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The Journal of Immunology, 2007, 178: 3208-3218.
Copyright © 2007 by The American Association of Immunologists, Inc.

Retinoid-Related Orphan Receptor {gamma} Controls Immunoglobulin Production and Th1/Th2 Cytokine Balance in the Adaptive Immune Response to Allergen1

Stephen L. Tilley2,*, Maisa Jaradat{ddagger}, Cliona Stapleton{ddagger}, Darlene Dixon§, Xiaoyang Hua*, Christopher J. Erikson*, Joshua G. McCaskill*, Kelly D. Chason*, Grace Liao{ddagger}, Leigh Jania{dagger}, Beverly H. Koller{dagger} and Anton M. Jetten{ddagger}

* Department of Medicine, Division of Pulmonary and Critical Care Medicine, and {dagger} Department of Genetics University of North Carolina, Chapel Hill, NC 27599; and {ddagger} Laboratory of Respiratory Biology, Cell Biology Section, and § Laboratory of Experimental Pathology, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The retinoid-related orphan receptors (ROR) comprise a distinct subfamily of nuclear receptors with the capacity to act as both repressors and activators of transcription. ROR{gamma}, the most recently identified member of the ROR family, has been shown to be important for the development of normal lymphocyte compartments as well as organogenesis of some lymphoid organs. In this report, we examine the capacity of ROR{gamma}-deficient mice to develop an adaptive immune response to Ag using OVA-induced inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking ROR{gamma}, low-grade pulmonary inflammation was observed and characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal Ig activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, reduced numbers of CD4+ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN-{gamma} and IL-10 production was markedly greater in splenocytes from ROR{gamma}-deficient mice following in vitro restimulation with OVA compared with wild-type splenocytes, and a shift toward a Th1 immune response was observed in sensitized/challenged ROR{gamma}-deficient animals in vivo. These data reveal a critical role for ROR{gamma} in the regulation of Ig production and Th1/Th2 balance in adaptive immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nuclear receptors constitute a superfamily of ligand-dependent transcription factors that include receptors for steroid hormones, retinoic acid, and thyroid hormone; and orphan receptors for which the ligands have yet to be identified (1). Several studies have demonstrated a role for a number of nuclear receptors in the regulation of inflammation. These include the glucocorticoid receptor (GR),3 estrogen receptor, vitamin D receptor, retinoic acid receptors, peroxisome proliferator-activated receptors, and several orphan receptors, including members of the retinoid-related orphan receptor (ROR) subfamily (2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The anti-inflammatory action of glucocorticoids, ligands for the GR, is well established, and these agonists form the first line of treatment in asthma.

The ROR, ROR{alpha}, beta, and {gamma}, constitute a subfamily of nuclear orphan receptors whose roles in inflammation are less well understood. Similar to other nuclear receptors, ROR are believed to be activated by small lipophilic molecules that bind to the receptor, induce a conformational change resulting in the dissociation of corepressor complexes, and facilitating translocation to the nucleus where gene expression is modulated. In addition to their capacity to directly promote gene transcription, an anti-inflammatory role for ROR has been suggested based on findings that they can positively regulate the expression of I{kappa}B{alpha} (12). Thus, similar to GR, ROR can act as both ligand-dependent transcription activators as well as ligand-dependent negative regulators of other transcription factors. Due in part to this complexity, the precise role of these receptors in inflammation has been difficult to predict.

The newest member of the ROR subfamily, ROR{gamma}, was first identified in skeletal muscle by its homology to retinoic acid receptor but speculated to play a role in inflammation based upon its abundant expression in CD4+CD8+ double-positive thymocytes (13, 14). Forced expression of ROR{gamma} in T cell hybridomas resulted in inhibition of TCR-induced proliferation and cell death, suggesting an important role for this ROR subtype in T cell apoptosis (15, 16). Indeed, disruption of the ROR{gamma} gene in mice resulted in a markedly increased rate of apoptosis in CD4+CD8+ double-positive thymocytes, confirming the physiological importance of ROR{gamma} in T cell biology in vivo (17, 18). Due to this accelerated rate of apoptosis, the numbers of peripheral blood T lymphocytes in ROR{gamma}-deficient mice were decreased 6-fold, with a 10-fold reduction in CD4+ cells and a 3-fold reduction in CD8+ cells (17). Despite a lower thymic output, T cells from ROR{gamma}-deficient mice appear to be exported normally to the periphery and have normal proliferative function (18). The numbers of CD4+ and CD8+ lymphocytes are normal to marginally increased in the spleens of ROR{gamma}-deficient mice, whereas the B cell compartment is substantially expanded, ~3-fold the numbers found in wild-type (wt) controls (19). In addition to these abnormalities in lymphocyte homeostasis, ROR{gamma}-deficient mice fail to develop lymph nodes and Peyer’s patches (17, 18). Based on these observations of abnormalities in T cell homeostasis and lymphoid organogenesis, we surmised that ROR{gamma}-deficient mice might have an impaired capacity to generate an adaptive immune response to Ag. To test this hypothesis, we compared the induction of inflammatory responses between wt and ROR{gamma}-deficient mice using OVA-induced inflammation as a model system for allergic asthma.


