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The Journal of Immunology, 2007, 179, 6325-6335
Copyright © 2007 by The American Association of Immunologists, Inc.
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Macrophages from 11beta-Hydroxysteroid Dehydrogenase Type 1-Deficient Mice Exhibit an Increased Sensitivity to Lipopolysaccharide Stimulation Due to TGF-beta-Mediated Up-Regulation of SHIP1 Expression

Tian Y. Zhang and Raymond A. Daynes1

Department of Pathology, School of Medicine, University of Utah, Salt Lake City, UT 84132


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
11beta-Hydroxysteroid dehydrogenase type 1 (11betaHSD1) performs end-organ metabolism of glucocorticoids (GCs) by catalyzing the conversion of C11-keto-GCs to C11-hydroxy-GCs, thereby generating activating ligands for the GC receptor. In this study, we report that 11betaHSD1–/– mice are more susceptible to endotoxemia, evidenced by increased weight loss and serum TNF-{alpha}, IL-6, and IL-12p40 levels following LPS challenge in vivo. Peritoneal and splenic macrophage (splnM{phi}) from these genetically altered mice overproduce inflammatory cytokines following LPS stimulation in vitro. Inflammatory cytokine overexpression by 11betaHSD1–/– splnM{phi} results from an increased activation of NF-{kappa}B- and MAPK-signaling cascades and an attenuated PI3K-dependent Akt activation. The expression of SHIP1 is augmented in 11betaHSD1–/– M{phi} and contributes to inflammatory cytokine production because overexpression of SHIP1 in primary bone marrow M{phi} (BMM{phi}) leads to a similar type of hyperresponsiveness to subsequent LPS stimulation. 11betaHSD1+/+ and 11betaHSD1–/– BMM{phi} responded to LPS similarly. However, 11betaHSD1–/– BMM{phi} derived in the presence of elevated GC levels up-regulated SHIP1 expression and increased their capacity to produce inflammatory cytokines following their activation with LPS. These observations suggest the hyperresponsiveness of 11betaHSD1–/– splnM{phi} results from myeloid cell differentiation in the presence of moderately elevated GC levels found within 11betaHSD1–/– mice. GC-conditioning of BMM{phi} enhanced SHIP1 expression via up-regulation of bioactive TGF-beta. Consistently, TGF-beta protein expression was increased in unstimulated CD11b– cells residing in the BM and spleen of 11betaHSD1–/– mice. Our results suggest that modest elevations in plasma GC levels can modify the LPS responsiveness of M{phi} by augmenting SHIP1 expression through a TGF-beta-dependent mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Glucocorticoids (GCs2; cortisol and corticosterone) are steroid hormones endogenously produced by the adrenal glands. Mammalian adrenal output of GCs is regulated by the hypothalamic pituitary adrenal axis under the influence of environmental cues, physical/psychological stress, and various insults (1). It is now appreciated that corticotrophin-releasing hormone produced by the hypothalamus triggers the release of the adrenalcorticotropin hormone by the pituitary gland, which in turn results in GC synthesis by cells of the zona fasciculata and zona reticularis of the adrenal cortex (2). In humans, plasma GC levels peak right before waking and gradually decline throughout the day until a nadir is reached in the early evening (3). Falling plasma GC levels releases the negative feedback regulation in the hypothalamus and allows more GCs to be synthesized. This oscillation in plasma GC levels has been termed the diurnal rhythm of GCs. The encounter of psychological and physical stress can override the GC diurnal rhythm and induce as much as a 10-fold increase in plasma GC levels in an acute manner (1).

Additional mechanisms in controlling plasma bioactive GC availability include albumin- and corticosteroid-binding globulins, which function as carrier proteins to buffer the rate of cellular entry of bioactive GCs (4, 5). As much as 90% of bioactive GCs in the circulation can be found bound to these carrier proteins (4, 5). Bioactive GCs unassociated with carrier proteins can freely diffuse through plasma membranes and associate with their cognate receptors, the GC receptors (GRs). GRs are ligand-activated transcription factors belonging to the bigger family of nuclear hormone receptors (6). Intracellularly, the expression of GRs and receptor-specific coactivators/corepressors determine hormone-specific responsiveness within a particular cell type (7). GRs undergo nuclear translocation following ligand binding, form homodimers, and become associated with GC response elements (GREs) in the promoter region of GC-responsive genes (7). Thus, GC-mediated effects can be mediated by direct transactivation/transrepression of gene expression. Alternatively, GC/GR complexes can associate with transcription factors, including NF-{kappa}B and AP-1, and thereby modulate gene expression via transcriptional interference (8).

End-organ metabolism of GCs by 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) exerts additional control over bioactive GC levels at the tissue and cellular level (9). 11betaHSD1 is a reductase that catalyzes the reactivation of C11-keto-GCs (cortisone and 11-dehydrocorticosterone) to C11-hydroxy-GCs (cortisol and corticosterone) (9). Mice deficient in 11betaHSD1 lack the ability to reactivate C11-keto-GCs to bioactive GCs, indicating 11betaHSD1 as the sole reductase in vivo (10). Surprisingly, 11betaHSD1-deficient animals exhibit a variety of advantageous phenotypes, including increased insulin sensitivity and resistance to stress-induced hypoglycemia (10). Furthermore, 11betaHSD1–/– mice do not undergo an age-associated decline in cognitive function (11). However, minor abnormalities have been reported. 11betaHSD1–/– neonates exhibit delayed lung maturation associated with decreased lung surfactant synthesis following birth (12, 13). 11betaHSD1–/– mice also exhibit elevated plasma GC levels at the diurnal nadir (14). This dysregulation in circulating GC levels has been hypothesized to result from insufficient negative feedback regulation due to the lack of end-organ C11-keto-GC reactivation in the hypothalamus of 11betaHSD1–/– mice (14).

In this study, we provide evidence that 11betaHSD1–/– mice are more susceptible to endotoxemia, as indicated by the increased weight loss and elevated serum TNF-{alpha}, IL-6, and IL-12p40 levels following LPS challenge in vivo. 11betaHSD1–/– splenic macrophages (splnM{phi}) and B cells were found to overproduce these proinflammatory cytokines in response to LPS stimulation in vitro. The overexpression of LPS-induced inflammatory cytokines resulted from augmented activation of the NF-{kappa}B- and MAPK-signaling cascades. Modified activation of these signaling pathways resulted from attenuated PI3K-dependent Akt activation due to significantly increased SHIP1 expression in 11betaHSD1–/– M{phi}. GC-conditioning of normal bone marrow M{phi} (BMM{phi}) resulted in a similar increase in SHIP1 expression via up-regulation of TGF-beta expression in these cells. Thus, differentiation under the influence of elevated plasma GC levels in 11betaHSD1–/– hosts modified the sensitivity of 11betaHSD1–/– M{phi} to LPS stimulation via a TGF-beta-mediated up-regulation of SHIP1 expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Experimental animals

11betaHSD1+/+ C57BL/6 wild-type (WT) mice were obtained from the National Cancer Institute. Breeding pairs of 11betaHSD1–/– mice on the C57BL/6 background were provided by Dr. J. R. Seckl (University of Edinburgh, Edinburgh, U.K.). All animals were bred and maintained in microisolator units and provided with sterile food and water ad libitum. The University of Utah Comparative Medicine Department guarantees strict compliance with the Animal Welfare Act.

Cell culture and reagents

Mink lung epithelial cells (Mv1Lu; CCL-64; American Type Culture Collection), single-cell suspensions of splenocytes, B220+ B cells, CD11b+ splnM{phi}, and peritoneal-elicited M{phi} (PEM{phi}) from C57BL/6 WT and 11betaHSD1–/– animals were prepared and maintained in GClow (3 x 10–10 M) complete medium (1% complete medium; RPMI 1640 (CellGro) supplemented with 1% FBS (HyClone), 2 mM L-glutamine, 100 U/ml gentamicin, and 50 µM 2-ME). LPS (Escherichia coli strain O111:B4) and [3H]thymidine (1.0 mCi, specific activity 2.0 Ci/mM) was purchased from Sigma-Aldrich. Abs specific for murine p-Ser473-Akt, p-p38, p-Erk44/42, and I{kappa}B{alpha} were obtained from Cell Signaling Technology. Abs specific for total Akt were purchased from eBioscience. A monoclonal anti-murine SHIP-1 Ab was purchased from Santa Cruz Biotechnology. Neutralizing Ab to murine TGF-beta1, TGF-beta2, TGF-beta3 was purchased from R&D Systems for use in TGF-beta neutralization experiments and Western blot analysis. Rabbit-anti-mouse HRP was obtained from Sigma-Aldrich. Goat anti-rabbit-HRP Ab was obtained from Bio-Rad. Murine rIFN-{gamma} and all purified and biotinylated Abs specific for various murine cytokines were purchased from BD Pharmingen/BD Biosciences.

