Enhanced susceptibility of staggerer (ROR{alpha}sg/sg) mice to lipopolysaccharide-induced lung inflammation

Cliona M. Stapleton,1,* Maisa Jaradat,1,* Darlene Dixon,2 Hong Soon Kang,1 Seong-Chul Kim,1 Grace Liao,1 Michelle A. Carey,3 Joey Cristiano,1 Michael P. Moorman,4 and Anton M. Jetten1

Division of Intramural Research, Laboratory of Respiratory Biology, 1Cell Biology Section and 3Molecular and Cellular Biology Section, 2Laboratory of Pathology, and 4Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

Submitted 16 September 2004 ; accepted in final form 14 March 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The retinoid-related orphan receptor {alpha} (ROR{alpha}), a member of the ROR subfamily of nuclear receptors, has been implicated in the control of a number of physiological processes, including the regulation of several immune functions. To study the potential role of ROR{alpha} in the regulation of innate immune responses in vivo, we analyzed the induction of airway inflammation in response to lipopolysaccharide (LPS) challenge in wild-type and staggerer (ROR{alpha}sg/sg) mice, a natural mutant strain lacking ROR{alpha} expression. Examination of hematoxylin and eosin-stained lung sections showed that ROR{alpha}sg/sg mice displayed a higher degree of LPS-induced inflammation than wild-type mice. Bronchoalveolar lavage (BAL) was performed at 3, 16, and 24 h after LPS exposure to monitor the increase in inflammatory cells and the level of several cytokines/chemokines. The increased susceptibility of ROR{alpha}sg/sg mice to LPS-induced airway inflammation correlated with a higher number of total cells and neutrophils in BAL fluids from LPS-treated ROR{alpha}sg/sg mice compared with those from LPS-treated wild-type mice. In addition, IL-1{beta}, IL-6, and macrophage inflammatory protein-2 were appreciably more elevated in BAL fluids from LPS-treated ROR{alpha}sg/sg mice compared with those from LPS-treated wild-type mice. The enhanced susceptibility of ROR{alpha}sg/sg mice appeared not to be due to a repression of I{kappa}B{alpha} expression. Our observations indicate that ROR{alpha}sg/sg mice are more susceptible to LPS-induced airway inflammation and are in agreement with the hypothesis that ROR{alpha} functions as a negative regulator of LPS-induced inflammatory responses.

retinoid-related orphan receptor {alpha}; innate immune response; nuclear receptor


NUCLEAR RECEPTORS MAKE UP a superfamily of ligand-dependent transcription factors (1). The retinoid-related orphan receptor (ROR) subfamily of nuclear receptors consists of ROR{alpha}, ROR{beta}, and ROR{gamma} (named NR1F1 to NR1F3 and RORA to RORC by the Nuclear Receptor Nomenclature Committee and the Human Gene Nomenclature Committee, respectively) (11, 20, 22, 23). ROR regulate transcription by binding as monomers to ROR response elements (RORE) consisting of the consensus sequence AGGTCA preceded by a 6-bp A/T-rich region in the promoter region of target genes (cited in Ref. 22). Typical of nuclear receptors, ROR are structurally composed of an amino-terminal domain, a DNA binding domain, a hinge region, and a ligand-binding domain. Through alternative splicing and promoter usage, each ROR gene generates two or more isoforms that differ only in their amino terminus. The utilization of different promoters results in a cell type-specific expression of certain ROR isoforms. As a consequence, these isoforms regulate different physiological processes and target genes (11, 16, 24). Recently, cholesterol and cholesterol sulfate have been identified as ligands of ROR{alpha}, whereas several retinoids have been reported to bind ROR{beta} and ROR{gamma} (22, 26, 44).

ROR{alpha} is expressed in a variety of tissues, including brain (e.g., cerebellum), kidney, lung, testis, and liver (4, 9, 15, 18, 22, 23, 33, 45). A deletion in the ROR{alpha} gene that results in the lack of ROR{alpha} expression was found to be responsible for the phenotype in staggerer (ROR{alpha}sg/sg) mice, a natural mutant mouse strain (9, 18, 45). Mice deficient in ROR{alpha} expression display an ataxic phenotype caused by severe cerebellar neurodegeneration (9, 16, 18, 42, 45). ROR{alpha} has been further implicated in atherosclerosis, osteogenesis, myogenesis, and angiogenesis (22, 31, 33, 35).

Several studies have indicated a critical role for ROR{alpha} in the regulation of a number of immune functions (21, 22). In vitro stimulation of peritoneal macrophages from ROR{alpha}sg/sg and ROR{alpha}–/– mice by lipopolysaccharide (LPS) results in a hyperinduction of IL-1{alpha}, IL-1{beta}, and TNF-{alpha} (10, 29), whereas overexpression of ROR{alpha} in human primary smooth muscle cells inhibits TNF-{alpha}-induced expression of IL-6, IL-8, and cyclooxygenase (COX)-2 (8). The latter has been linked to an upregulation of I{kappa}B{alpha}, an inhibitor of NF-{kappa}B-mediated transcriptional activation. Collectively, these studies suggest that ROR{alpha} may have a protective role during inflammation.

LPS is a structural component of the cell wall of gram-negative bacteria and a potent proinflammatory agent (19). It is often present at high levels in organic dusts and air pollution particulate matter, and chronic exposure is associated with the development of occupational lung disease (3, 41). Moreover, exposure to LPS is an important factor in determining the severity of asthma (36). The aim of this study was to examine the potential role of ROR{alpha} in the pulmonary, innate immune response. We therefore compared the induction of several well-established events in the inflammatory cascade, including the degree of airway inflammation, accumulation of bronchoalveolar lavage (BAL) fluid neutrophils, and induction of proinflammatory cytokines/chemokines [e.g., IL-6, IL-1{beta}, and macrophage inflammatory protein-2 (MIP-2)] between LPS-exposed wild-type (WT) and ROR{alpha}sg/sg mice. Our results demonstrate that ROR{alpha}sg/sg mice are significantly more susceptible to LPS-induced airway inflammation than WT mice, suggesting that ROR{alpha} functions as a negative regulator of the innate immune response in the airways. We provide evidence indicating that this increased susceptibility is not related to an effect on the NF-{kappa}B signaling or suppression of I{kappa}B{alpha} expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals. Heterozygous (ROR{alpha}+/sg) male and female C57/BL6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). ROR{alpha}+/sg pairs were bred at the National Institute of Environmental Health Sciences (NIEHS), and offspring was genotyped by PCR of tail DNA according to the instructions provided by Jackson Laboratories. ROR{alpha}sg/sg mice were also easily identifiable by their staggerer phenotype. WT littermates were used as control mice. All animal studies followed guidelines outlined by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and protocols were approved by the Institutional Animal Care and Use Committee at NIEHS. NIH-31 feed and water were supplied throughout the experiments.