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

ROR{gamma}-deficient mice (10, 17) were backcrossed four generations to the C57BL/6 background. Heterozygotes were intercrossed to produce ROR{gamma}–/– N4C57BL/6 (hereafter referred to as ROR{gamma}–/–) and ROR{gamma}+/+ N4C57BL/6 wt, littermate controls. Mice were genotyped by PCR analysis of DNA from tail biopsy as described previously (17). All mice were used between 9 and 22 wk of age, and within experiments, mutant and wt mice were matched for age and sex. For some experiments, ROR{gamma}–/– N4BALB/c and ROR{gamma}+/+ N4BALB/c littermate controls were used. These animals were generated by intercrossing heterozygotes after four generations of backcrossing to the BALB/c background. All experiments were conducted in accordance with institutional animal care and use guidelines of the University of North Carolina at Chapel Hill and the National Institute of Environmental Health Sciences.

Induction of allergic inflammation

Mice were sensitized with an i.p. injection of 20 µg of chicken egg OVA (grade V; Sigma-Aldrich) emulsified in 200 µl of aluminum hydroxide (alum) adjuvant (Alhydrogel; Accurate Chemical & Scientific). Fourteen days later, mice were challenged with either aerosolized 1% OVA or saline for 30 min on 5 consecutive days in a whole-body exposure chamber. Twenty-four hours following the last exposure, physiological, pathological, and biochemical phenotyping was performed.

Whole-lung lavage

The chest was opened, and lungs were lavaged by instillation as described previously (11). Lavage fluid was kept on ice and centrifuged at 360 x g for 10 min at 4°C. The supernatants were aliquoted, 10% FBS was added to each aliquot, and samples subsequently frozen at –80°C for cytokine analysis. Cell pellets were resuspended in 1 ml of HBSS and counted with a hemocytometer. Slides of lavage fluid cells were prepared using a Cytospin-3 centrifuge (Thermo Shandon) and stained with Mema-3 and Fast Green (eosinophil stain) for determination of cellular differentials.

Lung histopathology

Immediately following whole-lung lavage, the lungs were inflated with 10% neutral-buffered formalin under 20 cm of pressure, and the trachea was tied off. The lungs were then removed en bloc, immersed in 10% formalin, and dehydrated with ethanol before paraffin embedding. Five- to 6-µm serial sections were cut through the right lung lobes and stained with H&E for general morphology and periodic acid-Schiff (PAS)/Alcian blue for mucus.

Histopathology score

The H&E-stained sections were semiquantitatively scored by a pathologist for the degree of inflammation. Seventy to 100 fields were evaluated and scored in a blinded fashion from 0 to 4. A score of 0 indicated the absence of inflammation in the perivascular, peribronchial/bronchiolar, and intra-alveolar regions of the lung. Two inflammatory scores were assigned. One was based on perivascular infiltrates of lymphocytes only and the other on peribronchial/bronchiolar infiltrates of mixtures of polymorphonuclear neutrophils (PMN), eosinophils (EOS), and lymphocytes. For the perivascular lymphocytic infiltrates, the scores were assigned as follows: 1, minimal: few scattered perivascular infiltrates of lymphocytes involving <15% of the blood vessels; no perivascular cuffing of the lymphocytes; 2, mild: infiltrates of lymphocytes with formation of perivascular cuffs (consisting of fewer than five layers of cells) involving 15–25% of the blood vessels, with few adjacent foci of associated lymphocytes; 3, moderate: infiltrates of lymphocytes with formation of perivascular cuffs (consisting of 5–10 layers of cells) involving >25 but <75% of the blood vessels with some adjacent foci of lymphocytes; and 4, severe: infiltrates of lymphocytes with formation of perivascular cuffs (consisting of >10 layers of cells) involving >75% of the blood vessels, with many adjacent foci of lymphocytes. For the peribronchial/bronchiolar infiltrates of mixtures of PMN, EOS, and lymphocytes, the scores were assigned as follows: 1, minimal: inflammatory infiltrates of primarily lymphocytic cells mixed with a few PMN/EOS within the tissue surrounding the bronchi and bronchioles, involving <15% of the airways; 2, mild: peribronchial/bronchiolar infiltrates of PMN and EOS mixed with lymphocytes involving 15–25% of the airways; 3, moderate: peribronchiolar/bronchial infiltrates of predominantly EOS and PMN mixed with lymphocytes involving >25 but <75% of the airways; and 4, severe: peribronchiolar infiltrates of predominantly PMN and EOS mixed with lymphocytes involving >75% of the airways. In lung tissue sections that were scored moderate or severe, the mixed inflammatory infiltrates often also involved the perivascular and intra-alveolar regions. In cases of intra-alveolar infiltrates, there were alveolar macrophages present in addition to the mixed aggregates of EOS, PMN, and lymphocytes. Scores were subsequently grouped and averaged according to genotype and treatment.

