissue and Y-1 cells was isolated (RNeasy Mini Kit; QIAGEN, Hilden, Germany), including a DNase digestion step (RNase-free DNase set; QIAGEN). RNA concentration was determined by absorbance at 260 nm. Until further processing, RNA was dissolved in 30 µl RNase-free water and stored at –80 C.
One-step RT-PCR
RT-PCR was performed using a QIAGEN OneStep RT-PCR kit. Primers for tested genes were as follows: TLR-9 (product size: 297 bp): forward, 5'-TGC AGG AGC TGA ACA TGA AC-3', reverse, 5'-TAG AAG CAG GGG TGC TCA GT-3'; and ß-actin (product size: 392 bp): forward, 5'-TGT TAC CAA CTG GGA CGA CA-3'; reverse, 5'-TCT CAG CTG TGG TGG TGA AG-3'. ß-Actin as a housekeeping gene was used to ensure the purity of the cDNA. Quantitative analysis of TLR-9 mRNA levels was performed by determination of the optical density using Scion Image software (Scion Corp., Frederick, MD) and normalized to ß-actin mRNA expression levels.
Western blotting
Mouse adrenal glands and Y-1 cells were lysed as previously described (17). The following antibodies were used: TLR-9 (1:1000; IMG-431; Imgenex Corp., San Diego, CA); horseradish peroxidase-conjugated goat antirabbit IgG (1:5000; 20301; Imgenex Corp.); or goat antimouse IgG (1:5000; sc-2031; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Bands were detected using enhanced chemiluminescence reagents (Santa Cruz Biotechnology, Inc.) and normalized to ß-actin.
Immunostaining of tissue
TLR-9 staining was performed on paraffin-embedded adrenal tissue from WT mice and human tissue (fetal and adult) using the following materials: normal goat serum (Vector Laboratories, Peterborough, UK); rabbit antimouse TLR-9 antibody (1:100; Imgenex Corp.); rabbit polyclonal TLR-9 antibodies (H-100, Santa Cruz Biotechnology, Inc.; or Cell Signaling Technology, Inc., Danvers, MA); horseradish peroxidase system (DakoCytomation, Cambridgeshire, UK); 3,3'-diaminobenzidine (DakoCytomation); and Cy3-conjugated goat antirabbit IgG (H+L) (1:250; Jackson ImmunoResearch, Cambridgeshire, UK).
Immunofluorescence staining of cells
Expression of TLR-9 within Y-1 cells was tested by intracellular staining. Y-1 cells were seeded on glass cover slips and then treated with CpG ODN (2 µmol) for 24 h. Cells were stained with antimouse TLR-9 antibody (1:200; Imgenex Corp.) and Cy3-conjugated goat antirabbit IgG (1:1000; Jackson ImmunoResearch), and photographed.
Flow cytometry
The intracellular expression of murine TLR-9 in Y-1 cells was determined using FITC-labeled antimouse TLR-9 and appropriate rat IgG2a isotype control (eBioscience, San Diego, CA). Cells were fixed in 2% formaldehyde/1X PBS and then permeabilized with FACS buffer (1X PBS, 2% BSA). Cells were incubated with TLR-9 antibody or isotype control 1 h in the dark. Cells were resuspended in FACS buffer and analyzed on FACScalibur (Becton Dickinson), collecting 10,000 events. Data acquisition and analysis were performed using CellQuest software (Becton Dickinson).
EMSA
Nuclear extracts from adrenal glands were subjected to EMSA as previously described (16, 17). Quantitative analysis of nuclear factor (NF)-
B activation was performed by determination of the OD using Scion Image software to support the visual impression.
Morphometric analysis of the adrenal gland
Mouse adrenal glands were embedded in paraffin. Tissue sections (3 µm) were stained with hematoxylin, and adrenal size was planimetrically evaluated using SigmaScan Pro (SPSS, Inc., Chicago, IL) image measurement software (16, 17).
Corticosterone/ACTH plasma levels
Corticosterone and ACTH levels in mouse plasma and cell culture supernatants were measured by RIA (Diagnostic Systems Laboratories, Webster, TX) as previously described (16, 17).