Experimental protocol. Two- to five-month-old mice were intratracheally instilled with either 50 µl of sterile saline solution or 2 µg of Escherichia coli 0111:B4 LPS (Sigma, St. Louis, MO) diluted in 50 µl of sterile saline. Animals were first lightly anesthetized by placing them in a closed induction chamber connected to an isoflurane vaporizer. The anesthetized animal was quickly removed from the chamber and positioned on a restraint apparatus and the animal’s upper and lower incisors were suspended with rubber bands. The restraint apparatus held the animal with its head tilted on a 60° incline. The inside of the mouth was illuminated using a Nicholas illuminator. With the use of forceps, the tongue was gently pulled to one side of the bottom of the mouth, and the light source was positioned so that the glottis could be seen. The blunt, slightly bent needle (24-gauge x 1 inch) was gently inserted past the glottis and into the trachea. The tracheal rings could be felt as the tip of the needle was gently inserted. The tip of the blunt needle was in a position that was between the midpoint of the trachea and the bifurcation of the trachea. Saline, or the suspension of LPS, was slowly injected into the trachea. On completion of the instillation, the animal was returned to its housing cage and placed under a warming lamp until it had regained consciousness. Mice were killed at 3, 16, and 24 h following treatment with saline or LPS. The chest was opened and lungs were lavaged by instillation and withdrawal of 1 x 1 ml of Hanks' balanced salt solution (Sigma). The recovery of the instilled fluid was measured. In almost all instances, 90–96% of the volume was retrieved. In a few mice, the recovery was less; these mice were excluded from the study. After BAL, the right lobe of the lung was inflated and fixed with 4% neutral buffered paraformaldehyde, processed, and embedded in paraffin as described previously (14).

Histopathological analysis of the lung. Serial sections (5 to 6 µm) were stained with hematoxylin and eosin and subsequently semiquantitatively scored by a pathologist for the degree of inflammation. Sections were scored in an unbiased fashion from 0 to 4. A score of 0 indicated the absence of inflammation; 1, minimal perivascular infiltrates of lymphocytes without involvement of the peribronchiolar or alveolar regions; 2, mild perivascular and peribronchiolar infiltrates of lymphocytes mixed with fewer neutrophils; 3, moderate perivascular and peribronchiolar infiltrates of predominantly neutrophils mixed with fewer lymphocytes, and infiltrates of neutrophils within the alveolar sacs with increased alveolar macrophages; and 4, severe perivascular and peribronchiolar infiltrates of predominantly neutrophils mixed with lymphocytes, alveolar sac accumulations of neutrophils and fewer lymphocytes, and increased alveolar macrophages. Although lavage may influence the alveolar inflammatory infiltrate content, the observed histological differences between treated vs. control mice and WT vs. ROR{alpha}sg/sg mice were consistent and significant. Scores were subsequently grouped and averaged according to genotype and treatment.

BAL fluid processing and analysis. BAL fluid was kept on ice and centrifuged at 1,000 g for 10 min at 4°C. The supernatants were aliquoted and 10% fetal bovine serum was added to each aliquot, which was then frozen at –70°C for chemokine and cytokine analysis. Cell pellets were resuspended in 1 ml of Hanks' balanced salt solution, and cells were counted (Coulter Electronics, Hialeah, FL). Slides of cells from the BAL fluid were prepared using a Cytospin-3 centrifuge (Shandon, Pittsburgh, PA) and stained with Wright-Giemsa (Fisher Scientific, Pittsburgh, PA) for an unbiased differential cell count.

Cytokine and chemokine analysis. Levels of IL-6, IL-1{beta}, and MIP-2 in BAL fluid were measured using ELISA assays (R&D Systems, Minneapolis, MN).

Statistical analysis of data. Statistical analysis was carried out using one-way ANOVA followed by the Bonferroni method (multiple comparison) when comparing among groups and Student's t-test for comparison within the group using SigmaStat 2.0 software (Jandel). All values are expressed as averages + SE.

White blood cell analysis. Blood was obtained from saphenous vein, red blood cells were lysed with ZAP-OGLOBIN II lytic reagent, and total white blood cells were counted using a Coulter Counter (Coulter, Miami, FL). In addition, cells were differentially counted in an unbiased manner using a Hema-Tek II Slide Stainer (Bayer, Tarrytown, NY).

Northern blot analysis. Total RNA from tissue and cells was isolated using TriReagent (Sigma) and then examined by Northern blot analysis using radiolabeled probes for ROR{alpha}, I{kappa}B{alpha}, and COX-2.