Immunohistochemistry

To detect CD3, frozen mouse lungs sectioned on slides were thawed 5 min on ice, then dipped in ice cold acetone for 2 min. Slides were dipped in 0.3% hydrogen peroxide in methanol for 10 min, then blocked with the Avidin/Biotin Blocking kit (Vector Laboratories), according to the manufacturer’s instructions. CD3 Ab (hamster anti-mouse; Biolegend), 1/10 in Ab diluent (BD Pharmingen), was applied, and sections were incubated in a humid chamber overnight at room temperature (RT). Anti-hamster secondary Ab, 1/2000 in Ab diluent, was applied, and sections incubated for 1 h in a humid chamber at room temperature. Streptavidin-HRP (BD Pharmingen) was applied for 45 min in a humid chamber. The DAB Substrate kit (BD Pharmingen) was used for detection following the manufacturer’s instructions. To detect CD45, paraffin-embedded lungs were sectioned on slides. Sections were deparaffinized in xylene for 5 min and rehydrated through an ethanol series. Slides were heated in 10 mM sodium citrate to boiling in a microwave and allowed to stand in the hot solution for 30 min. Slides were dipped in 0.3% hydrogen peroxide in PBS for 15 min and dipped in acetone for 30 s. Sections were otherwise treated the same as frozen slides, except CD45 Ab (rat anti-mouse; BD Pharmingen), 1/25, was used as the primary Ab and anti-rat Ab, 1/2000, as the secondary Ab. PBS was used to wash the sections between each step. Control sections of each type were treated exactly as experimental sections, except for incubation with PBS rather than primary Ab in Ab diluent. Sections were counterstained with methylene green, dehydrated through alcohols, cleared in xylene, and coverslipped using a permanent mounting medium.

Flow cytometric analysis

Lung lavage fluid lymphocytes expressing CD3, CD4, CD8, and B220 were stained as described previously (17). Briefly, 5 x 105 cells were incubated with a combination of FITC- or PE-conjugated CD3, CD4, CD8, and B220 Abs (BD Pharmingen). Cell surface markers were sorted using a LSR flow cytometer (BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences).

Cytokine assays

Lavage fluid supernatants were stored at –80°C until analysis. Samples were thawed on ice, and levels of IL-13 were measured with a commercially available ELISA kit according to the manufacturer’s instructions (R&D Systems). Other cytokines were assayed with Bio-Plex multiplex cytokine assay system (Bio-Rad) using the Luminex 100 instrument (Luminex) according to the manufacturer’s instructions.

Real-time quantitative RT-PCR analysis

RNA was isolated from the lungs of mice of each genotype and treatment group, reverse transcribed, and analyzed for the expression of CCL-17 (Tarc) and CCL-24 (eotaxin-2) by real-time quantitative RT-PCR. The PCR were conducted in triplicate using a 7300 Real-Time PCR system using the TaqMan One-Step RT-PCR mix (Applied Biosystems).

Splenocyte cultures

Spleens were removed and homogenized with a pellet pestle (Kontes) in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10 mM HEPES (Mediatech) and 10% FBS (Invitrogen Life Technologies). Cell suspension was collected and centrifuged 370 x g at 4°C for 10 min. The pellet was resuspended in 10 ml of ice-cold RBC lysis solution (0.14 M NH4Cl and 0.017 M Tris (pH 7.2)), incubated on ice for 5 min, 5 ml of RPMI-HEPES-FBS added, and centrifuged again. Lysis was repeated if RBC were apparent in the pellet. Once RBC lysis was complete, cells were washed twice with RPMI 1640 medium and strained through a cell strainer (BD Biosciences). Cells were counted, pelleted, and resuspended in RPMI 1640 medium with 10% FBS, 10 U/ml penicillin, and 0.1 mg/ml streptomycin (Invitrogen Life Technologies) at 37°C at a density of 5 x 106 cells/ml in medium alone or medium supplemented with OVA (100 µg/ml). Culture supernatants were harvested after 72 h and stored at –80°C until analysis.