Thioglycolate-elicited peritoneal M{phi}

Activated peritoneal M{phi} were elicited by the injection of 1 ml of 4% Bacto Brewer’s Thioglycolate (Difco Laboratories) into the peritoneal cavity of C57BL/6 WT and 11betaHSD1–/– mice. The animals were sacrificed on day 4 following the injection of thioglycolate and the elicited M{phi} lavaged by i.p. injection of PBS (5 ml, 4°C). PEM{phi} were washed in 1% complete medium (three times), resuspended at the density of 0.5 x 106 and rested for ≥5 h. At the indicated time points, C57BL/6 WT and 11betaHSD1–/– PEM{phi} were pelleted. The supernatants were collected at each time point to evaluate the levels of cytokines present by ELISA and the cell pellets were lysed and the total cellular proteins extracted for western blot analysis.

BM-derived M{phi}

To generate murine BMM{phi}, total BM cells were flushed out from the tibias and femurs of 11betaHSD1+/+ and 11betaHSD1–/– animals. Single-cell suspensions of total bone marrow (0.5 x 106/ml) were cultured in vitro in BMM{phi} medium (RPMI 1640 supplemented with 20% L929 conditioned medium (as a source of M-CSF), 30% equine serum (HyClone), 2 mM L-glutamine, 100 U/ml gentamicin, and 50 µM 2-ME). Media was changed on day 4 of culture. After 6 days in culture, adherent M{phi} were detached from bacteriologic petri plates, washed three times, and resuspended at a density of 0.5 x 106 cells/ml in 1% complete medium. GC-BMM{phi} were generated under identical conditions in the presence of added corticosterone (20 nM). For stimulations, 200 µl of cells were plated in triplicate in 96-well plates, rested for ≥5 h followed by the addition of 10 ng/ml LPS. Viability of cells were determined at the time of plating using the trypan blue exclusion method and was consistently ≥99%.

Purification of specific cell populations

CD11b+ M{phi} were purified by from the peritoneal exudates, spleens, and BM of C57BL/6 WT and 11betaHSD1–/– animals by positive selection using magnetic bead sorting kits specific for CD11b (Miltenyi Biotec). B220+ B cells were purified from the spleen of C57BL/6 WT and 11betaHSD1–/– animals by negative selection using magnetic bead sorting kits specific for B220 (Miltenyi Biotec). Purity of the selected cell population was determined using flow cytometric analysis and was routinely shown to be >95%. The unlabeled negative fractions of the splenocytes were referred to as the CD11b– population where appropriate.

Western blot analysis

BMM{phi}, CD11b+ splnM{phi}, and PEM{phi} were rested overnight (0.5 x 106 cells/ml) in 1% complete medium and subsequently stimulated by adding 10 ng/ml LPS. At the indicated time points, cells were pelleted, lysed (2 mM Tris-HCl, 1 mM NaCl, 1 mM KCl, 0.3 mM MgCl2 plus complete protease inhibitor mixture tablets (Roche) supplemented with 20 mM NaV3O4, 25 mM NaF, 5 mM Na2P4O7, and 1% Triton X-100) and the protein concentration determined by the BCA (BCA kit; Pierce) method. An equal amount of protein (15–20 µg) was mixed with SDS sample buffer, boiled for 5 min followed by SDS-PAGE. Proteins were then transferred onto polyvinylidene difluoride membranes, blocked (1% nonfat dry milk, 1% BSA in TBS-0.1% Tween 20), and probed for various proteins with the indicated specific Abs. Following probing with the primary Ab specific for the protein of interest, blots were washed and subsequently probed with appropriate HRP-conjugated secondary Abs. Proteins of interest were detected using the ECL method (Amersham) followed by exposure to x-ray film for the appropriate exposure times. Western blots were quantitated using ImageJ Software (version 1.37; W. S. Rasband, National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gove.ij/). The intensity of the protein-of-interest bands were normalized to the appropriate total protein loading controls and the resulting ratio presented in numerical format below individual blots.

ELISA

All quantitatively analysis of cytokines were performed using rat-anti-murine mAbs (purified and biotinylated anti-murine TNF-{alpha}, IL-6, IL-12, and IL-10 Abs) for capture and detection, followed by development with HRP-conjugated streptavidin, and ABTS as substrate. The detection limit for all ELISA was <16 pg/ml.

Transient transfections

BMM{phi} were generated as described above. At the end of the 6-day culture period, BMM{phi} were collected, washed, and resuspended in Nucleofector Solution at a density of 2 x 106/100 µl containing vector only or p150SHIP1 (2.5 µg/106 cells, purified by CsCl gradient). Transfections were conducted using the Amaxa Nucleofector (program Y-001). Following transfection, the cells were immediately placed in 10% complete medium (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml gentamicin, and 50 µM 2-ME prewarmed to 37°C) at a density of 0.5 x 106/ml and rested for the predetermined optimal time of 30 h for p150SHIP1. Following the resting period, transfected cells were either pelleted and lysates prepared for Western blot analysis or resuspended in 1% complete medium (0.5 x 106/ml) and used for LPS stimulations. Transfection efficiency was determined using a pmaxGFP expression construct supplied by Amaxa. The percentage of GFP+-BMM{phi} was determined using flow cytometric analysis 16 h following transfection and was consistently ≥85%.

Quantitative real-time PCR

RNA was extracted from pelleted cells using chloroform/phenol followed by ethanol precipitation (Chomczynski method). One microgram of RNA from each sample was used to synthesize cDNA by reverse transcription. The expression levels of various genes of interest were determined using the following primer pairs: murine TGF-beta1: upper 5'-ATGATGCTAAAGAGGTCACCCGC-3', lower 5'-CCAAGGTAACGCCAGGAATT-3'; murine TGF-beta2: upper 5'-CTTAACATCTCCCACCCAGC-3', lower 5'-TCACCACTGGCATATGTAGA-3'; murine TGF-beta3: upper 5'-TCTGCCCCAAAGGAATTACC-3', lower 5'-CTCCATTGGGCTGAAAGGTG-3'; murine SHIP1: upper 5'-GAGGATGCTATTGATGAGGC-3', lower 5'-CTTCTTCAGGTGAGGGAGGT-3'. All samples were amplified using optimized protocols for 40 cycles using a Roche Light Cycler.

TGF-beta-mediated inhibition of cell proliferation of MvLu1 cells

MvLu1 cells were cultured initially in complete medium containing 10% FBS and were gradually weaned to complete medium containing 2% FBS before their use in the following assay. Single-cell suspensions of MvLu1 cells were plated 5 x 104/100 µl/well in 96-well plates. Supernatants collected from BMM{phi} cultures generated in the presence or absence of 20 nM corticosterone from days 1 to 5 were added to individual wells in triplicates (100 µl/well) in the presence or absence of neutralizing Abs to TGF-beta (2 µg/ml). MvLu1 cells were coincubated with supernatants for 24 h at 37°C, 5% CO2 before [3H]thymidine was added. After an 18-h pulse with [3H]thymidine (1 µCi), [3H]thymidine-containing fractions of cell lysates are collected on to glass filters, dried, and the radioactivity quantitated by scintillation counting. The results represent average values of triplicate samples ± SD.

LPS challenge in vivo

11betaHSD1–/– and C57BL/6 WT animals were injected with 10 µg of LPS in 100 µl of PBS i.p. At various times following the injection of LPS, blood was drawn from all animals and allowed to clot. Serum was separated and stored at –70°C until further use. Each animal was weighted daily to followed the severity of LPS response and as indicator of the recovery of all animals.

Statistics

Statistical analysis was performed with the EZAnalyze Freeware. The p values were determined by Student’s t test (unpaired).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PEM{phi} from 11betaHSD1–/– animals are hyperresponsive to LPS stimulation

It was recently reported that LPS-inducible IL-6 production by thioglycolate-elicited M{phi} from the peritoneal cavity (PEM{phi}) of 11betaHSD1–/– mice are elevated compared with PEM{phi} from 11betaHSD1+/+ mice (15). Our laboratory has recently shown that GC-mediated up-regulation of SHIP1 expression in primary M{phi} can result in the up-regulation of inflammatory cytokine production following TLR activation, including IL-6 (16). Because 11betaHSD1–/– mice are known to have increased circulating GC levels at the diurnal nadir, we wondered whether increased IL-6 production by 11betaHSD1–/– PEM{phi} was due to increased SHIP1 expression. PEM{phi} were elicited by the injection of 4% thioglycolate into the peritoneal cavity of 11betaHSD1+/+ and 11betaHSD1–/– animals. Four days following the injection, PEM{phi} were collected from the peritoneal cavity of 11betaHSD1+/+ and 11betaHSD1–/– animals, washed, and a single-cell suspension was prepared for simulation with LPS (10 ng/ml). At various time points following LPS stimulation, cells were pelleted and total cellular proteins were extracted for Western blot analysis of SHIP1 expression. Supernatants were collected to determine inflammatory cytokine production by ELISA. As shown in Fig. 1A, SHIP1 protein expression was elevated in 11betaHSD1–/– PEM{phi} compared with 11betaHSD1+/+ PEM{phi}. Consistent with this observation and with data recently reported by Chapman and colleagues (15), LPS stimulated IL-6 production by 11betaHSD1–/– PEM{phi} was elevated in comparison to 11betaHSD1+/+ PEM{phi}. Additionally, we found an increase in IL-12 production in 11betaHSD1–/– PEM{phi} compared with 11betaHSD1+/+ PEM{phi} (Fig. 1, B and C).