Retrovirus infection. ROR{alpha}1 and the dominant negative mutant ROR{alpha}1({Delta}AF2), lacking the activation function 2, were cloned into the expression vector p3XFlag-CMV-7.1 (Sigma). The p3X-Flag-ROR fragments were then inserted into the retroviral vector pLXIN (BD Biosciences, Palo Alto, CA). The pLXIN plasmids were verified by DNA sequencing and restriction mapping. pLXIN plasmid DNA was transfected into the packaging cell line pT67 using Fugene 6 transfection reagent (Roche, Indianapolis, IN). Stable cell lines were established after G418 selection and conditioned medium containing the retrovirus was obtained. The macrophage-like RAW 264.7 cells were subsequently infected with the retrovirus in the presence of polybrene (8 µg/ml). After G418 selection, stable RAW 264.7 cell lines containing pLXIN-3XFlag, pLXIN-3XFlag-ROR{alpha}1, or pLXIN-3XFlag-ROR{alpha}1({Delta}AF2) were established. Expression of ROR{alpha}1 was verified by Northern and Western blot analyses using the mouse anti-Flag M2 antibody (Sigma).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased susceptibility of ROR{alpha}sg/sg mice to LPS-induced airway inflammation. To examine the potential role of ROR{alpha} in the modulation of the pulmonary innate immune response, we compared LPS-induced acute airway inflammation in WT and ROR{alpha}sg/sg mice. Previous studies have shown that in rodents and in humans, exposure to LPS induces a severe inflammatory response that is accompanied by activation of macrophages, release of several cytokines/chemokines, and infiltration of neutrophils into the airways (13, 40, 46, 48, 51). WT and ROR{alpha}sg/sg mice were exposed to 2 µg of LPS or saline via intratracheal instillation, and BAL fluids and lungs were collected at 3, 16, and 24 h following initial exposure. Hematoxylin and eosin-stained sections of lungs were obtained at these time points and scored in an unbiased fashion by a semiquantitative, histopathological scoring system. Histopathological changes were examined in lungs of both WT and ROR{alpha}sg/sg mice. No histological differences were observed between lungs of unexposed WT and ROR{alpha}sg/sg mice. At 3 h after LPS exposure, a minimal-to-mild accumulation of red blood cells into the perivascular spaces was noted; few neutrophils and lymphocytes were present (Fig. 1). No histopathological differences were observed between LPS-exposed ROR{alpha}sg/sg and WT mice. At 16 h after LPS exposure, histopathological changes observed in lungs of LPS-treated WT and ROR{alpha}sg/sg mice were more severe. Histopathological changes in lungs of LPS-treated WT mice scored an average of 1.7 (Fig. 1) and were characterized by mild perivascular, intravascular, and peribronchiolar infiltrates of lymphatic cells mixed with neutrophils (Fig. 2B). However, histopathological abnormalities in lungs of LPS-treated ROR{alpha}sg/sg mice scored an average of 2.7 (Fig. 1). These lungs exhibited a moderate perivascular, intravascular, and peribronchiolar infiltration of neutrophils and lymphocytes (Fig. 2D). Histopathological changes in lungs 24 h postdosing were similar to those observed at 16 h (Fig. 1). Both WT and ROR{alpha}sg/sg saline-treated mice suffered minimal perivascular infiltrates of lymphocytic cells as shown in Fig. 2. Our observations indicate that ROR{alpha}sg/sg mice are significantly more susceptible to LPS-induced airway inflammation than WT mice. This difference in susceptibility appeared not to be due to structural changes in the lung since histopathological examination did not reveal any apparent structural differences between lungs of WT and ROR{alpha}sg/sg mice.



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Fig. 1. Staggerer (ROR{alpha}sg/sg) mice are more susceptible to LPS-induced airway inflammation than wild-type (WT) mice. Hematoxylin and eosin (H&E)-stained sections of lungs from WT and ROR{alpha}sg/sg mice (n = 11–13/group) 3 (A), 16 (B), and 24 (C) h following saline or LPS instillations were scored in an unbiased manner from 0 to 4, as described in MATERIALS AND METHODS; 0–1 indicates no or little inflammation, and 4 represents severe inflammation. The average histology inflammatory scores from LPS-treated and untreated WT and ROR{alpha}sg/sg mice were calculated and plotted. ROR{alpha}sg/sg mice instilled with LPS suffered a more severe inflammatory response than LPS-instilled WT mice (*P < 0.001). Significant differences between saline- and LPS-exposed WT (~P < 0.001) and between saline- and LPS-exposed ROR{alpha}sg/sg mice (^P < 0.001) are indicated.

 


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Fig. 2. Enhanced LPS-induced inflammation in lungs of ROR{alpha}sg/sg mice compared with those of WT mice. A: representative H&E section of lung from saline-instilled WT mice showing no evidence of inflammation. B: representative section of lung from LPS-exposed WT mice with a score of 1.7, indicating minimal-to-mild inflammation. Perivascular infiltrates of a few lymphocytic cells are observed. C: representative section of lung from saline-instilled ROR{alpha}sg/sg mice with no evidence of inflammation. D: representative section of lung from LPS-exposed ROR{alpha}sg/sg mice with a score of 2.5. This score corresponds to mild-to-moderate perivascular, intravascular, and peribronchiolar infiltrates of neutrophils (PMN) and lymphocytes (arrows). Intra-alveolar infiltrates of PMN (arrowheads) and lymphocytes and an increased number of alveolar macrophages were also observed. Sections shown here were taken from lungs of mice 16 h after exposure.

 
Increased susceptibility of ROR{alpha}sg/sg mice to LPS-induced airway inflammation is associated with an enhancement in the total number of BAL fluid inflammatory cells. At 3 h following either LPS or saline exposure, LPS-treated WT mice showed a significantly higher total cell count compared with LPS-treated ROR{alpha}sg/sg mice (P < 0.05; Fig. 3). In contrast, at 16 h following exposure, total BAL cell count was dramatically increased in both LPS-treated ROR{alpha}sg/sg and WT mice; however, this increase was much more pronounced in ROR{alpha}sg/sg mice (P < 0.0001). At 24 h, LPS-treated ROR{alpha}sg/sg mice continued to display a higher number of cells in BAL fluid compared with LPS-treated WT mice. During the inflammatory response in the lung, alveolar macrophages become activated and release cytokines and chemokines that are responsible for recruiting neutrophils from the bloodstream (32, 46). The neutrophils recruited to the lung provide auxiliary defenses against invading pathogens (30, 47). Figure 4A shows that there was no significant difference in the influx of neutrophils between LPS-treated WT and ROR{alpha}sg/sg mice at 3 h after exposure. However, a large increase in the recruitment of neutrophils to the airways was observed in WT and ROR{alpha}sg/sg mice at 16 and 24 h following exposure to LPS. Interestingly, at 16 h (Fig. 4B), the number of neutrophils in the BAL fluid from ROR{alpha}sg/sg mice was approximately threefold higher than in that from WT mice (P < 0.0001). At 24 h, the number of neutrophils continued to be higher in LPS-treated ROR{alpha}sg/sg mice compared with LPS-treated WT mice (Fig. 4C). These observations are in agreement with our conclusion that ROR{alpha}sg/sg mice exhibit an enhanced susceptibility to LPS-induced airway inflammation.