Measurements of Ig isotypes

Blood was obtained from the aorta or heart while mice were deeply anesthetized with pentobarbital. Blood was placed in an Eppendorf tube and allowed to coagulate. Samples were centrifuged at 6000 x g for 2 min, and the serum was removed and stored at –80°C. The samples were thawed on ice, and the levels of several mouse Ig isotypes were determined by ELISA using biotinylated rat anti-mouse IgE (R35-92), IgG1, IgG2a and b, IgG3, IgA, and IgM according to the manufacturer’s instructions (BD Pharmingen). OVA-specific IgE was measured by the same ELISA with the following modification: the 96-well plate was coated with OVA (100 µl of 20 mg/ml OVA in the coating buffer provided by the manufacturer). Samples, standard, and controls were incubated overnight at 4°C. For OVA-specific IgG1 and IgG2c, 96-well high-binding microplates (Fisher Scientific) were coated with 100 µl/well of 1 mg/ml OVA (Sigma-Aldrich) in PBS at 37°C for 2 h, followed by incubation with 200 µl/well blocking buffer (1% BSA in PBS) for 1 h at room temperature. OVA-specific IgG2c measurements were made on serum samples diluted 1/300 and/or serially to 1/3000 in blocking buffer. For OVA-specific IgG1 measurements, samples were serially diluted to 1/5,000 and/or 1/500,000. A standard curve for each assay was prepared by diluting pooled sera that had been previously tested as highly concentrated in OVA-specific IgG1 or IgG2c. One hundred microliters of each standard or sample dilution was applied to the wells in triplicate and incubated overnight at 4°C. One hundred microliters of biotinylated anti-mouse IgG1 (BD Biosciences) (2 µg/ml in blocking buffer) and 100 µl of biotinylated anti-mouse IgG2c (Southern Biotechnology Associates) (1/5000 in blocking buffer) were added to each well and incubated at room temperature for 30 min. One hundred microliters of streptavidin-HRP (BD Biosciences) (1/1000 in blocking buffer) was applied to each well and incubated for 30 min at room temperature, followed by the addition of HRP substrate (ABTS; Sigma-Aldrich). Color was developed for 30 min, and absorbances were read at 405 nm. Calculations were made using the serum dilution that fell closest to the middle of the linear part of the standard curve. Concentrations are expressed as arbitrary units.

Airway physiology

Airway mechanics were evaluated in anesthetized, paralyzed, mechanically ventilated mice using a computer-controlled mouse mechanical ventilator with software specifically designed for evaluating respiratory mechanics (Scireq). Mice were anesthetized with pentobarbital (70–90 mg/kg), tracheostomized with a 19-gauge canula, and ventilated at a frequency of 300 breaths/min, 150 µl of tidal volume, and 4 cm/H2O PEEP. After paralysis with pancuronium bromide (0.8 mg/kg), airway resistance (Raw) was measured using the forced oscillation technique, where an 8-s oscillatory flow waveform is delivered to the murine airway. Using this methodology, Raw is determined by fitting input impedance to the constant phase model. Following these measurements, dynamic resistance (RL) was determined before and following aerosols of methacholine by measuring pressure changes at the airway opening during the delivery of a 1-Hz sinusoidal breath of 150 µl and by fitting the data to the linear single-compartment model using multiple linear regression. After baseline measurements of resistance, aerosols of methacholine were delivered for 20 s each through a side-port in the ventilator circuit, and RL was measured every 10 s postchallenge.

Statistical analysis of data

Statistical analysis was conducted using one-way ANOVA, followed by Bonferroni method (multiple comparison) when comparing between groups and Student’s t test for comparison within the group using the Sigmastat 2.0 software (Jandel). All values are expressed as averages ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lung inflammation in ROR{gamma}–/– mice

To determine the role of ROR{gamma} in the development of allergic inflammation in the lung, we evaluated changes in lung lavage fluid cellularity and lung histology in response to OVA. Interestingly, while saline-treated wt mice showed predominantly macrophages and a few lymphocytes in the lavage fluid, a significant number of saline-treated ROR{gamma}–/– mice had increased numbers of inflammatory cells present (Fig. 1A). Cytospins of this fluid revealed that, while most of the cells were macrophages, lymphocytes and granulocytes were also present in significantly greater numbers than wt mice (Fig. 1, A, C, and D). Fast green stain (specific for EOS granules) revealed that approximately half of these granulocytes were EOS.