Plasma cytokines
Assays were performed using a mouse Cytokine Multiplex Antibody Bead Kit (Biosource, Nivelles, Belgium) for Luminex Laserassay as previously described (16). Granulocyte macrophage colony stimulating factor (GM-CSF), IL-1ß, -2, -4, -5, -6, -10, and -12, IFN-, and TNF- were measured. Assays were analyzed with STarStation software (Applied Cytometry Systems, Sheffield, UK).
Statistical evaluation
Results are presented as mean ± SEM. Data were analyzed by the Student’s t test, ANOVA, followed by Bonferroni’s multiple post tests or one-sample t test where appropriate (GraphPad Prism 4.0; GraphPad Software, Inc., San Diego, CA). Statistical significance was considered at P < 0.05.
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Results |
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We show for the first time that murine adrenal glands expressed TLR-9 both at mRNA and protein level as demonstrated by RT-PCR (Fig. 1A
) and Western blot analysis (Fig. 1B). However, TLR-9 protein was absent in TLR-9–/– mice. Furthermore, both immunohistochemical and immunofluorescence staining for TLR-9 in the adrenal gland of untreated WT mice revealed that TLR-9 was expressed in the adrenal cortex, but not in the adrenal medulla. No staining was observed in negative controls where no specific TLR-9 antibody was used (Fig. 1C). As depicted in Fig. 1, D and E, the cortical zona fasciculata exhibited prominent staining for TLR-9. Morphometric analysis of the adrenal gland displayed no difference in adrenal size between WT and TLR-9–/– mice, as shown in Fig. 1, F and G. Furthermore, in untreated mice, corticosterone and ACTH plasma levels were similar in WT and TLR-9–/– mice (Fig. 1, H and I).
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Plasma levels of corticosterone (Fig. 2A) and ACTH (Fig. 2B) were similar in WT and TLR-9–/– mice under control physiological conditions. After CpG-ODN challenge (30 min after injection of D-GalN), WT mice demonstrated a 3-fold increase in corticosterone plasma levels after 2 h and back to baseline values after 6 h. In contrast, CpG-ODN effects were abolished in TLR-9–/– mice (Fig. 2A). CpG-ODN had only a mild effect on ACTH plasma levels in WT animals, which was not statistically significant. Again, no effects were observed in TLR-9–/– mice (Fig. 2B).
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WT mice were challenged with CpG-ODN (30 min after injection of D-GalN) and killed at several time points (15–360 min). Using RT-PCR and Western blotting, TLR-9 expression was studied. Figure 3A depicts a typical autoradiograph in which adrenal glands were analyzed using RT-PCR. TLR-9 band intensities were normalized to ß-actin levels in the same sample. When compared with control, CpG-ODN challenge did not influence levels of TLR-9 mRNA over time (Fig. 3B). Similar results were obtained in Western blot studies (Fig. 3, C and D) demonstrating no significant change in TLR-9 protein expression pattern after CpG-ODN challenge. Analysis of corticosterone and ACTH plasma levels revealed that CpG-ODN only caused a significant increase of corticosterone levels at 2 h (Fig. 3E). This same peak had also been observed previously in an independent experiment (Fig. 2A
). CpG-ODN did not affect ACTH plasma levels at any time point (Fig. 3F).
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B activity
We determined NF-B activity in adrenal glands after a CpG-ODN challenge. As depicted in Fig. 4, A (autoradiograph) and B (densitometry), CpG-ODN had no detectable effect after 2 or 6 h on NF-B activity in WT or TLR-9–/– mice. In another set of experiments, WT mice were challenged with CpG-ODN for 15–360 min. When compared with control, no significant alterations of NF-B activity were detected [Fig. 4, C (autoradiograph) and D (densitometry)]. However, there was a 30% increase of NF-B-DNA binding activity after 30 min, which was of no statistical significance (P < 0.0538).