Figure 1
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  		FIGURE 1. Thioglycolate-elicited peritoneal M{phi} from 11betaHSD1–/– mice exhibit hyperresponsiveness to LPS stimulation. Thioglycolate-elicited PEM{phi} from 11betaHSD1+/+ and 11betaHSD1–/– mice were cultured (0.5 x 106/ml) in 1% complete medium and stimulated with LPS (10 ng/ml). A, At the indicated times, cell lysates were prepared for Western blot analysis of SHIP1 expression using an Ab specific for murine SHIP1. The blot was stripped and reprobed for actin to determine equal protein loading. B and C, Supernatants were collected at the indicated time points to quantitate IL-6 and IL-12p40 levels by ELISA. All results are representative of more than or equal to three independent experiments. Error bars represent mean ± SD. *, p < 0.001; **, p < 0.05.

 
11betaHSD1–/– splnM{phi} and B cells are hyperresponsive to LPS stimulation

Thioglycolate-elicited PEM{phi} are considered to be inflammatory M{phi} that have been activated following their recruitment into the peritoneal cavity. To determine whether nonactivated 11betaHSD1–/– immune cells are hyperresponsive to LPS stimulation, splenocytes from 11betaHSD1+/+ and 11betaHSD1–/– animals were stimulated with LPS. As shown in Fig. 2, A and B, we found more IL-6 and IL-12p40 in the supernatants of 11betaHSD1–/– splenocytes compared with 11betaHSD1+/+ splenocytes. TNF-{alpha} was undetectable in the supernatant of LPS-stimulated total splenocytes at all time points examined. M{phi} and B cells express cell surface TLR4/CD14 and thus represent the major LPS-responsive cellular constituents of the spleen (17). To determine whether splnM{phi} and/or B cells are responsible for the augmentation in cytokine production, we purified CD11b+ M{phi} and B220+ B cells from the spleens of 11betaHSD1+/+ and 11betaHSD1–/– mice followed by their stimulation with the a single low dose of LPS. As shown in Fig. 2, C–H, 11betaHSD1–/– splnM{phi} and B cells produced increased amounts of inflammatory cytokines compared with 11betaHSD1+/+ splnM{phi} and B cells at all the time points examined.


Figure 2
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  		FIGURE 2. CD11b+ M{phi} and B220+ B cells from spleens of 11betaHSD1–/– mice produce elevated levels of inflammatory cytokines following LPS stimulation. A and B, Single-cell suspensions of total splenocytes (5 x 106/ml) from 11betaHSD1+/+ and 11betaHSD1–/– mice were stimulated with LPS (10 ng/ml). (C–E) CD11b+ M{phi} and (F–H) B220+ B cells were purified from spleens of 11betaHSD1+/+ and 11betaHSD1–/– mice, resuspended (0.5 x 106/ml) in 1% complete medium and stimulated with LPS (10 ng/ml). At the indicated time points, supernatants were collected and the levels of TNF-{alpha}, IL-6, and IL-12p40 were quantitated by ELISA. All results are representative of more than or equal to three independent experiments. Error bars represent mean ± SD. *, p < 0.001; **, p < 0.05.

 
To determine whether 11betaHSD1–/– mice are hyperresponsive to LPS challenge in vivo, 11betaHSD1–/– and 11betaHSD1+/+ animals were given LPS via i.p. injection. Each animal was weighed daily to follow weight loss as an indicator of the severity of the inflammatory response and recovery kinetics. Blood was collected at the indicated times following LPS injection to determine serum cytokine levels. As shown in Fig. 3A, 11betaHSD1–/– hosts lost more weight by day 3 following LPS administration and took longer to recover from the LPS challenge compared with 11betaHSD1+/+ mice. Consistent with our in vitro data, serum TNF-{alpha}, IL-6, and IL-12p40 levels were elevated in LPS exposed 11betaHSD1–/– animals compared with the 11betaHSD1+/+ counterparts (Fig. 3, B–D). Thus, 11betaHSD1–/– animals exhibit an increased sensitivity to LPS stimulation in vivo.


Figure 3
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  		FIGURE 3. 11betaHSD1–/– mice exhibit accelerated weight loss and elevated serum proinflammatory cytokines following endotoxin challenge in vivo. 11betaHSD1+/+ and 11betaHSD1–/– mice (in groups of five) were given LPS (1 mg/kg) i.p. A, Individual mice were weighed daily at 1000 h to monitor disease severity and recovery. B–D, Blood was drawn at the indicated time points and allowed to clot. Serum was separated and used to determine circulating levels of TNF-{alpha}, IL-6, and IL-12p40 by ELISA. All results are representative of more than or equal to three independent experiments. Error bars represent mean ± SD. *, p < 0.05; **, p < 0.001.

 
11betaHSD1–/– and 11betaHSD1+/+ BMM{phi} exhibit hyperresponsiveness to LPS stimulation, but only following a GC conditioning

To determine whether the hyperresponsiveness of 11betaHSD1–/– splnM{phi} and PEM{phi} are the result of the genetic deficiency in 11betaHSD1 expression within the M{phi} themselves, we generated BMM{phi} from the BM of 11betaHSD1+/+ and 11betaHSD1–/– mice. 11betaHSD1+/+and 11betaHSD1–/– total BM were cultured in M-CSF-containing medium for 6 days. At the end of the culture period, all adherent BMM{phi} were detached, washed, and resuspended for LPS stimulation in the presence or absence of rIFN-{gamma}. As shown in Fig. 4, A–C, no differences were observed in any of the LPS-inducible proinflammatory cytokines examined whether IFN-{gamma} was present in the culture system or not. Thus, the lack of 11betaHSD1 in the LPS-responsive M{phi} population was not responsible for the hyperresponsiveness of 11betaHSD1–/– BMM{phi}.


Figure 4
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  		FIGURE 4. 11betaHSD1+/+ and 11betaH SD1–/– BMM{phi} exhibit hyperresponsiveness to LPS stimulation following GC conditioning. A–C, Single-cell suspensions of 11betaHSD1–/– and 11betaHSD1+/+ BMM{phi} (0.5 x 106/ml) were stimulated with LPS (1–10 ng/ml) in the presence or absence of murine rIFN-{gamma} (1–10 ng/ml). Supernatants were collected at 24 h to determine TNF-{alpha}, IL-6, and IL-12p40 levels by ELISA. D, Blood was drawn from 11betaHSD1+/+ and 11betaHSD1–/– mice at 8 a.m. and 8 p.m. and allowed to clot. Serum was separated and used to determine circulating corticosterone levels using a modified ELISA. E–G, BMM{phi} were generated from 11betaHSD1+/+ and 11betaHSD1–/– mice in the presence or absence of corticosterone (20 nM) and stimulated with LPS (10 ng/ml). Supernatants were collected at 24 h to quantitate TNF-{alpha}, IL-6, and IL-12p40 levels by ELISA. All results are representative of more than or equal to three independent experiments. Error bars represent mean ± SD. *, p < 0.05; **, p < 0.001.

 
Consistent with previous reports, 11betaHSD1–/– mice on the C57BL/6 background were confirmed to have elevated plasma GC levels at the nadir of the diurnal rhythm (Fig. 4D). GC-conditioning of myeloid progenitors during M{phi} differentiation was recently reported by us to exert permissive effects on the ability of BMM{phi} to respond to LPS stimulation (16). These observations led us to hypothesize that differentiation of myeloid progenitors under the influence of elevated circulating GC levels within the 11betaHSD1–/– mice might be responsible for modifying their responsiveness to LPS stimulation. To evaluate this possibility, BMM{phi} were generated from the BM of 11betaHSD1–/– or 11betaHSD1+/+ mice in the presence or absence of 20 nM corticosterone. GC-conditioned and nonconditioned 11betaHSD1+/+ and 11betaHSD1–/– BMM{phi} were stimulated with LPS and supernatant cytokine levels were quantitated by ELISA. As shown in Fig. 4, E–G, 11betaHSD1–/– BMM{phi} derived in the presences of elevated corticosterone levels produced increased levels of proinflammatory cytokines (TNF-{alpha}, 2.13-fold; IL-6, 2.04-fold; IL-12p40, 2.87-fold) following LPS stimulation. Consistent with our previous observations, proinflammatory cytokines were also increased in 11betaHSD1+/+ BMM{phi} generated in the presence of added corticosterone compared with normally derived 11betaHSD1+/+ BMM{phi} (Fig. 4, E–G). Thus, 11betaHSD1–/– myeloid progenitors differentiating in the presence of elevated GC levels results in a hyperresponsiveness to LPS stimulation.