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Fig. 3. Increased susceptibility of ROR{alpha}sg/sg mice to LPS-induced airway inflammation is associated with an enhancement in the total number of bronchoalveolar lavage fluid (BALF) cells. After saline and LPS exposure, WT and ROR{alpha}sg/sg were killed at 3 (n = 14–16 animals/group, A), 16 (n = 15–17 animals/group, B), and 24 h (n = 8 animals/group, C). Subsequently, BALF were collected, and the total number of cells was determined using a Coulter Counter. The experiments at 3 and 16 h were carried out twice with similar results. Results shown are a compilation of the data from the 2 independent experiments. The results at 24 h are from 1 experiment. Cell count was expressed as cells x 105/ml. Significant differences in cell numbers were observed between LPS-exposed WT vs. ROR{alpha}sg/sg mice at 3 (*P < 0.05) and 16 h (*P < 0.0001). Significant differences between saline- and LPS-exposed WT (~P < 0.05) and between saline- and LPS-exposed ROR{alpha}sg/sg mice (^P < 0.005) are indicated.

 


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Fig. 4. Increased susceptibility of ROR{alpha}sg/sg mice to LPS-induced airway inflammation correlates with a prolonged accumulation of BALF neutrophils. After saline or LPS exposure, WT and ROR{alpha}sg/sg mice were killed at 3 (n = 14–16 animals/group, A and D), 16 (n = 13–16 animals/group, B and E), and 24 h (n = 8 animals/group, C and F). Subsequently, BALF were collected, and the percentage of neutrophils (A–C) and macrophages (D–F) was determined as described in MATERIALS AND METHODS. Significant differences in neutrophils were observed between LPS-exposed WT vs. ROR{alpha}sg/sg mice at 16 h (*P < 0.0001). Significant differences between saline- and LPS-exposed WT (~P < 0.005) and between saline- and LPS-exposed ROR{alpha}sg/sg mice (^P < 0.005) are indicated.

 
As the neutrophils are recruited to the lung, depletion of macrophages begins (32). As shown in Fig. 4, D–F, the number of macrophages in the BAL fluid of both LPS-treated WT and ROR{alpha}sg/sg mice decreased over the 24-h time period. At 3, 16, and 24 h, the number of macrophages in LPS-treated ROR{alpha}sg/sg mice tended to be lower than that of LPS-treated WT mice.

Comparison of different white blood cell populations in WT and ROR{alpha}sg/sg mice did not reveal significant differences in the number of total white blood cells in the circulation or in the percentage of neutrophils, lymphocytes, monocytes, and eosinophils (Table 1).


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Table 1. WBC in WT vs. ROR{alpha}sg/sg mice

 
Increased susceptibility of ROR{alpha}sg/sg mice to LPS-induced airway inflammation correlates with increased cytokine and chemokine release. LPS exposure induces activation of macrophages and release of a number of different cytokines/chemokines (13, 46, 48). We therefore compared the effect of LPS exposure on the level of the cytokines IL-1{beta} and IL-6 and the chemokine MIP-2 in the BAL fluid from WT and ROR{alpha}sg/sg mice. As shown in Fig. 5, A–C, IL-1{beta} levels were dramatically increased in BAL fluid from both WT and ROR{alpha}sg/sg mice 16 and 24 h after LPS exposure. Interestingly, at 16 h the levels of IL-1{beta} were significantly (3-fold) higher in BAL fluid from LPS-treated ROR{alpha}sg/sg mice compared with those of LPS-exposed WT mice (P < 0.0001), whereas at 24 h, the levels remained twofold higher (P < 0.0001). IL-6 levels in BAL fluid were highest at 3 h after LPS exposure (Fig. 5, D–F). At both 3 and 16 h, IL-6 levels were significantly higher in BAL fluid from LPS-exposed ROR{alpha}sg/sg mice compared with those of LPS-exposed WT mice (P < 0.0001), whereas little difference was observed at 24 h.



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Fig. 5. Increased susceptibility of ROR{alpha}sg/sg mice to LPS-induced airway inflammation correlates with prolonged cytokine release. After saline or LPS exposure, WT and ROR{alpha}sg/sg mice were killed at 3 (n = 15–16, A and D), 16 (n = 15–17, B and E), and 24 h (n = 8, C and F). Subsequently, BALF were collected, and levels of IL-1{beta} (pg/ml, A–C) and IL-6 (pg/ml, D–F) were determined by ELISA. Significant differences in IL-1{beta} and IL-6 levels were observed between LPS-exposed WT vs. ROR{alpha}sg/sg mice at 16 and 24 h and at 3 and 16 h, respectively (*P < 0.0001). Significant differences between saline- and LPS-exposed WT (~P < 0.002, except E ~P < 0.05) and between saline- and LPS-exposed ROR{alpha}sg/sg mice (^P < 0.002, except A ^P < 0.05) are indicated.

 
Next, the levels of the chemokine MIP-2 were measured in BAL fluid of mice treated with either saline or LPS. Little difference in MIP-2 levels was observed between saline-treated WT and ROR{alpha}sg/sg mice. In LPS-treated mice, MIP-2 levels were highest at 3 h after LPS exposure (Fig. 6). At this time point, MIP-2 was significantly higher (130%) in BAL fluid from LPS-treated ROR{alpha}sg/sg mice than in those of LPS-treated WT mice (P < 0.0001). Although the amount of MIP-2 present in BAL fluid had dramatically dropped by 16 and 24 h, levels present in BAL fluid from LPS-exposed ROR{alpha}sg/sg mice remained significantly (210%) higher (P < 0.005) than those detected in BAL fluid from LPS-exposed WT mice. These observations support the conclusion that ROR{alpha}sg/sg mice exhibit a greater sensitivity to LPS-induced airway inflammation than WT mice.



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Fig. 6. Increased susceptibility of ROR{alpha}sg/sg mice to LPS-induced airway inflammation correlates with enhanced levels of the chemokine macrophage inflammatory protein-2 (MIP-2). After saline or LPS exposure, WT and ROR{alpha}sg/sg mice were killed at 3 (n = 15–16, A), 16 (n = 15–17, B), and 24 h (n = 8, C). Subsequently, BALF were collected, and levels of MIP-2 (pg/ml) were determined by ELISA. Significant differences in MIP-2 levels were observed between LPS-exposed WT vs. ROR{alpha}sg/sg mice at 3, 16, and 24 h (*P < 0.005). Significant differences between saline- and LPS-exposed WT (~P < 0.0001, 3 h; ~P < 0.05, 16 h) and between saline- and LPS-exposed ROR{alpha}sg/sg mice (^P < 0.001, 3 and 16 h; ^P < 0.05, 24 h) are indicated.