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We next focused on the plasma activity of various cytokines in WT and TLR-9–/– mice. Mice were either challenged with saline or CpG-ODN (120 or 360 min). As shown in Table 1, under control physiological conditions, WT mice had similar levels of cytokines as control TLR-9–/– mice. In contrast, CpG-ODN challenge (120 min) in WT mice caused a marked and significant increase in all cytokines (except IL-1ß and IL-2), ranging from 5- to 213-fold. Plasma levels of IL-6 (213-fold), IL-10 (37-fold), IL-12 (29-fold), IFN- (100-fold), and TNF- (30-fold) were elevated in particular, as compared with controls. After 360 min, levels of GM-CSF, IL-5, IL-6, IL-10, IL-12, and IFN- remained significantly increased. In contrast, CpG-ODN mediated effects in WT mice were abolished in TLR-9–/– mice. Only IL-6 levels increased within 120 min, but 31-fold less than in WT animals. In addition, IL-6 levels were back to baseline after 360 min in TLR-9–/– mice and still up-regulated in WT mice.
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We further investigated the effects of CpG-ODN challenge on plasma cytokine expression at different time points (15–360 min). As summarized in Table 1, all cytokines were significantly up-regulated between 45 and 60 min (IL-1ß approximately by 61-fold, IL-6 by 232-fold, IL-10 by 99-fold, IL-12 by 39-fold, GM-CSF by 6-fold, and TNF- by 288-fold), and plasma levels of IL-2, IL-5, IL-10, IL-12, and GM-CSF remained significantly high at 360 min. In contrast, IL-1ß, IL-4, IL-6, and TNF- levels declined to baseline already after 120 min.
TLR-9 expression in mouse adrenocortical Y-1 cells
TLR-9 expression in Y-1 cells was carried out using intracellular immunofluorescence staining (Fig. 5, A–D), RT-PCR (Fig. 5, E and F), Western blotting (Fig. 5, G and H), and FACS analysis (data not shown). As depicted in Fig. 5A, there was strong staining for TLR-9 detectable in mouse macrophages (RAW264.7; positive control). When cells were stained without the specific TLR-9 antibody, no staining could be observed (negative control). Untreated/unstimulated Y-1 cells showed a weak staining of TLR-9, indicating constitutive expression (Fig. 5C). However, when Y-1 cells were challenged with CpG-ODN, there was a strong intracellular TLR-9 staining detectable (Fig. 5D).
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, E and F) and protein (Fig. 5, G and H) were expressed in Y-1 cells (control). When Y-1 cells were challenged with CpG-ODN at various time points (1–72 h), a time-dependent and significant increase in levels of TLR-9 mRNA could be detected (Fig. 5E depicts an autoradiograph and Fig. 5F the respective densitometry). After 72 h, TLR-9 mRNA levels were back to baseline (Fig. 5F). RAW264.7 cells were used as a control for TLR-9 mRNA expression (Fig. 5E).
When Y-1 cells were challenged with CpG-ODN at various time points (1–72 h), a 50% decrease of TLR-9 protein expression was observed after 1 h, returning to baseline levels after 4 and 24 h (Fig. 5G depicts an autoradiograph and Fig. 5H the respective densitometry). However, after 24 and 72 h, protein levels decreased significantly when compared with control.
Effects of CpG-ODN on cytokine levels in Y-1 cells
Y-1 cells were challenged with CpG-ODN at various time points (1–72 h). IL-5 and IFN- levels were not detectable at any time point (Table 2). GM-CSF, IL-1ß, IL-6, and TNF- levels increased in a time-dependent fashion: GM-CSF by 33-fold, IL-1ß by 11-fold, IL-6 by 144-fold, and TNF- by 24-fold. In comparison, there was only a mild increase in the levels of IL-2, IL-4, and IL-10 detectable. IL-12 levels increased dramatically after 24 h (by 183-fold), 48 h (by 818-fold), and 72 h (2856-fold), respectively.
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Immunohistochemical analysis of normal human fetal and adult adrenal glands revealed TLR-9 expression in the adrenal cortex, but not in the adrenal medulla. As depicted in Fig. 6A, there was no staining for TLR-9 detectable when the adrenal sections were stained without the specific TLR-9 antibody (negative control). As in mouse adrenal glands, there was a staining of TLR-9 in zona fasciculata of both fetal (Fig. 6B) and adult (Fig. 6C) adrenal glands, indicating constitutive expression of TLR-9. Furthermore, we detected TLR-9 protein in the human adrenal cell line NCI-H295R (Western blotting); however, the expression levels were extremely low (data not shown). Forskolin-stimulated NCI-H295R cells alone or in combination with CpG-ODN caused a significant increase in cortisol secretion (data not shown). When CpG-ODN was given alone, no significant increase in cortisol secretion was observed (data not shown).