Hyperresponsiveness of 11betaHSD1–/– splnM{phi} to LPS stimulation results from altered LPS signaling

LPS stimulation of M{phi} results in the activation of major signaling cascades that culminate in the transcriptional activation of genes encoding proinflammatory mediators (17). Activation of the NF-{kappa}B-signaling pathway and the MAPK-signaling cascades in murine M{phi} are exquisitely regulated for the induction of inflammatory cytokines following LPS stimulation (17, 18). To gain a better understanding of the molecular mechanism responsible for the overexpression of proinflammatory cytokines by 11betaHSD1–/– M{phi} following LPS stimulation, we characterized the activation status of these major signaling pathways in 11betaHSD1+/+ and 11betaHSD1–/– splnM{phi}. CD11b+ 11betaHSD1+/+ and 11betaHSD1–/– splnM{phi} were purified and stimulated with LPS for the indicated time points. Cells were pelleted, lysed, and total cellular protein extracts prepared for Western blot analysis. Interestingly, I{kappa}B{alpha} degradation was observed to be accelerated in LPS-treated 11betaHSD1–/– splnM{phi} in comparison to 11betaHSD1+/+ counterparts (Fig. 5A). In addition, p-p38 and p-Erk42/44 levels were elevated in 11betaHSD1–/– splnM{phi} following LPS addition compared with 11betaHSD1+/+ splnM{phi} (Fig. 5, B and C). Thus, an increased MAPK and NF-{kappa}B activation could be responsible for the observed overexpression of proinflammatory cytokines by 11betaHSD1–/– splnM{phi}.


Figure 5
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  		FIGURE 5. 11betaHSD1–/– splnM{phi} exhibit altered LPS signaling. A–E, CD11b+ M{phi} were purified from the spleens of 11betaHSD1+/+ and 11betaHSD1–/– mice. Single-cell suspensions (0.5 x 106/ml) were prepared and stimulated with LPS (10 ng/ml). F–J, Normally derived 11betaHSD1+/+ and 11betaHSD1–/– BMM{phi} were stimulated with LPS (10 ng/ml). At the indicated times, cell lysates were prepared for Western blot analysis using Abs specific for I{kappa}B{alpha}, p-p38, p-Erk44/42, p-Akt, and SHIP1. Blots were stripped and reprobed using the appropriate Abs to determine equal protein loading. All results are representative of more than or equal to three independent experiments.

 
Hyperresponsiveness of 11betaHSD1–/– splnM{phi} is associated with depressed Akt activation and augmented SHIP1 expression

LPS stimulation of M{phi} results in the generation of PI(3,4,5)P3 (PIP3) via the activation of PI3K (19). The presence of PIP3 facilitates the recruitment and activation of pleckstrin homology domain-containing kinases, such as Akt, a known negative regulator of NF-{kappa}B and MAPK activation (20, 21). When we questioned the Akt activation status in 11betaHSD1+/+ and 11betaHSD1–/– splnM{phi}, pSer473-Akt levels were found to be attenuated in 11betaHSD1–/– splnM{phi} following their LPS stimulation in comparison to 11betaHSD1+/+ splnM{phi} (Fig. 5D).

The activation of many signaling pathways are dependent upon the PIP3 generated by PI3K activation. Hemopoietic cell-specific SHIP1 exerts negative control over the activation of PIP3-dependent signaling pathways via its 5'-phosphatase activity, dephosphorylating PI(3,4,5)P3 to PI(3,4)P2 (22, 23, 24). To determine whether altered SHIP1 expression played a role in modifying PI3K-dependent Akt activation in 11betaHSD1–/– splnM{phi}, 11betaHSD1+/+, and 11betaHSD1–/– splnM{phi} were stimulated with LPS. SHIP1 expression was determined in the lysates of LPS-stimulated 11betaHSD1+/+ and 11betaHSD1–/– splnM{phi} by Western blot analysis. As shown in Fig. 5E, SHIP1 expression was increased in 11betaHSD1–/– splnM{phi} in comparison to 11betaHSD1+/+ splnM{phi}. It is therefore possible that an augmented level of SHIP1 expression could be responsible for depressed Akt activation observed in 11betaHSD1–/– splnM{phi}.

To determine whether increased activation of the NF-{kappa}B- and MAPK-signaling pathways correlated with enhanced cytokine production, we characterized BMM{phi} from 11betaHSD1+/+ and 11betaHSD1–/– mice. BMM{phi} were generated as described from 11betaHSD1-deficient and WT animals followed by their stimulation with LPS. Western blot analysis of cellular lysate prepared from 11betaHSD1+/+ and 11betaHSD1–/– BMM{phi} at the indicated time points revealed no differences in the activation of NF-{kappa}B-, MAPK-, and PI3K-signaling pathways in these two populations of cells following LPS stimulation (Fig. 5, F–I). Additionally, SHIP1 was also similarly expressed in 11betaHSD1+/+ and 11betaHSD1–/– BMM{phi} (Fig. 5J).

To solidify a role for SHIP1 in the regulation of proinflammatory cytokine production in murine M{phi} following LPS stimulation, we overexpressed a WT full-length SHIP1 plasmid (p150SHIP1) in BMM{phi} derived from C57BL/6 WT mice. As indicated in Fig. 6A, an overexpression of SHIP1 protein was observed in WT-BMM{phi} ~36 h following transfection of the expression plasmid. BMM{phi} overexpressing SHIP1, or not, were stimulated with LPS and the supernatants were collect at 24 h to determine the cytokines present. Consistent with SHIP1 playing a positive regulatory role in LPS signaling, we found that BMM{phi} overexpressing SHIP1 produced significantly more proinflammatory cytokines following the addition of LPS (TNF-{alpha}, 4.13-fold; IL-6, 17.1-fold; IL-12p40, 4.61-fold) compared with the control BMM{phi} transfected with the empty vector (Fig. 6, B–D).


Figure 6
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  		FIGURE 6. SHIP1 overexpression in primary BMM{phi} results in hyperresponsiveness to LPS stimulation. A, Normally derived BMM{phi} were mock transfected, or transfected with a plasmid encoding full-length SHIP1 (p150-SHIP1) or the empty vector. At 30 h following transfection, cell lysates were used to determine transfection success by Western blot analysis using Abs specific for the hemagglutinin (HA) tag and SHIP1. B–D, Parallel cell fractions were stimulated with LPS (10 ng/ml). Supernatants were collected at 18 h to determine the levels of TNF-{alpha}, IL-6, and IL-12p40 by ELISA. All results are representative of more than or equal to independent experiments. Error bars represent mean ± SD. *, p < 0.001; **, p < 0.05.

 
GCs augment SHIP1 expression via TGF-beta

GCs are endogenous ligands for the GR. Ligand activation of the GR induces nuclear translocation of the ligand/receptor complex and its association with GREs in the promoter region of a wide variety of genes, thereby modifying their transcriptional regulation (6). Promoter scanning of a ~100-kb region upstream of the TATA box of the SHIP1 gene did not reveal canonical GRE elements (our own observation). Although this does not rule out the possibility that GREs are present further upstream or downstream from the transcription initiation site, functioning as enhancers of SHIP1 transcription, we thought it might be possible that GCs are enhancing SHIP1 expression indirectly. Published literature revealed TGF-beta to be the only factor with the capacity to increase SHIP1 expression in M{phi} (25). Because GCs have already been established to regulate TGF-beta expression transcriptionally, translationally, and posttranslationally, we hypothesized that GCs may be inducing SHIP1 expression via a TGF-beta-dependent manner (22, 26, 27).

Using our in vitro BMM{phi} culture to determine whether GCs can increase SHIP1 expression, RNA was extracted from adherent BMM{phi} generated in the absence or presence of corticosterone from days 0 to 5. Quantitative real-time PCR was performed to determine TGF-beta1, TGF-beta2, and TGF-beta3 mRNA expression. This experiment revealed a gradual increase in TGF-beta1 and TGF-beta2 mRNA that continued throughout the BMM{phi} culture period in the presence of corticosterone (Fig. 7, A and B). TGF-beta3 was not detected in any of the samples. In addition, quantitative real-time PCR analysis showed increased SHIP1 transcript levels with parallel kinetics to those observed for TGF-beta (Fig. 7C).