Figure 1
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  		FIGURE 1. Lung lavage fluid cellularity in wt and ROR{gamma}–/– mice. Total cells and differentials were evaluated in sensitized mice 24 h following the last of five daily aerosol challenges with saline (A) or OVA (B). Saline-exposed ROR{gamma}–/– ({square}, n = 15), saline-exposed wt ({blacksquare}, n = 13), OVA-exposed ROR{gamma}–/– ({square}, n = 14), and OVA-exposed wt ({blacksquare}, n = 16). Data represent mean cell number ± SEM. *, p < 0.05. C, Representative cytospin from saline-exposed wt mouse showing macrophages and a few lymphocytes. D, Representative cytospin from saline-exposed ROR{gamma}–/– mouse showing granulocytes, lymphocytes, and macrophages. Slides were stained with Hema-3.

 
As expected, OVA-exposed wt mice developed a robust inflammatory cell influx into the lung with significant increases in numbers of EOS, lymphocytes, macrophages, and PMN in the lung lavage fluid compared with saline-treated wt controls. While ROR{gamma}–/– mice also developed increased lung lavage fluid cellularity in response to OVA, the numbers of EOS and lymphocytes were significantly lower than OVA-exposed wt mice (Fig. 1B). Similar reductions in the proportion of EOS were also observed in ROR{gamma}–/– N4BALB/c mice (data not shown).

Histologically, the lungs from saline-treated ROR{gamma}–/– mice showed perivascular accumulation of lymphocytes (Fig. 2C). Saline-treated wt mice did not show this lymphocytic accumulation around blood vessels (Fig. 2A). Wt mice challenged with OVA displayed changes typical of allergen-induced inflammation: infiltration of many peribronchial/vascular EOS, PMN, and lymphocytes as well as moderate amounts of these cells in the alveolar sacs (Fig. 2B). Sections of lungs were scored by a semiquantitative, histopathological scoring system in a blinded fashion using two distinctive criteria: 1) the extent of perivascular infiltration of lymphocytes; and 2) the extent of peribronchial/bronchiolar infiltration of mixtures of PMN, EOS, and lymphocytes. The average peribronchiolar inflammation score for OVA-exposed wt mice was 3.2, indicating moderate inflammation (Fig. 3). Lungs from ROR{gamma}–/– mice challenged with OVA revealed mild peribronchial/bronchiolar infiltration of EOS and PMN (Fig. 2D). The average score for peribronchial/bronchiolar infiltration of inflammatory cells for OVA-exposed ROR{gamma}–/– was 1.4, indicating mild inflammation (Fig. 3). The perivascular infiltration of lymphocytes observed in saline-treated ROR{gamma}–/– mice was further enhanced following OVA treatment (Figs. 2D). In OVA-treated wt mice, a perivascular infiltration of a mixed population of inflammatory cells was seen, consisting largely of EOS and PMN, similar to the peribronchial/bronchiolar infiltrates. The primary lymphocytic perivascular infiltrate found in ROR{gamma}–/– mice was not observed in the saline or OVA-treated wt mice. A similar marked reduction in pulmonary inflammation was also seen in OVA-exposed ROR{gamma}–/– N4BALB/c mice (data not shown).


Figure 2
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  		FIGURE 2. OVA-induced lung inflammation in wt and ROR{gamma}–/– mice. Twenty-four hours following the last of five daily aerosol challenges, lungs were evaluated in sensitized wt and ROR{gamma}–/– mice. Saline-exposed H&E (A and C), OVA-exposed H&E (B and D) mice, and oVa-exposed PAS/Alcian blue for mucus (E and F) in wt (A, B, and E) and ROR{gamma}–/– (C, D, and F) mice. Arrowheads denote perivascular lymphoid aggregates; arrows denote peribronchovascular inflammatory infiltrates. b, bronchus;v, vessel.

 

Figure 3
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  		FIGURE 3. Inflammatory scores for wt and ROR{gamma}–/– mice. H&E-stained sections of lungs from sensitized wt and ROR{gamma}–/– mice challenged with either saline (–) or OVA (+) were scored from 0 to 4 for the extent of inflammatory cell infiltration, as described in Materials and Methods. Perivascular lymphocytes only (Figure 3) and mixed peribronchial/bronchiolar infiltrates ({blacksquare}) were scored separately. Zero to 1 indicates no or little inflammation, whereas 4 represents severe inflammation. Data represent mean inflammatory score ± SEM. *, p < 0.05 compared with saline-exposed group of the same genotype. +, p < 0.05 compared with OVA-exposed wt mice.