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Discussion |
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During sepsis, relative adrenal insufficiency occurs in a substantial number of patients and is responsible for increased mortality. Substitution therapy with low-dose hydrocortisone during septic shock or acute respiratory distress syndrome has improved survival (21, 22). However, the molecular mechanisms underlying an inadequate adrenal function during inflammation still remain to be elucidated. The role of TLRs provides a new exciting approach in understanding adrenal gland function during sepsis.
Here, we demonstrate for the first time that TLR-9 mRNA and protein are present in the murine adrenal gland as well as in the murine adrenocortical tumor cell line Y-1. To study TLR-9 expression in vivo, tissue samples from healthy, nontreated animals (normal leukocyte blood count) were taken. Because adrenal gland histology revealed only few leukocytes present in this organ, we can exclude that leukocyte-derived TLR-9 mRNA and protein is contributing to the PCR and Western blot signal in adrenal glands. We also can show that TLR-9 is present in the human adrenal cortex as well as in the human adrenocortical cell line NCI-H295R (very low levels).
These findings are in line with a previous report that TLR-9 mRNA is expressed in human adrenal glands (18). Furthermore, it has been shown that TLR-9 is present in murine spleen, kidney, liver, lung, and brain, among others (23, 24), and in various human tissues (18, 25). However, role and function of TLR-9 in the adrenal gland have not been studied.
In addition, we investigated adrenal morphology and function in WT and TLR-9–/– mice under control physiological conditions. Neither adrenal size nor plasma levels of corticosterone or ACTH differed between animals. This is in contrast to our previous findings in which TLR-2 or TLR-4 deficient mice demonstrated marked differences in the aforementioned parameters when compared with WT animals (16, 17). Although baseline function was abnormal in TLR-2 or -4 mice, this did not influence the phenotype. Therefore, it was of great interest to study mice under conditions of inflammation, in which an appropriate adrenal function is highly important to combat disease.
Here, we investigated the effects of systemic inflammation on the HPA axis in WT and TLR-9–/– mice in a systemic inflammatory response syndrome model using the TLR-9 agonist CpG-ODN. CpG-ODN caused a 3-fold increase of corticosterone levels in WT mice, comparable with the effects of lipopolysaccharide in a similar model (16). This effect was absent in TLR-9–/– mice, indicating that TLR-9 contributed either directly or indirectly to this hormonal response.
CpG-ODN had no effect on plasma levels of ACTH, either in WT or in TLR-9–/– mice. Although this fits with the lack of expression of TLR-9 in the murine pituitary gland (24), we cannot exclude a small indirect (IL-6 driven) stimulatory effect in WT mice. The HPA axis can be influenced by both pituitary (ACTH) and extrapituitary mechanisms (26, 27). For example, lipopolysaccharide, a component of gram-negative bacterial cell walls, increases ACTH and corticosterone plasma levels (28), as well as the release of various cytokines. The latter fact mediates the production of CRH in the hypothalamus. In turn, CRH stimulates the release of ACTH from the pituitary gland, which then increases glucocorticoid synthesis in the adrenal gland (29). Based on our in vivo results, one could argue that corticosterone production/release was mediated by an extrapituitary mechanism.