Figure 7
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  		FIGURE 7. GCs augment SHIP1 expression in BMM{phi} via an up-regulation of TGF-beta expression. A and B, BMM{phi} were generated in the presence or absence of corticosterone (20 nM). Cells were collected on the indicated days for analysis of TGF-beta1, TGF-beta2, and TGF-beta3 mRNA expression by quantitative real-time PCR. C, Supernatants from normally derived (ND-BMM{phi}) or GC-conditioned (20 nM corticosterone) (GC-BMM{phi}) BMM{phi} cultures were incubated with MvLu1 cells for 24 h in the presence or absence of neutralizing Abs for TGF-beta (2 µg/ml). RPMI 1640 was used to control for the effects of BMM{phi} medium on the proliferation of MvLu1 cells. All cells were then pulsed with [3H]thymidine for an additional 14 h before collection and scintillation counting to quantitate [3H]thymidine incorporation. D, SHIP1 mRNA levels were determined by quantitative real-time PCR in the same samples described in A and B. E, BMM{phi} were derived normally (ND-BMM{phi}), or in the presence of added corticosterone (20 nM, GC-BMM{phi}), human rTGF-beta (20–40 ng/ml), neutralizing Abs to TGF-beta (2 µg/ml) or with the combination of corticosterone and anti-TGF-beta Abs. SHIP1 expression was determined by Western blot analysis using cell lysates prepared from these populations of cells. All results are representative of more than or equal to three independent experiments. Error bars represent mean ± SD. *, p < 0.05; **, p < 0.001.

 
Taking advantage of Mv1Lu cells, a mink lung epithelial cell line exquisitely sensitive to TGF-beta induced arrest in cellular proliferation, we analyzed whether the increase in TGF-beta transcripts led to an increase in the presence of bioactive TGF-beta in culture. Supernatants collected from BMM{phi} derived in the presence or absence of corticosterone on days 1–5 were added in triplicate onto monolayered MvLu1 cells in 96-well plates with or without neutralizing Abs to TGF-beta. [3H]Thymidine was added to each well following a 24-h coincubation as an indicator of DNA synthesis. MvLu1 cells in triplicate wells were collected 16 h following the addition of the [3H]thymidine and the amount of [3H]thymidine incorporated quantitated by scintillation counting. We found that MvLu1 cells cocultured with supernatants collected from corticosterone-conditioned BMM{phi} cultures did not proliferate as well as MvLu1 cells cocultured in parallel with supernatants collected from BMM{phi} cultures without exogenously added corticosterone (Fig. 7D). The inhibitory effects of supernatants collected from GC-BMM{phi} on the proliferation of MvLu1 cells were abrogated by the addition of neutralizing Abs to TGF-beta (Fig. 7D), indicting that the inhibitory effects are TGF-beta mediated. Our results are consistent with an increased TGF-beta expression as the result of GC conditioning.

To directly implicate TGF-beta in the indirect augmentation of SHIP1 expression in BMM{phi} by GC conditioning, we used neutralizing Abs to TGF-beta (all three isoforms). Fig. 7E clearly shows that when TGF-beta activity is neutralized during the conditioning period, SHIP1 expression became almost undetectable. Consistent with our previously reported findings, SHIP1 protein expression was increased ~2-fold when corticosterone (10–20 nM) was added during the differentiation of BMM{phi}.

TGF-beta expression is elevated in 11betaHSD1–/– bone marrow cells and splenocytes

We reasoned that differentiation of myeloid progenitors into M{phi} in the presence of elevated corticosterone levels, such as the microenvironment of the 11betaHSD1–/– host, would lead to increased SHIP1 expression. Total BM and splenocytes from 11betaHSD1+/+ and 11betaHSD1–/– mice were separated into two fractions based the expression of CD11b. Quantitative real-time PCR was performed to determine the expression of all three isoforms of TGF-beta (TGF-beta1, TGF-beta2, TGF-beta3). As shown in Fig. 8, A and B, TGF-beta1 and TGF-beta2 mRNA levels were not significantly different among 11betaHSD1+/+ and 11betaHSD1–/– BM cells and splenocytes, regardless of CD11b expression. Again, TGF-beta3 was not detected in any of the populations tested. Surprisingly, when an Ab with pan-specificity for all three isoforms of TGF-beta was used to quantitate active TGF-beta homo/heterodimers, we found that TGF-beta expression was enhanced in the CD11b– fraction of BM cells and splenocytes from 11betaHSD1–/– mice (Fig. 8C). Finally, we examined SHIP1 transcript levels in CD11b+ M{phi} from 11betaHSD1+/+ and 11betaHSD1–/– mice. As shown in Fig. 8D, SHIP1 transcript levels were enhanced in CD11b+ M{phi} from the BM (2-fold) and spleen (2.24-fold) of 11betaHSD1–/– mice. Consistently, SHIP1 protein was also increased in 11betaHSD1–/– CD11b+ M{phi} from the BM (Fig. 8E).


Figure 8
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  		FIGURE 8. TGF-beta expression is enhanced in 11betaHSD1–/– mice in vivo. Cells from the BM and spleen of 11betaHSD1+/+ and 11betaHSD1–/– mice were separated into CD11b+ and CD11b– fractions by magnetic bead sorting to determine (A and B) TGF-beta1, TGF-beta2, TGF-beta3 mRNA levels by quantitative real-time PCR. C, Bioactive TGF-beta protein expression by Western blot analysis under nonreducing conditions using an Ab specific for TGF-beta. D, SHIP1 mRNA expression by quantitative real-time PCR. E, SHIP1 protein expression by Western blot analysis using a SHIP1-specific Ab. All results are representative of more than or equal to three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
11betaHSD1–/– mice possess a number of surprisingly desirable qualities, such as stress-induced insulin resistance and resistance to peripheral obesity, as well as resistance to age-associated declines in cognitive functions (10, 11). The administration of inhibitors of 11betaHSD1 enzymatic activity have been reported to improve insulin sensitivity in patients with type II diabetes and improve cognitive functions in the elderly (28). These benefits associated with the generalized depression of 11betaHSD1 activities represent the primary driving force behind the recent search for 11betaHSD1-specific inhibitors (29, 30, 31).

An 11betaHSD1 deficiency is also associated with adverse phenotypes (32). 11betaHSD1–/– murine neonates have been shown to exhibit delayed lung surfactant synthesis following birth (12, 13). Moreover, plasma corticosterone levels in 11betaHSD1–/– mice are 2- to 3-fold higher at the diurnal nadir compared with normal animals probably due to dysregulated feedback inhibition of corticotropin-releasing hormone synthesis in the hypothalamus (14). Both of these findings are hypothesized to be due to the lack of end-organ reactivation of C11-keto-GCs by 11betaHSD1 in the lung and hypothalamus of these animals (12, 14). More recently, Chapman et al. (15) demonstrated that PEM{phi} from 11betaHSD1–/– mice exhibit a decreased phagocytosis of apoptotic neutrophils and overproduce IL-6 following LPS stimulation. These findings are surprising, because the classical paradigm of suppressive influences of GCs would predict that elevated GC levels would promote phagocytosis by professional phagocytes while potently inhibiting inflammatory cytokine synthesis (33, 34, 35).

In this study, we report that thioglycolate-elicited M{phi} from the peritoneal cavity of 11betaHSD1+/+ and 11betaHSD1–/– mice indeed exhibit differential responsiveness to LPS stimulation. LPS-activated 11betaHSD1–/– PEM{phi} produced more IL-6 and IL-12p40 compared with 11betaHSD1+/+ PEM{phi}. A more thorough characterization revealed that 11betaHSD1–/– splenic CD11b+ M{phi} and B220+ B cells both contribute to the increase in TNF-{alpha}, IL-6, and IL-12p40 production by 11betaHSD1–/– splenocytes following LPS stimulation. Similar to 11betaHSD1–/– PEM{phi}, SHIP1 expression was significantly elevated in 11betaHSD1–/– splnM{phi}, suggesting that 11betaHSD1–/– monocytes may already have elevated SHIP1 levels before their recruitment to the peritoneal cavity. Moreover, serum TNF-{alpha}, IL-6, and IL-12p40 levels were augmented in 11betaHSD1–/– mice post-LPS challenge in vivo, indicating that these animals may be more susceptible to endotoxemia. Consistent with this hypothesis, 11betaHSD1–/– mice exhibit increased rapid weight loss upon infection with Salmonella typhimurium (data not shown). Interestingly, 11betaHSD1–/– B cells also expressed increased levels of SHIP1 (data not shown), it is therefore possible that SHIP1 plays a similar positive regulatory role in B cell LPS responses.