 
Mucus metaplasia is another characteristic feature of OVA-induced inflammation. As shown in Fig. 2E, wt mice sensitized and challenged with OVA showed a large number of PAS/Alcian blue-positive staining goblet cells in the respiratory epithelium. In contrast, similarly treated ROR{gamma}–/– mice showed significantly less PAS/Alcian blue-positive goblet cells (Fig. 2F). Both wt and ROR{gamma}–/– saline-treated controls showed absence of PAS/Alcian blue-positive goblet cells in the airway epithelium (data not shown). The attenuation of allergic inflammation observed in OVA-treated ROR{gamma}–/– mice on two different genetic backgrounds suggests that ROR{gamma} is critical for the development of a normal adaptive immune response to allergen.

Lymphocyte phenotyping

To further characterize the perivascular lymphocyte accumulation observed in ROR{gamma}–/– animals, immunohistochemistry was performed using mAbs against CD3 and CD45/B220. As shown in Fig. 4, these perivascular lesions were composed of a mixed population of B and T lymphocytes. To further evaluate changes in lymphocyte populations following OVA challenge, flow cytometry was performed on lavage fluid lymphocytes from saline and OVA-exposed ROR{gamma}–/– and wt animals. Significant increases in CD3+, B220+, CD4+, and CD8+ cells were observed in both wt and ROR{gamma}–/– mice compared with their respective saline-exposed controls (Fig. 4E). A trend toward lower numbers of CD4+ (p = 0.054) and CD8+ (p = 0.069) lymphocytes was observed in the OVA-treated ROR{gamma}–/– group when compared with similarly exposed wt mice. Equally, a trend toward lower numbers of B cells was also observed (p = 0.13). However, T/B cell ratios and CD4+/CD8+ ratios in ROR{gamma}–/– mice were similar to wt controls, and CD4+ T cells constituted the majority of the T cell population in OVA-treated animals of both genotypes.


Figure 4
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  		FIGURE 4. Lymphocyte populations in the lungs of ROR{gamma}–/– mice. Frozen and paraffin-embedded sections from saline-exposed ROR{gamma}–/– mice were used for immunohistochemistry with CD3 and CD45 Abs, respectively. A and C, Controls with secondary Ab only. B, CD3 mAb. D, CD45/B220 mAb. E, Lung lavage fluid was collected from sensitized mice 24 h following the last of five daily aerosol challenges with saline (–) or OVA (+). Lymphocytes were sorted by flow cytometry after Ab labeling of CD3 ({blacksquare}), B220 ({square}), CD4 (Figure 4), and CD8 (Figure 4). Data represent mean cell number ± SEM. n = 6–8 mice/group; *, p < 0.05 compared with respective saline-treated group.

 
Reduced induction of Th2 chemokine mRNA in ROR{gamma}–/– mice

Eotaxin-2/CCL-24 and TARC/CCL-17 are high-affinity ligands for chemokine receptors (CCR3 and CCR4) selectively expressed on cells present in Th2-mediated inflammation (EOS, basophils, and Th2 lymphocytes). To evaluate the expression of these chemokines, RNA from lungs of OVA- and saline-challenged wt and ROR{gamma}–/– mice were examined using quantitative real-time RT-PCR. As shown in Fig. 5, the expression of both eotaxin-2/CCL-24 and TARC/CCL-17 was markedly lower in the lungs of ROR{gamma}–/– mice than in wt animals.


Figure 5
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  		FIGURE 5. Th2 chemokine expression in the lungs of wt and ROR{gamma}–/– mice. Eotaxin/CCL-24 (A) and TARC/CCL-17 (B) mRNA expression were examined by quantitative real-time PCR in OVA-exposed animals and compared with expression in saline-exposed mice of the same genotype. *, p < 0.01.

 
Aberrant cytokine production in ROR{gamma}–/– mice

To further evaluate the immune response to OVA, Th1 and Th2 cytokines were examined in the lung lavage fluid from saline and OVA-exposed wt and ROR{gamma}–/– mice. As shown in Fig. 6, A, C, and E, robust induction of IL-4, IL-5, and IL-13 was observed in wt animals. In contrast, modest induction of these Th2 cytokines was detected in ROR{gamma}–/– mice. ROR{gamma}–/– mice showed trends toward paradoxical elevations in IL-2 and IFN-{gamma} after OVA challenge (p = 0.072, p = 0.061), as well as significant elevations in TNF-{alpha} (Fig. 6, B, F, and G). These results show that ROR{gamma}–/– mice produce an aberrant cytokine response to OVA, which might contribute to the reduced lung inflammation observed in these animals.