 
ROR{alpha} does not affect I{kappa}B{alpha} expression. A previous study reported that ROR{alpha}1 positively regulates the expression of I{kappa}B{alpha} mRNA (8). It has been suggested therefore that ROR{alpha} may function as a negative regulator of inflammation, and, inversely, inflammation may be enhanced in mice deficient in ROR{alpha}. To further investigate the potential role of I{kappa}B{alpha}, we examined the induction of I{kappa}B{alpha} mRNA in lungs from control and LPS-exposed WT and ROR{alpha}sg/sg mice. As shown in Fig. 7A, no difference in the induction of I{kappa}B{alpha} mRNA was observed in lungs from control and LPS-exposed WT and ROR{alpha}sg/sg mice. These results do not support a role for I{kappa}B{alpha} in the enhanced inflammatory response in ROR{alpha}sg/sg mice. This was supported by experiments examining the effects of ROR{alpha}1 and ROR{alpha}1({Delta}AF2), a mutant of ROR{alpha}1 that lacks the activation function 2 and functions as a dominant negative receptor in macrophage RAW 264.7 cells. Expression of ROR{alpha}1 and ROR{alpha}1({Delta}AF2) mRNA in RAW 264.7 cells was confirmed by Northern blot analysis (Fig. 7B) and Western blot analysis (not shown). Treatment of RAW 264.7 cells with LPS caused a dramatic increase in the expression of I{kappa}B{alpha} and COX-2 mRNA that is mediated through activation of the NF-{kappa}B signaling pathway. Little difference was observed in the induction of these genes between LPS-treated parental RAW 264.7 cells and cells expressing either pLXIN empty vector, pLXIN-ROR{alpha}1, or pLXIN-ROR{alpha}1({Delta}AF2). These results suggest that in this cell system, expression of ROR{alpha}1 and ROR{alpha}1({Delta}AF2) does not influence either the NF-{kappa}B signaling pathway or the expression of I{kappa}B{alpha}.



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Fig. 7. Expression of ROR{alpha} does not affect the induction of I{kappa}B{alpha} mRNA in lungs of LPS-treated mice or in LPS-treated macrophage RAW 264.7 cells. A: total RNA was isolated from lungs of WT and ROR{alpha}sg/sg mice exposed to either saline or LPS. RNA was then examined by Northern blot analysis for the expression of I{kappa}B{alpha} mRNA. B: parental macrophage RAW 264.7 cells and cells expressing either the empty retroviral vector pLXIN, pLXIN-ROR{alpha}1, or pLXIN-ROR{alpha}1({Delta}AF2) were treated with and without LPS for 16 h before total RNA was isolated. RNA was then examined by Northern blot analysis for the expression of ROR{alpha}, I{kappa}B{alpha}, and cyclooxygenase (COX)-2 mRNA.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Initial characterization of ROR{alpha}sg/sg mice showed that splenocytes derived from ROR{alpha}sg/sg mice exhibit a delay in terminating their immune response (49), whereas studies with smooth muscle cells and macrophages provided evidence suggesting a negative role for ROR{alpha} in the regulation of inflammation (8, 10, 29). Based on these observations, we hypothesized that ROR{alpha}sg/sg mice (18) may undergo an enhanced or prolonged inflammatory response in the lung and that ROR{alpha} may have a protective role in airway inflammation. To examine the potential negative modulatory role of ROR{alpha} in LPS-induced airway inflammation in vivo, we compared the induction of several inflammatory responses, including recruitment of inflammatory cells and cytokine/chemokine secretion in WT and ROR{alpha}sg/sg mice.

At 3 h after LPS exposure, no significant histopathological differences were observed between lungs of WT and ROR{alpha}sg/sg mice. Examination of lungs at 16 and 24 h after exposure to LPS showed that both WT and ROR{alpha}sg/sg mice exhibit several histopathological abnormalities characteristic for acute airway inflammation that, however, differed in severity. LPS-exposed WT mice showed mild abnormalities, whereas ROR{alpha}sg/sg mice exhibited a moderate perivascular, intravascular, and peribronchiolar infiltration of neutrophils and lymphocytes. In addition, lungs of LPS-exposed ROR{alpha}sg/sg mice exhibited greater intra-alveolar infiltrates of neutrophils and lymphocytes and an increased number of alveolar macrophages. These results suggest that lack of ROR{alpha} expression has a proinflammatory effect in this model.

Stimulation of the innate immune response is associated with the induction of several inflammatory mediators, such as IL-1{beta}, IL-6, and MIP-2 (2, 28, 46). Our results demonstrated that 16 and 24 h after LPS exposure, the levels of IL-1{beta} in BAL fluid from ROR{alpha}sg/sg mice were, respectively, three- and twofold higher than that from WT mice. The levels of MIP-2 and IL-6 were dramatically increased in both WT and ROR{alpha}sg/sg mice 3 h after LPS exposure and diminished by 16 and 24 h. Interestingly, at 16 h, the levels of IL-6 in BAL fluid from ROR{alpha}sg/sg mice remained significantly elevated compared with those in WT mice, whereas MIP-2 levels remained significantly elevated at both the 16- and 24-h time points. MIP-2 and other chemokines have been reported to function as chemoattractants for neutrophils (38). Examination of BAL fluid demonstrated that the number of neutrophils was enhanced in both WT and ROR{alpha}sg/sg mice; however, at 16 h, the number of neutrophils in ROR{alpha}sg/sg mice was 2.5-fold higher than that in BAL fluid from WT mice and remained significantly higher at 24 h. These collective observations suggest that ROR{alpha}sg/sg mice exhibit a more prolonged inflammatory response and are in agreement with the hypothesis that ROR{alpha} has a negative modulatory role in LPS-induced innate immune response in the airways. Our results are consistent with previously reported studies showing a greatly enhanced induction of cytokines in response to LPS in isolated ROR{alpha}sg/sg and ROR{alpha}–/– macrophages compared with WT macrophages (10, 29). ROR{alpha} was shown to be highly expressed in resting macrophages and downregulated during LPS activation. These observations support the concept that ROR{alpha} functions as a suppressor of the LPS-induced inflammatory response. Based on these results, one might predict that the host defense system against bacteria is diminished in ROR{alpha}sg/sg mice and that these mice therefore might be more susceptible to bacterial infections. Experiments are underway to investigate this.