M{phi} responsiveness to LPS stimulation is mediated through the TLR4/CD14/MD2-signaling complex (17, 36). Following recognition of LPS by CD14, TLR4 and MD2 are recruited and result in the activation of a myriad of signaling pathways (17). Activation of NF-{kappa}B- and MAPK-signaling cascades ultimately leads to the induction of inflammatory cytokine synthesis, including TNF-{alpha}, IL-6, and IL-12 (17, 18). During our characterization of the activation status of these signaling pathway, we found NF-{kappa}B and MAPK activation were enhanced in 11betaHSD1–/– splnM{phi}. In contrast, PI3K-dependent Akt activation was attenuated in the same population of cells. The phosphorylation of Akt on Ser473 is essential in the activation of Akt kinase activity, which has been demonstrated to potently antagonize the activation of NF-{kappa}B, p38, and Erk44/42 via phosphorylation of inhibitory residues on these signaling molecules (37, 38, 39, 40). Thus, diminished Akt activation could result in the augmentation in NF-{kappa}B, p38, and Erk44/42 activation observed in 11betaHSD1–/– splnM{phi}.

The elevated SHIP1 expression in 11betaHSD1–/– peritoneal and splnM{phi} suggests that SHIP1 may contribute to the attenuation of PI3K-dependent Akt activation and ultimately the overexpression of inflammatory cytokines. In support of this hypothesis, SHIP1 has been implicated in the positive regulation of LPS signaling in murine M{phi}, because TNF-{alpha}, IL-6, and IL-12p40 production by SHIP1–/– BMM{phi} are significantly attenuated following LPS stimulation (41). Similarly, small interfering RNA-mediated knockdown of SHIP1 in primary murine BMM{phi} was recently found to behave similarly to SHIP1–/– BMM{phi} (16). Krystal et al. (42, 43) has clearly demonstrated the skewing of SHIP1–/– BMM{phi} toward a M2 phenotype. In contrast, the overexpression of SHIP1 in RAW264.7 cells results in increased production of the above-mentioned inflammatory cytokines subsequent to stimulation (41).

Although strong evidence suggests positive regulation of inflammatory cytokine expression by SHIP1, a negative regulatory role has also been suggested. LPS-induced IL-6 and IL-12p40 expression is enhanced in SHIP1–/– BMM{phi} (25, 44, 45). Additionally, small interfering RNA-mediated knockdown of SHIP1 expression in RAW264.7 cells also resulted in promoted inflammatory cytokine production (44). In our experiments, augmented SHIP1 expression in PEM{phi} and splnM{phi} and its overexpression in primary BMM{phi} correlated with enhanced inflammatory cytokine production. Therefore, increased SHIP1 levels appear to contribute to proinflammatory cytokine production in a positive manner in primary murine M{phi}.

The generation of PIP3 via PI3K activities controls a variety of cellular processes in addition to TLR signaling, including cellular trafficking/chemotaxis, phagocytosis by professional phagocytes, and the proliferation of a variety of cell types (19, 46). Increased SHIP1 levels in splnM{phi} and PEM{phi} of 11betaHSD1–/– mice imply other PIP3-mediated processes may be affected. Consistent with this hypothesis, the total number of cells obtained from the BM and spleen of 11betaHSD1–/– mice are consistently elevated ~20% compared with WT mice (data not shown), perhaps reflecting a modest influence of increased SHIP1 expression on the proliferation of hemopoietic cells in these hosts. Moreover, the reported defect in the phagocytic capacity of 11betaHSD1–/– PEM{phi} may somehow be associated with an elevated SHIP1 expression in these cells (15).

Increased production of IL-6 by 11betaHSD1–/– PEM{phi} has been suggested to result from the lack of generation of bioactive GCs by 11betaHSD1, which then functions to limit inflammatory cytokine production (15). This explanation seems unlikely, however, because no C11-keto-GCs were present to function as substrate for 11betaHSD1 enzymatic activity in the in vitro culture experiments used to establish IL-6 overproduction (15). 11betaHSD1+/+ and 11betaHSD1–/– BMM{phi} respond similarly to LPS challenge in the presence or absence of IFN-{gamma}, clearly indicating that the hyperresponsiveness of 11betaHSD1–/– splnM{phi} did not result from genetic deficiency of 11betaHSD1 in these cells. Our experiments indicate that 11betaHSD1+/+ and 11betaHSD1–/– myeloid progenitors that are differentiated under identical conditions possess similar responsiveness to subsequent LPS stimulation. We therefore hypothesized that differences in the maturation of myeloid progenitors within the 11betaHSD1–/– host environment may be responsible for creating their hyperresponsive phenotype. This hypothesis begged the question of what difference(s) within the 11betaHSD1–/– host microenvironment may be responsible for the differential differentiation of 11betaHSD1–/– M{phi}.

The only plasma parameter known to be different between 11betaHSD1–/– mice and their WT counterparts under steady-state conditions is the elevated circulating corticosterone levels found in 11betaHSD1–/– animals. The diurnal rhythm of GCs is entrained according to environmental conditions, primarily the 24-h light/dark cycle. To be certain observed differences in plasma GCs levels were not due to differences in husbandry conditions between our facility and those at Edinburgh, we characterized the serum corticosterone levels at the nadir and zenith of the diurnal cycle. Indeed, animals in our colony of 11betaHSD1–/– mice exhibited 2- to 3-fold elevations in circulating GC levels at both of the time points examined. We recently reported the permissive effect of moderately elevated GC levels on BMM{phi} (16). Conditioning of BM-derived M{phi} by the addition of low levels of corticosterone resulted in increased production of inflammatory cytokines (TNF-{alpha}, IL-6, and IL-12p40) following their LPS stimulation. We therefore hypothesized that increased plasma GC levels in 11betaHSD1–/– mice may be responsible for creating the proinflammatory phenotype of M{phi} from the spleens of these animals. Our hypothesis was supported by the observation that GC-conditioning of 11betaHSD1–/– BMM{phi} resulted in increased LPS-inducible cytokine production by ~2-fold.

During GC conditioning of BMM{phi}, TGF-beta1 and TGF-beta2 mRNA and protein expression were found to increase in parallel to SHIP1 expression. Furthermore, the addition of neutralizing Abs to TGF-betas abrogated GC-mediated increases in SHIP1 protein expression in these BMM{phi}, suggesting the concept that GCs enhanced SHIP1 expression via induction of TGF-beta. When the role of TGF-beta in augmenting SHIP1 expression was examined in vivo, we found more TGF-beta protein in the CD11b– (non-M{phi}) population of cells within the BM and spleen of 11betaHSD1–/– mice. These results represent bioactive homo or heterodimers of TGF-betas (molecular mass ~25 kDa) expressed within these cells, because nonreducing conditions were used to detect TGF-beta by Western blot analysis. Therefore, it appears that bioactive TGF-betas produced by non-M{phi} within the BM and spleen enhanced SHIP1 levels in developing 11betaHSD1–/– splnM{phi} and PEM{phi} via a paracrine manner. TGF-beta mRNA expression in CD11b– BM and CD11b– splenocytes did not correlate with increased TGF-beta protein expression in these cells, however, and may reflect differences of posttranscriptional/translational regulation of TGF-beta in vitro (in BMM{phi} cultures) and in vivo (in the BM and spleen). This observation is preceded by examples of discrepancies in TGF-beta transcript/protein levels in a variety of cell types and reflects the complexities in the regulation of TGF-beta expression in vivo (47, 48). TGF-beta expression by hemopoietic cells is ubiquitous, thus a number of cell type(s) could be responsible for the observed increase in bioactive TGF-beta expression. We are currently characterizing the 11betaHSD1–/– immune cell type(s) responsible for this activity.

The differentiation of M{phi} from a granulocyte/monocyte progenitor occurs according to discreet steps under the influence of hormones, growth factors, and cytokines within the host microenvironment. Granulocyte/monocyte progenitors reside in the BM compartment and commit to the myeloid lineage before their exit into peripheral circulation as blood monocytes (49). Circulating blood monocytes extravasate into tissues and further differentiate into tissue-resident M{phi}, where they exhibit varying degrees of self-renewal capacity depending on their anatomical location. For example, alveolar M{phi} are known to have a long half-life and exhibit self-renewal abilities (50, 51, 52). splnM{phi}, in contrast, have a relatively short half-life and do not undergo proliferation in the spleen (53, 54). We propose that dynamic control over bioactive GC availability via 11betaHSD1 can influence the developmental/differentiation programs of myeloid progenitors and thereby modify their subsequent responsiveness to exogenous stimulation. For example, monocytes and M{phi} residing in a tissue where local bioactive GCs levels are elevated may display hyperresponsiveness to LPS stimulation. What other cellular processes are modified by the permissive influences of elevated GC levels remains to be determined.

The permissive roles played by GCs were postulated by the noted physiologist Hans Selye in the 1950s (55, 56, 57). Our data described herein clearly suggest a permissive role of GCs in enhancing the M{phi} inflammatory response elicited by LPS both in vitro and in vivo via the up-regulation of SHIP1. In vivo LPS challenge has been shown to activate the hypothalamic pituitary adrenal axis via the induction of inflammatory cytokines, such as IL-1 and IL-6. Moreover, stress-induced adrenal output of GCs is somewhat exaggerated in 11betaHSD1–/– mice (10). It is therefore possible that elevated IL-6 production following in vivo LPS challenge would lead to further increases in adrenal output of GCs in 11betaHSD1–/– mice. Thus, consistent with the permissive influence of GCs, 11betaHSD1–/– animals seem to mount a greater proinflammatory response to LPS despite elevated GC levels in vivo.