Figure 6
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  		FIGURE 6. Cytokines in lung lavage fluid of wt and ROR{gamma}–/– mice. Lung lavage fluid supernatant was collected from sensitized mice 24 h following the last of five daily aerosol challenges with saline (Figure 6, –) or OVA ({blacksquare}, +), and IL-4 (A), IL-2 (B), IL-5 (C), IL-12 (D), IL-13 (E), IFN-{gamma} (F), TNF-{alpha} (G), and IL-10 (H) protein levels were measured. Data represent mean cytokine level ± SEM. *, p < 0.05 compared with saline-exposed group of the same genotype. #, p < 0.05 compared with similarly exposed wt mice. n = 8 mice/group.

 
To further examine the influence of ROR{gamma} deficiency on Ag-induced cytokine production, OVA-sensitized splenocytes were examined following in vitro restimulation. As shown in Fig. 7, splenocytes from ROR{gamma}–/– mice showed marked increases in all cytokines examined following stimulation with OVA, suggesting that these animals are capable of generating a robust adaptive immune response to Ag. Significant increases in IFN-{gamma} levels were observed following OVA stimulation of ROR{gamma}–/– splenocytes (p = 0.03) at 6.8 times higher than levels produced by wt OVA-stimulated cells (p = 0.06) (Fig. 7D). Production of the T regulatory cytokine IL-10 was also examined following in vitro restimulation. OVA-stimulated splenocytes from ROR{gamma}–/– mice produced five times greater amounts of IL-10 than similarly treated wt splenocytes (p = 0.04) (Fig. 7E). Taken together, these studies suggest that ROR{gamma} can significantly influence the cytokine profile induced by Ag.


Figure 7
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  		FIGURE 7. Cytokine production by splenocytes from wt and ROR{gamma}–/– mice. Splenocytes were cultured at 37°C at a density of 5 x 106 cells/ml in RPMI 1640 medium alone (Figure 7, –) or medium supplemented with OVA (100 µg/ml) ({blacksquare}, +). Culture supernatants were harvested after 72 h, and protein levels of IL-4 (A), IL-2 (B), IL-5 (C), IFN-{gamma} (D), and IL-10 (E) were measured. Data represent mean cytokine level ± SEM. *, p < 0.05 compared with saline-exposed group of the same genotype. #, p < 0.05 compared with similarly exposed wt mice. n = 6–8 mice/group.

 
Spontaneous polyclonal Ig production in ROR{gamma}–/– mice

Ig production by ROR{gamma}–/– mice was first examined by measuring total IgE in sera. As expected, total IgE levels increased significantly in wt mice following OVA challenge (Fig. 8A). Total IgE levels in saline-treated ROR{gamma}–/– mice were significantly greater than saline-treated wt mice and were as high as levels observed in OVA-treated wt animals. Total IgE did not change significantly in ROR{gamma}–/– mice after OVA treatment (p = 0.78). To determine whether dysregulated Ig production by ROR{gamma}–/– mice was restricted to the IgE class, IgM, IgA, and IgG subclasses were measured. As shown in Fig. 8B, IgM and all IgG subclasses trended higher in ROR{gamma}–/– mice compared with wt controls.


Figure 8
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  		FIGURE 8. Ig levels in the serum of wt and ROR{gamma}–/– mice. Serum was collected from sensitized mice 24 h following the last of five daily aerosol challenges with saline (Figure 8, –) or OVA ({blacksquare}, +). Total IgE (A), IgA, IgM, and IgG (B), and OVA-specific IgE (C), IgG1 (D), and IgG2c (E) were measured by ELISA. Data are presented as mean serum Ig level ± SEM. *, p < 0.05 compared with saline-exposed group of the same genotype. +, p < 0.05 compared with similarly exposed wt mice. n = 8 mice/group.

 
OVA-specific Ig production has Th1 bias in ROR{gamma}–/– mice

In contrast to total IgE, OVA-specific IgE levels were low in saline-treated ROR{gamma}–/– mice but rose substantially following OVA exposure (Fig. 8C). Next, IgG1 (Th2)- and IgG2c (Th1)-specific Igs were measured in sera from wt and ROR{gamma}–/– animals. As shown in Fig. 8D, IgG1 levels increased substantially in wt animals following OVA treatment. A much more modest induction of IgG1 was observed in ROR{gamma}–/– mice following OVA challenge, and levels were significantly lower than those from OVA-treated wt controls. In contrast, many OVA-exposed ROR{gamma}–/– animals showed marked elevations of IgG2c, whereas levels of this Th1 Ig tended to be lower in wt mice following OVA exposure (109 ± 51 vs 4.5 ± 1.3 U, p = 0.08) (Fig. 8E).