The underlying mechanisms by which ROR{alpha} modulates LPS-induced inflammatory responses have yet to be determined. Understanding the mechanism by which ROR{alpha} regulates inflammation will also enable us to answer the question of whether lack of ROR{alpha} expression enhances the susceptibility to LPS-induced inflammation or increases the severity of inflammation. LPS has been reported to act through Toll-like receptors (TLRs), transmembrane proteins that are expressed in many cell types. Triggering of the TLR4 signaling pathway leads to activation of the NF-{kappa}B cascade as well as of c-Jun amino-terminal kinase and the mitogen-activated protein kinase p38 (17, 19, 27, 46). Activation of these kinase signaling pathways lead to changes in the expression of a number of cell adhesion and proinflammatory genes, including TNF-{alpha} and IL-1{beta} (13, 46, 48). These inflammatory mediators amplify the inflammatory response and induce events downstream in the inflammatory cascade. Induction and release of MIP-2, and other chemokines that function as chemoattractants for neutrophils, lead to the accumulation of these cells in BAL fluid (38). ROR{alpha} may affect any of these step(s) in the inflammatory cascade. A recent study has demonstrated that ROR{alpha} positively regulates the expression of I{kappa}B{alpha} in cultured smooth muscle cells possibly by interacting with an RORE in the promoter regulatory region of the I{kappa}B{alpha} gene (8). Induction of I{kappa}B{alpha} after LPS exposure has been suggested to be involved in the downregulation of cytokine/chemokine expression after induction by LPS (19, 27). Therefore, aberrant regulation of I{kappa}B{alpha} in ROR{alpha}sg/sg mice could at least in part be responsible for the prolonged expression of cytokines/chemokines observed in ROR{alpha}sg/sg mice and as a consequence for the observed increased inflammation. It is interesting to note that the anti-inflammatory action of glucocorticoids involves an inhibition of the NF-{kappa}B signaling pathway (34). This effect is mediated by a direct interaction of the glucocorticoid receptor (GR) with the p65 subunit of NF-{kappa}B. In contrast, the anti-inflammatory action of the peroxisome proliferators-activated receptors (PPAR) appears to occur through an NF-{kappa}B-independent mechanism (50). Comparison of the induction of I{kappa}B{alpha} mRNA expression in lungs from WT and ROR{alpha}sg/sg mice after LPS exposure did not reveal a difference in the level of I{kappa}B{alpha} mRNA expression, suggesting that lack of ROR{alpha} did not influence I{kappa}B{alpha} expression. In addition, ectopic expression of ROR{alpha}1 and ROR{alpha}1({Delta}AF2) in the macrophage cell line RAW 264.7 showed little difference in the induction of I{kappa}B{alpha} and COX-2 mRNA by LPS. Previous studies have shown that induction of both of these genes is dependent on the activation of the NF-{kappa}B signaling pathway (7, 8). Our observations therefore suggest that, under the conditions tested, ROR{alpha} does not appear to affect the NF-{kappa}B signaling pathway and I{kappa}B{alpha} expression. The increased susceptibility to LPS-induced inflammation in ROR{alpha}sg/sg mice is likely to be mediated by a different mechanism. Although histological analysis did not detect structural differences between lungs of WT and ROR{alpha}sg/sg mice, we cannot rule out that changes in susceptibility may be related to developmental effects on the respiratory or immune system. ROR{alpha}sg/sg mice exhibit cerebellar degeneration due to defects in Purkinje cell differentiation (9, 18, 45), and it has been suggested that abnormalities in innervation by the nervous system may account for some of the changes in the immune responses (49).

Recently, Kallen et al. (25) demonstrated that cholesterol and several cholesterol derivatives function as agonists for the ROR{alpha} receptor. Generation of synthetic agonists and antagonists that either activate or inhibit the ROR{alpha} signaling pathway might therefore lead to the development of novel therapeutic strategies for human (inflammatory) disease. Agonists for a number of nuclear receptors, including GR and PPAR, have been shown to have anti-inflammatory actions. The role of glucocorticoids as anti-inflammatory drugs is well established (6, 12, 34, 37, 39, 50). Although glucocorticoids only partially affect LPS-mediated effects, dexamethasone significantly reduces the induction of cytokines and the increase in BAL fluid neutrophils in LPS-induced acute airway inflammation (12, 37). Thiazolidinediones, which function as agonists for PPAR, are used against diabetes and have also been reported to antagonize signaling pathways activated by proinflammatory mediators (5, 43). Because ROR{alpha} appears to have a protective role in LPS-induced airway inflammation as well as in atherosclerosis (21, 33), its anti-inflammatory action may be enhanced by ROR{alpha} ligands. Therefore, it would be interesting to examine in future studies the potential anti-inflammatory effects of synthetic high-affinity ROR{alpha} agonists.


    ACKNOWLEDGMENTS
 
We thank Drs. Stephen Tilley, Dan Morgan, and Jeff Card for valuable comments and advice regarding this manuscript. Thanks also to Jesse DeGraff, Herman Price, Clark Colegrove, and Sandy Ward for technical assistance throughout this project.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Jetten, Laboratory of Respiratory Biology, Cell Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Dr., Research Triangle Park, NC 27709 (E-mail: jetten{at}niehs.nih.gov)