Many other conditions both natural and pathological are known to be associated with elevated plasma GC levels. For example, during chronic stress and the aging process, circulating GCs are known to become elevated (58, 59, 60, 61, 62). Additionally, it is known that patients with metastatic breast cancer, rheumatoid arthritis, and HIV infections also exhibit elevated plasma GCs levels (63, 64, 65). It is possible that a modest increase in GC levels associated with these conditions may play a role in modifying inflammatory responsiveness in these patients. Characterizing the molecular basis for the altered inflammatory responses of patients with these conditions would broaden our understanding of the extent that permissive effects of GCs have on immune function.


   Acknowledgment
 
We thank Dr. Jonathan R. Seckl from the University of Edinburgh for generously providing us with the 11betaHSD1–/– mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Raymond A. Daynes, Department of Pathology, 5B412 School of Medicine, University of Utah, 30 North 1900 East, Salt Lake City, UT 84132-2501. E-mail address: daynes.office{at}path.utah.edu Back

2 Abbreviations used in this paper: GC, glucocorticoid; GR, GC receptor; GRE, GC response element; 11BHSD1, 11beta-hydroxysteroid dehydrogenase type 1; M{phi}, macrophage; BM, bone marrow; WT, wild type; PEM{phi}, peritoneal-elicited M{phi}; splnM{phi}, splenic M{phi}; PIP3, PI(3,4,5)P3. Back