CpG-ODN challenge did not affect the expression of TLR-9 mRNA and protein in adrenal glands of WT mice. One of the reasons could be the dose of CpG-ODN administered (1 nmol/g). Several studies on the distribution of phosphorothioate-ODNs in rodents have shown that the majority of iv injected phosphorothioate-ODNs (96%) are cleared from the circulation as a result of rapid redistribution over the body fluid (30). It has been demonstrated that 4 min after injection, 50% of 125I-CpGs were eliminated from the bloodstream with a very high uptake in liver and kidney (23, 30, 31, 32). Together, these data suggest that the amount of CpG-ODN reaching the mouse adrenal gland is probably low. On the other side, we observed that TLR-9 mRNA levels in Y-1 cells markedly increased after CpG-ODN challenge, and protein levels decreased over time. This could be due to a higher concentration of CpG-ODN used in cell culture (2000-fold) and lack of clearance. It is unclear why protein levels decreased, however, we cannot exclude whether high levels of CpG-ODN initially (1 h) occupied receptor binding sites and, therefore, interfered with the protein detection system. After 4 and 24 h, TLR-9 protein levels were back to baseline and decreased at 48 and 72 h. Again, it is possible that CpG-ODN degradation products may influence TLR-9 detection.
CpG-ODN challenge was associated with high-plasma levels of corticosterone and various cytokines in the mouse. We profiled the cytokine expression (after CpG-ODN challenge) in the mouse for every 15 min for the first hour, and then at 2 and 6 h. Interestingly, most cytokines were already significantly up-regulated after 45–60 min (see time course in Table 1). Inflammatory cytokines, such as IL-1, IL-6, and TNF-, have been identified as important modulators of HPA axis function (6, 7). Plasma levels of these three cytokines were increased by more than 10-fold compared with baseline, supporting the concept of cytokine-mediated high-glucocorticoid output and a change from neuron-endocrine to immune-endocrine function (8). There is good evidence that CpG-ODN leads in vitro as well as in vivo to stimulation of immune competent cells, resulting in the release of various cytokines (11, 33, 34, 35, 36). In a model of CpG-DNA induced liver injury (D-GalN-sensitized mice), the proinflammatory cytokines IL-1ß, -6, and –12, and TNF- were generated within 1 h (37). However, after 4 h, levels of these cytokines returned back to baseline. These findings correspond to our observation in WT mice (Table 1). Cytokine levels were significantly increased between 45 and 60 min. After 2 h, levels of IL-1ß, IL-6, and TNF- declined to baseline, and only IL-12 levels remained high. We cannot exclude whether the cytokine profile measured in our study is, at least in part, due to CpG-ODN induced liver injury.
To date, 20 TLR-9 polymorphisms have been identified (38). A possible association of TLR-9 polymorphism has been suggested for asthma (38) and Crohn’s disease (39). There is currently no information available whether TLR-9 polymorphism is associated with adrenal insufficiency during inflammatory conditions.
TLR-9 is somehow involved in the adrenal stress response after CpG-ODN challenge. The absence of TLR-9 abolishes the corticosterone and cytokine response, supporting the concept that CpG-ODN mediates its effects on adrenal cells via TLR-9. However, this does not appear to compromise the phenotype of TLR-9–/– mice. Whether this effect is mediated directly (via adrenal TLR-9) or indirectly (via blood cell TLR-9 and, consequently, cytokine release), or a combination of both pathways, remains open for further studies. However, we have at least strong evidence for a direct effect: 30% increase in adrenal NF-B activity after 30 min, significant plasma cytokine response after 45 min, and significant corticosterone levels are detectable at 120 min. Together with our previous findings, TLR-9 activation contributes to an endocrine stress system response.
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online May 1, 2007
1 N.T. and A.K. contributed equally to this work. 
Abbreviations: CpG, Cytosine-phosphate-guanine; D-GalN, D-galactosamine; GM-CSF, granulocyte macrophage colony stimulating factor; HPA, hypothalamic-pituitary-adrenal; IFN, interferon; NF, nuclear factor; ODN, oligodeoxynucleotide; TLR, Toll-like receptor; WT, wild type.
Received December 6, 2006.
Accepted April 24, 2007.
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-mediated shock. Eur J Immunol 27:1671–1679[Medline]This article has been cited by other articles:
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  E. Sanchez-Lemus, Y. Murakami, I. M. Larrayoz-Roldan, A. J. Moughamian, J. Pavel, T. Nishioku, and J. M. Saavedra Angiotensin II AT1 Receptor Blockade Decreases Lipopolysaccharide-Induced Inflammation in the Rat Adrenal Gland Endocrinology, October 1, 2008; 149(10): 5177 - 5188. [Abstract] [Full Text] [PDF]
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, P < 0.05.