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

* C. M. Stapleton and M. Jaradat contributed equally to this work. Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aranda A and Pascual A. Nuclear hormone receptors and gene expression. Physiol Rev 81: 1269–1304, 2001.[Abstract/Free Full Text]
  2. Bastos K, Marinho CR, Barboza R, Russo M, Alvarez JM, and D'Imperio Lima M. What kind of message does IL-12/IL-23 bring to macrophages and dendritic cells? Microbes Infect 6: 630–636, 2004.[CrossRef][ISI][Medline]
  3. Becker S, Soukup JM, Gilmour MI, and Devlin RB. Stimulation of human and rat alveolar macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol Appl Pharmacol 141: 637–648, 1996.[CrossRef][ISI][Medline]
  4. Becker-Andre M, Andre E, and DeLamarter JF. Identification of nuclear receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif sequences. Biochem Biophys Res Commun 194: 1371–1379, 1993.[CrossRef][ISI][Medline]
  5. Chinetti G, Fruchart JC, and Staels B. Peroxisome proliferator-activated receptors and inflammation: from basic science to clinical applications. Int J Obes Relat Metab Disord 27, Suppl 3: S41–S45, 2003.
  6. Corbel M, Lagente V, Theret N, Germain N, Clement B, and Boichot E. Comparative effects of betamethasone, cyclosporin and nedocromil sodium in acute pulmonary inflammation and metalloproteinase activities in bronchoalveolar lavage fluid from mice exposed to lipopolysaccharide. Pulm Pharmacol Ther 12: 165–171, 1999.[CrossRef][ISI][Medline]
  7. D'Acquisto F, Iuvone T, Rombola L, Sautebin L, Di Rosa M, and Carnuccio R. Involvement of NF-{kappa}B in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Lett 418: 175–178, 1997.[CrossRef][ISI][Medline]
  8. Delerive P, Monté D, Dubois G, Trottein F, Fruchart-Najib J, Mariani J, Fruchart JC, and Staels B. The orphan nuclear receptor ROR{alpha} is a negative regulator of the inflammatory response. EMBO Rep 2: 42–48, 2001.[Abstract/Free Full Text]
  9. Dussault I, Fawcett D, Matthyssen A, Bader JA, and Giguere V. Orphan nuclear receptor ROR{alpha}-deficient mice display the cerebellar defects of staggerer. Mech Dev 70: 147–153, 1998.[CrossRef][ISI][Medline]
  10. Dzhagalov I, Giguere V, and He YW. Lymphocyte development and function in the absence of retinoic acid-related orphan receptor {alpha}. J Immunol 173: 2952–2959, 2004.[Abstract/Free Full Text]
  11. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, and Littman DR. An essential function for the nuclear receptor ROR{gamma}(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immun 5: 64–73, 2004.[CrossRef][ISI]
  12. Ek A, Larsson K, Siljerud S, and Palmberg L. Fluticasone and budesonide inhibit cytokine release in human lung epithelial cells and alveolar macrophages. Allergy 54: 691–699, 1999.[CrossRef][ISI][Medline]
  13. Ferretti S, Bonneau O, Dubois GR, Jones CE, and Trifilieff A. IL-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger. J Immunol 170: 2106–2112, 2003.[Abstract/Free Full Text]
  14. Fujimoto W, Nakanishi G, Arata J, and Jetten AM. Differential expression of human cornifin {alpha} and {beta} in squamous differentiating epithelial tissues and several skin lesions. J Invest Dermatol 108: 200–204, 1997.[CrossRef][ISI][Medline]
  15. Giguere V, Tini M, Flock G, Ong E, Evans RM, and Otulakowski G. Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR{alpha}, a novel family of orphan hormone nuclear receptors. Genes Dev 8: 538–553, 1994.[Abstract]
  16. Gold DA, Baek SH, Schork NJ, Rose DW, Larsen DD, Sachs BD, Rosenfeld MG, and Hamilton BA. ROR{alpha} coordinates reciprocal signaling in cerebellar development through sonic hedgehog and calcium-dependent pathways. Neuron 40: 1119–1131, 2003.[CrossRef][ISI][Medline]
  17. Haddad E, Birrell M, McCluskie K, Ling A, Webber SE, Foster ML, and Belvisi MG. Role of p38 MAP kinase in LPS-induced airway inflammation in the rat. Br J Pharmacol 132: 1715–1724, 2001.[CrossRef][ISI][Medline]
  18. Hamilton BA, Frankel WN, Kerrebrock AW, Hawkins TL, FitzHugh W, Kusumi K, Russell LB, Mueller KL, van Berkel V, Birren BW, Kruglyak L, and Lander ES. Disruption of the nuclear hormone receptor ROR{alpha} in staggerer mice. Nature 379: 736–739, 1996.[CrossRef][ISI][Medline]
  19. Hawiger J. Innate immunity and inflammation: a transcriptional paradigm. Immunol Res 23: 99–109, 2001.[CrossRef][ISI][Medline]
  20. He YW. Orphan nuclear receptors in T lymphocyte development. J Leukoc Biol 72: 440–446, 2002.[Abstract/Free Full Text]
  21. Jarvis CI, Staels B, Brugg B, Lemaigre-Dubreuil Y, Tedgui A, and Mariani J. Age-related phenotypes in the staggerer mouse expand the ROR{alpha} nuclear receptor's role beyond the cerebellum. Mol Cell Endocrinol 186: 1–5, 2002.[CrossRef][ISI][Medline]
  22. Jetten AM. Recent advances in the mechanisms of action and physiological functions of the retinoid-related orphan receptors (RORs). Curr Drug Targets Inflamm Allergy 3: 395–412, 2004.[CrossRef][Medline]
  23. Jetten AM, Kurebayashi S, and Ueda E. The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes. Prog Nucleic Acid Res Mol Biol 69: 205–247, 2001.[ISI][Medline]
  24. Jetten AM and Ueda E. Retinoid-related orphan receptors (RORs): roles in cell survival, differentiation and disease. Cell Death Differ 9: 1167–1171, 2002.[CrossRef][ISI][Medline]
  25. Kallen JA, Schlaeppi JM, Bitsch F, Delhon I, and Fournier B. Crystal structure of the human ROR{alpha} ligand binding domain in complex with cholesterol sulfate at 2.2. J Biol Chem 279: 14033–14038, 2004.[Abstract/Free Full Text]
  26. Kallen JA, Schlaeppi JM, Bitsch F, Geisse S, Geiser M, Delhon I, and Fournier B. X-ray structure of the hROR{alpha} LBD at 1.63: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of ROR{alpha}. Structure 10: 1697–1707, 2002.[CrossRef][ISI][Medline]