Received for publication June 27, 2007. Accepted for publication August 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Munck, A., P. M. Guyre, N. J. Holbrook. 1984. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr. Rev. 5: 25-44. [Abstract/Free Full Text]
  2. Jacobson, L., R. Sapolsky. 1991. The role of the hippocampus in feedback regulation of the hypothalamic-pituitary-adrenocortical axis. Endocr. Rev. 12: 118-134. [Abstract/Free Full Text]
  3. Wilkinson, C. W., E. R. Peskind, M. A. Raskind. 1997. Decreased hypothalamic-pituitary-adrenal axis sensitivity to cortisol feedback inhibition in human aging. Neuroendocrinology 65: 79-90. [Medline]
  4. Rosner, W.. 1991. Plasma steroid-binding proteins. Endocrinol. Metab. Clin. North Am. 20: 697-720. [Medline]
  5. Siiteri, P. K., J. T. Murai, G. L. Hammond, J. A. Nisker, W. J. Raymoure, R. W. Kuhn. 1982. The serum transport of steroid hormones. Recent Prog. Horm. Res. 38: 457-510. [Medline]
  6. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, R. M. Evans. 1995. The nuclear receptor superfamily: the second decade. Cell 83: 835-839. [Medline]
  7. Truss, M., M. Beato. 1993. Steroid hormone receptors: interaction with deoxyribonucleic acid and transcription factors. Endocr. Rev. 14: 459-479. [Abstract/Free Full Text]
  8. Beato, M., P. Herrlich, G. Schutz. 1995. Steroid hormone receptors: many actors in search of a plot. Cell 83: 851-857. [Medline]
  9. Tomlinson, J. W., E. A. Walker, I. J. Bujalska, N. Draper, G. G. Lavery, M. S. Cooper, M. Hewison, P. M. Stewart. 2004. 11beta-Hydroxysteroid dehydrogenase type 1: a tissue-specific regulator of glucocorticoid response. Endocr. Rev. 25: 831-866. [Abstract/Free Full Text]
  10. Kotelevtsev, Y., M. C. Holmes, A. Burchell, P. M. Houston, D. Schmoll, P. Jamieson, R. Best, R. Brown, C. R. Edwards, J. R. Seckl, J. J. Mullins. 1997. 11beta-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress. Proc. Natl. Acad. Sci. USA 94: 14924-14929. [Abstract/Free Full Text]
  11. Yau, J. L., J. Noble, C. J. Kenyon, C. Hibberd, Y. Kotelevtsev, J. J. Mullins, J. R. Seckl. 2001. Lack of tissue glucocorticoid reactivation in 11beta -hydroxysteroid dehydrogenase type 1 knockout mice ameliorates age-related learning impairments. Proc. Natl. Acad. Sci. USA 98: 4716-4721. [Abstract/Free Full Text]
  12. Hundertmark, S., A. Dill, H. Buhler, P. Stevens, K. Looman, V. Ragosch, J. R. Seckl, C. Lipka. 2002. 11beta-Hydroxysteroid dehydrogenase type 1: a new regulator of fetal lung maturation. Horm. Metab. Res. 34: 537-544. [Medline]
  13. Hundertmark, S., A. Dill, A. Ebert, B. Zimmermann, Y. V. Kotelevtsev, J. J. Mullins, J. R. Seckl. 2002. Fetal lung maturation in 11beta-hydroxysteroid dehydrogenase type 1 knockout mice. Horm. Metab. Res. 34: 545-549. [Medline]
  14. Harris, H. J., Y. Kotelevtsev, J. J. Mullins, J. R. Seckl, M. C. Holmes. 2001. Intracellular regeneration of glucocorticoids by 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-1 plays a key role in regulation of the hypothalamic-pituitary-adrenal axis: analysis of 11beta-HSD-1-deficient mice. Endocrinology 142: 114-120. [Abstract/Free Full Text]
  15. Gilmour, J. S., A. E. Coutinho, J. F. Cailhier, T. Y. Man, M. Clay, G. Thomas, H. J. Harris, J. J. Mullins, J. R. Seckl, J. S. Savill, K. E. Chapman. 2006. Local amplification of glucocorticoids by 11 beta-hydroxysteroid dehydrogenase type 1 promotes macrophage phagocytosis of apoptotic leukocytes. J. Immunol. 176: 7605-7611. [Abstract/Free Full Text]
  16. Zhang, T. Y., R. A. Daynes. 2007. Glucocorticoid conditioning of myeloid progenitors enhances TLR4 signaling via negative regulation of the phosphatidylinositol 3-kinase-Akt pathway. J. Immunol. 178: 2517-2526. [Abstract/Free Full Text]
  17. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511. [Medline]
  18. Seger, R., E. G. Krebs. 1995. The MAPK signaling cascade. FASEB J. 9: 726-735. [Abstract]
  19. Fruman, D. A., L. C. Cantley. 2002. Phosphoinositide 3-kinase in immunological systems. Semin. Immunol. 14: 7-18. [Medline]
  20. Fukao, T., S. Koyasu. 2003. PI3K and negative regulation of TLR signaling. Trends Immunol. 24: 358-363. [Medline]
  21. Alessi, D. R., S. R. James, C. P. Downes, A. B. Holmes, P. R. Gaffney, C. B. Reese, P. Cohen. 1997. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B{alpha}. Curr. Biol. 7: 261-269. [Medline]
  22. Rauh, M. J., J. Kalesnikoff, M. Hughes, L. Sly, V. Lam, G. Krystal. 2003. Role of Src homology 2-containing-inositol 5'-phosphatase (SHIP) in mast cells and macrophages. Biochem. Soc. Trans. 31: 286-291. [Medline]
  23. Sly, L. M., M. J. Rauh, J. Kalesnikoff, T. Buchse, G. Krystal. 2003. SHIP, SHIP2, and PTEN activities are regulated in vivo by modulation of their protein levels: SHIP is up-regulated in macrophages and mast cells by lipopolysaccharide. Exp. Hematol. 31: 1170-1181. [Medline]
  24. Damen, J. E., L. Liu, P. Rosten, R. K. Humphries, A. B. Jefferson, P. W. Majerus, G. Krystal. 1996. The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase. Proc. Natl. Acad. Sci. USA 93: 1689-1693. [Abstract/Free Full Text]
  25. Sly, L. M., M. J. Rauh, J. Kalesnikoff, C. H. Song, G. Krystal. 2004. LPS-induced upregulation of SHIP is essential for endotoxin tolerance. Immunity 21: 227-239. [Medline]
  26. Skurk, T., M. Birgel, Y. M. Lee, H. Hauner. 2006. Effect of troglitazone on tumor necrosis factor {alpha} and transforming growth factor beta expression and action in human adipocyte precursor cells in primary culture. Metabolism 55: 309-316. [Medline]
  27. Otsuka, M., K. Goto, S. Tsuchiya, Y. Aramaki. 2005. Phosphatidylserine-specific receptor contributes to TGF-beta production in macrophages through a MAP kinase, ERK. Biol. Pharm. Bull. 28: 1707-1710. [Medline]
  28. Sandeep, T. C., J. L. Yau, A. M. MacLullich, J. Noble, I. J. Deary, B. R. Walker, J. R. Seckl. 2004. 11beta-hydroxysteroid dehydrogenase inhibition improves cognitive function in healthy elderly men and type 2 diabetics. Proc. Natl. Acad. Sci. USA 101: 6734-6739. [Abstract/Free Full Text]
  29. Jean, D. J., Jr, C. Yuan, E. A. Bercot, R. Cupples, M. Chen, J. Fretland, C. Hale, R. W. Hungate, R. Komorowski, M. Veniant, et al 2007. 2-(S)-phenethylaminothiazolones as potent, orally efficacious inhibitors of 11beta-hydroxysteriod dehydrogenase type 1. J. Med. Chem. 50: 429-432. [Medline]
  30. Sorensen, B., M. Winn, J. Rohde, Q. Shuai, J. Wang, S. Fung, K. Monzon, W. Chiou, D. Stolarik, H. Imade, et al 2007. Adamantane sulfone and sulfonamide 11-beta-HSD1 inhibitors. Bioorg. Med. Chem. Lett. 17: 527-532. [Medline]
  31. Rohde, J. J., M. A. Pliushchev, B. K. Sorensen, D. Wodka, Q. Shuai, J. Wang, S. Fung, K. M. Monzon, W. J. Chiou, L. Pan, et al 2007. Discovery and metabolic stabilization of potent and selective 2-amino-N-(adamant-2-yl) acetamide 11beta-hydroxysteroid dehydrogenase type 1 inhibitors. J. Med. Chem. 50: 149-164. [Medline]
  32. Seckl, J. R., B. R. Walker. 2001. Minireview: 11beta-hydroxysteroid dehydrogenase type 1-a tissue-specific amplifier of glucocorticoid action. Endocrinology 142: 1371-1376. [Abstract/Free Full Text]
  33. Rook, G. A.. 1999. Glucocorticoids and immune function. Baillieres Best Pract. Res. Clin. Endocrinol. Metab. 13: 567-581. [Medline]
  34. Ramirez, F.. 1998. Glucocorticoids induce a Th2 response in vitro. Dev. Immunol. 6: 233-243. [Medline]
  35. Piemonti, L., P. Monti, P. Allavena, B. E. Leone, A. Caputo, V. Di Carlo. 1999. Glucocorticoids increase the endocytic activity of human dendritic cells. Int. Immunol. 11: 1519-1526. [Abstract/Free Full Text]
  36. Taylor, P. R., L. Martinez-Pomares, M. Stacey, H. H. Lin, G. D. Brown, S. Gordon. 2005. Macrophage receptors and immune recognition. Annu. Rev. Immunol. 23: 901-944. [Medline]
  37. Gratton, J. P., M. Morales-Ruiz, Y. Kureishi, D. Fulton, K. Walsh, W. C. Sessa. 2001. Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells. J. Biol. Chem. 276: 30359-30365. [Abstract/Free Full Text]
  38. Kim, A. H., G. Khursigara, X. Sun, T. F. Franke, M. V. Chao. 2001. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol. Cell. Biol. 21: 893-901. [Abstract/Free Full Text]
  39. Rommel, C., B. A. Clarke, S. Zimmermann, L. Nunez, R. Rossman, K. Reid, K. Moelling, G. D. Yancopoulos, D. J. Glass. 1999. Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt. Science 286: 1738-1741. [Abstract/Free Full Text]
  40. Liew, F. Y., D. Xu, E. K. Brint, L. A. O’Neill. 2005. Negative regulation of Toll-like receptor-mediated immune responses. Nat. Rev. Immunol 5: 446-458. [Medline]
  41. Fang, H., R. A. Pengal, X. Cao, L. P. Ganesan, M. D. Wewers, C. B. Marsh, S. Tridandapani. 2004. Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP. J. Immunol. 173: 360-366. [Abstract/Free Full Text]
  42. Rauh, M. J., V. Ho, C. Pereira, A. Sham, L. M. Sly, V. Lam, L. Huxham, A. I. Minchinton, A. Mui, G. Krystal. 2005. SHIP represses the generation of alternatively activated macrophages. Immunity 23: 361-374. [Medline]
  43. Sly, L. M., V. Ho, F. Antignano, J. Ruschmann, M. Hamilton, V. Lam, M. J. Rauh, G. Krystal. 2007. The role of SHIP in macrophages. Front. Biosci. 12: 2836-2848. [Medline]
  44. An, H., H. Xu, M. Zhang, J. Zhou, T. Feng, C. Qian, R. Qi, X. Cao. 2005. Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism. Blood 105: 4685-4692. [Abstract/Free Full Text]
  45. Rauh, M. J., L. M. Sly, J. Kalesnikoff, M. R. Hughes, L. P. Cao, V. Lam, G. Krystal. 2004. The role of SHIP1 in macrophage programming and activation. Biochem. Soc. Trans. 32: 785-788. [Medline]
  46. Fruman, D. A., R. E. Meyers, L. C. Cantley. 1998. Phosphoinositide kinases. Annu. Rev. Biochem. 67: 481-507. [Medline]
  47. Fortunel, N. O., A. Hatzfeld, J. A. Hatzfeld. 2000. Transforming growth factor-beta: pleiotropic role in the regulation of hematopoiesis. Blood 96: 2022-2036. [Abstract/Free Full Text]
  48. Dong, M., G. C. Blobe. 2006. Role of transforming growth factor-beta in hematologic malignancies. Blood 107: 4589-4596. [Abstract/Free Full Text]
  49. Akashi, K., D. Traver, T. Miyamoto, I. L. Weissman. 2000. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404: 193-197. [Medline]
  50. Gordon, S., P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5: 953-964. [Medline]
  51. Naito, M.. 1993. Macrophage heterogeneity in development and differentiation. Arch. Histol. Cytol. 56: 331-351. [Medline]
  52. Sawyer, R. T., P. H. Strausbauch, A. Volkman. 1982. Resident macrophage proliferation in mice depleted of blood monocytes by strontium-89. Lab. Invest. 46: 165-170. [Medline]
  53. Hume, D. A., I. L. Ross, S. R. Himes, R. T. Sasmono, C. A. Wells, T. Ravasi. 2002. The mononuclear phagocyte system revisited. J. Leukocyte Biol. 72: 621-627. [Abstract/Free Full Text]
  54. Wijffels, J. F., Z. de Rover, R. H. Beelen, G. Kraal, N. van Rooijen. 1994. Macrophage subpopulations in the mouse spleen renewed by local proliferation. Immunobiology 191: 52-64. [Medline]
  55. Selye, H.. 1955. Stress, hormones and inflammation. Am. J. Proctol. 6: 226-227. [Medline]
  56. Selye, H.. 1955. Stress and disease. Science 122: 625-631. [Free Full Text]
  57. Selye, H.. 1953. Stress and inflammation. Am. J. Proctol. 4: 229-230. [Medline]
  58. Dalm, S., L. Enthoven, O. C. Meijer, M. H. van der Mark, A. M. Karssen, E. R. de Kloet, M. S. Oitzl. 2005. Age-related changes in hypothalamic-pituitary-adrenal axis activity of male C57BL/6J mice. Neuroendocrinology 81: 372-380. [Medline]
  59. Bruunsgaard, H., B. K. Pedersen. 2003. Age-related inflammatory cytokines and disease. Immunol. Allergy Clin. North Am. 23: 15-39. [Medline]
  60. Chen, J., R. Mo, P. A. Lescure, D. E. Misek, S. Hanash, R. Rochford, M. Hobbs, R. L. Yung. 2003. Aging is associated with increased T-cell chemokine expression in C57BL/6 mice. J. Gerontol. A Biol. Sci. Med. Sci. 58: 975-983. [Medline]
  61. Rohleder, N., B. M. Kudielka, D. H. Hellhammer, J. M. Wolf, C. Kirschbaum. 2002. Age and sex steroid-related changes in glucocorticoid sensitivity of pro-inflammatory cytokine production after psychosocial stress. J. Neuroimmunol. 126: 69-77. [Medline]
  62. Roth, G.. 1980. Interaction of hormones with receptors and alterations of these processes with age. Adv. Exp. Med. Biol. 129: 157-169. [Medline]
  63. Spiegel, D., J. Giese-Davis, C. B. Taylor, H. Kraemer. 2006. Stress sensitivity in metastatic breast cancer: analysis of hypothalamic-pituitary-adrenal axis function. Psychoneuroendocrinology 31: 1231-1244. [Medline]
  64. Cutolo, M., A. Sulli, C. Pizzorni, M. E. Secchi, S. Soldano, B. Seriolo, R. H. Straub, K. Otsa, G. J. Maestroni. 2006. Circadian rhythms: glucocorticoids and arthritis. Ann. NY Acad. Sci. 1069: 289-299. [Medline]
  65. Bailey, M., H. Engler, J. Hunzeker, J. F. Sheridan. 2003. The hypothalamic-pituitary-adrenal axis and viral infection. Viral Immunol. 16: 141-157. [Medline]




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