  27. Karin M and Delhase M. The I{kappa}B kinase (IKK) and NF-{kappa}B: key elements of proinflammatory signalling. Semin Immunol 12: 85–98, 2000.[CrossRef][ISI][Medline]
  28. Knox KS and Twigg HL III. Immunologic and nonimmunologic lung defense mechanisms. In: Middleton's Allergy Principles and Practice (6th ed.), edited by Adkinson NF Jr., Yunginger JW, Busse WW, Bochner BS, Holgate ST, and Simons FER. Philadelphia, PA: Mosby, 2003, p. 687–709.
  29. Kopmels B, Mariani J, Delhaye-Bouchaud N, Audibert F, Fradelizi D, and Wollman EE. Evidence for a hyperexcitability state of staggerer mutant mice macrophages. J Neurochem 58: 192–199, 1992.[ISI][Medline]
  30. Larsen GL and Holt PG. The concept of airway inflammation. Am J Respir Crit Care Med 162: S2–S6, 2000.[Free Full Text]
  31. Lau P, Bailey P, Dowhan DH, and Muscat GE. Exogenous expression of a dominant negative ROR{alpha}1 vector in muscle cells impairs differentiation: ROR{alpha}1 directly interacts with p300 and myoD. Nucleic Acids Res 27: 411–420, 1999.[Abstract/Free Full Text]
  32. Madjpour C, Jewell UR, Kneller S, Ziegler U, Schwendener R, Booy C, Kläusli L, Pasch T, Schimmer RC, and Beck-Schimmer B. Decreased alveolar oxygen induces lung inflammation. Am J Physiol Lung Cell Mol Physiol 284: L360–L367, 2003.[Abstract/Free Full Text]
  33. Mamontova A, Séguret-Macé S, Esposito B, Chaniale C, Bouly M, Delhaye-Bouchard N, Luc G, Staels B, Duverger N, Mariani J, and Tedgui A. Severe atherosclerosis and hypoalphalipoproteinemia in the Staggerer mouse, a mutant of the nuclear receptor ROR{alpha}. Circulation 98: 2738–2743, 1998.[Abstract/Free Full Text]
  34. McKay LI and Cidlowski JA. Cross-talk between nuclear factor-{kappa}B and the steroid hormone receptors: mechanisms of mutual antagonism. Mol Endocrinol 12: 45–56, 1998.[Abstract/Free Full Text]
  35. Meyer T, Kneissel M, Mariani J, and Fournier B. In vitro and in vivo evidence for orphan nuclear receptor ROR{alpha} function in bone metabolism. Proc Natl Acad Sci USA 97: 9197–9202, 2000.[Abstract/Free Full Text]
  36. Michel O. Role of lipopolysaccharide (LPS) in asthma and other pulmonary conditions. J Endotoxin Res 9: 293–300, 2003.[CrossRef][ISI][Medline]
  37. O'Leary EC, Marder P, and Zuckerman SH. Glucocorticoid effects in an endotoxin-induced rat pulmonary inflammation model: differential effects on neutrophil influx, integrin expression, and inflammatory mediators. Am J Respir Cell Mol Biol 15: 97–106, 1996.[Abstract]
  38. Oppenheim JJ, Zachariae COC, Mukaida N, and Matsushima K. Properties of novel proinflammatory supergene "intercine" cytokine family. Annu Rev Immunol 9: 617–648, 1991.[CrossRef][ISI][Medline]
  39. Rocksen D, Lilliehook B, Larsson R, Johansson T, and Bucht A. Differential anti-inflammatory and anti-oxidative effects of dexamethasone and N-acetylcysteine in endotoxin-induced lung inflammation. Clin Exp Immunol 122: 249–256, 2000.[CrossRef][ISI][Medline]
  40. Savov JD, Gavett SH, Brass DM, Costa DL, and Schwartz DA. Neutrophils play a critical role in development of LPS-induced airway disease. Am J Physiol Lung Cell Mol Physiol 283: L952–L962, 2002.[Abstract/Free Full Text]
  41. Schenker MB, Christiani D, Cormier Y, Dimich-Ward H, Doekes G, Dosman J, Douwes J, Dowling K, Enarson D, Green F, Heederik D, Husman K, Kennedy S, Kullman G, Lacasse Y, Lawson B, Malmberg P, May J, McCurdy S, Merchant J, Myers J, Nieuwenhuijsen M, Olenchock S, Saiki C, Schwartz DA, Seiber J, Thorne P, Wagner G, White N, Xu X, and Chan-Yeung M. Respiratory health hazards in agriculture. Am J Respir Crit Care Med 158: S1–S76, 1998.[Free Full Text]
  42. Sidman RL, Lane PW, and Dickie MN. Staggerer, a new mutation in the mouse affecting the cerebellum. Science 137: 610–612, 1962.[ISI][Medline]
  43. Simonin MA, Bordji K, Boyault S, Bianchi A, Gouze E, Becuwe P, Dauca M, Netter P, and Terlain B. PPAR-{gamma} ligands modulate effects of LPS in stimulated rat synovial fibroblasts. Am J Physiol Cell Physiol 282: C125–C133, 2002.[Abstract/Free Full Text]
  44. Stehlin-Gaon C, Willmann D, Zeyer D, Sanglier S, Van Dorsselaer A, Renaud JP, Moras D, and Schule R. All-trans retinoic acid is a ligand for the orphan nuclear receptor ROR{beta}. Nat Struct Biol 10: 820–825, 2003.[CrossRef][ISI][Medline]
  45. Steinmayr M, André E, Conquet F, Rondi-Reig L, Delhaye-Bouchaud N, Auclair N, Daniel H, Crépel F, Mariani J, Sotelo C, and Becker-André M. Staggerer phenotype in retinoid-related orphan receptor {alpha}-deficient mice. Proc Natl Acad Sci USA 95: 3960–3965, 1998.[Abstract/Free Full Text]
  46. Strieter RM, Belperio JA, and Keane MP. Host innate defenses in the lung: the role of cytokines. Curr Opin Infect Dis 16: 193–198, 2003.[ISI][Medline]
  47. Sunil VR, Connor AJ, Zhou P, Gordon MK, Laskin JD, and Laskin DL. Activation of adherent vascular neutrophils in the lung during acute endotoxemia. Respir Res 3: 21–31, 2002.[CrossRef][Medline]
  48. Thorne PS, McCray PB, Howe TS, and O'Neill MA. Early-onset inflammatory responses in vivo to adenoviral vectors in the presence or absence of lipopolysaccharide-induced inflammation. Am J Respir Cell Mol Biol 20: 1155–1164, 1999.[Abstract/Free Full Text]
  49. Trenker E and Hoffmann M. Defective development of the thymus and immunological abnormalities in the neurological mouse mutation "staggerer". J Neurosci 6: 1733–1737, 1986.[Abstract]
  50. Trifilieff A, Bench A, Hanley M, Bayley D, Campbell E, and Whittaker P. PPAR-{alpha} and -{gamma} but not -{delta} agonists inhibit airway inflammation in a murine model of asthma: in vitro evidence for an NF-{kappa}B-independent effect. Br J Pharmacol 139: 163–171, 2003.[CrossRef][ISI][Medline]
  51. Zhang P, Quinton LJ, Bagby GJ, Summer WR, and Nelson S. Interferon-{gamma} enhances the pulmonary CXC chemokine response to intratracheal lipopolysaccharide challenge. J Infect Dis 187: 62–69, 2003.[CrossRef][ISI][Medline]




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