Lack of airflow obstruction in OVA-exposed ROR{gamma}–/– mice

To determine whether the attenuated allergic response in ROR{gamma}–/– mice impacted airway physiology, airway mechanics were evaluated in anesthetized, paralyzed, mechanically ventilated animals. OVA exposure resulted in significant increases in Raw in wt mice (Fig. 9A). In contrast, Raw in OVA-exposed ROR{gamma}–/– mice was no different from saline-treated controls. RL at baseline and following graded methacholine challenge was also no different between OVA-exposed and saline-exposed ROR{gamma}–/– mice (Fig. 9B). In contrast, RL in OVA-exposed wt mice was significantly greater than saline-exposed wt and OVA-exposed ROR{gamma}–/– mice at baseline and following most challenges with methacholine. Failure to detect characteristic physiological changes in the airways of OVA-exposed ROR{gamma}–/– mice is consistent with the markedly attenuated allergic inflammation observed in these animals.


Figure 9
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  		FIGURE 9. Airway physiology in wt and ROR{gamma}–/– mice. Twenty-four hours following the last of five daily exposures to saline ({square}) or OVA ({blacksquare}), baseline Raw (A) and resistance following challenge with aerosolized methacholine (B) was determined in anesthetized, paralyzed, and mechanically ventilated mice. A, Raw was measured by oscillatory mechanics. B, RL was measured during a single 1-hz breath. B, Baseline. • represents wt OVA-exposed mice (n = 14), {circ} represents OVA-exposed ROR{gamma}–/– mice (n = 10), {blacksquare} represents saline-exposed wt mice (n = 15), and {square} represents saline-exposed ROR{gamma}–/– mice (n = 12). For both A and B, data represent mean resistance ± SEM. *, p < 0.05 vs saline-exposed wt mice; #, p < 0.05 vs OVA-exposed ROR{gamma}–/– mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we examined the capacity of mice deficient in ROR{gamma} to develop allergic lung inflammation in response to OVA sensitization and challenge. Similar to the asthmatic airway, this murine response is dominated by Th2 cells and a corresponding compliment of cytokines. We show that allergic lung inflammation produced by this model is severely blunted in ROR{gamma}–/– mice and that cytokine profiles are altered in the absence of ROR{gamma}. While a number of immunological lesions have been described in this mouse line, our findings here suggest additional roles for ROR{gamma} in T cell homeostasis and response to allergen.

The most striking finding of the present study was the aberrant cytokine profile produced by ROR{gamma}–/– mice. In contrast to wt mice, which developed a robust Th2 immune response characterized by the induction of IL-4, IL-5, and IL-13, OVA-sensitized and -challenged ROR{gamma}–/– animals showed unexpected increases in TNF-{alpha}, IL-2, and IFN-{gamma} and reciprocal reductions in IL-4, IL-5, and IL-13. OVA-stimulated splenocytes from these animals also showed excessive production of IFN-{gamma}. Interestingly, ROR{gamma}–/– mice showed a trend toward greater IL-10 levels in the lung lavage fluid, and production of IL-10 by OVA-stimulated splenocytes was significantly greater than that produced by similarly treated splenocytes from wt animals. This enhanced IL-10 production by ROR{gamma}–/– mice is one potential mechanism for the suppression of allergic lung inflammation observed in our experiments. IL-10 is a major regulator of innate and adaptive immunity, and a number of studies have shown that this cytokine can inhibit allergic inflammation by several different mechanisms. IL-10 has been shown to reduce proinflammatory cytokine release by mast cells, down-regulate EOS function, inhibit cytokine production and chemokine receptor expression by immune cells, and inhibit T cell proliferation (20, 21, 22, 23, 24). In vivo, mice deficient in IL-10 demonstrate exaggerated allergic responses (25, 26, 27). Conversely, IL-10 administration to mice before allergen treatment induces Ag-specific T cell unresponsiveness (28).

Therefore, it is possible that the attenuated allergic inflammation observed in ROR{gamma}–/– mice is driven by excessive IL-10 production in response to Ag.

This shift in cytokine profiles, coupled with our findings of reduced IgG1 and elevated IgG2c levels in the sera of OVA-exposed ROR{gamma}–/– mice, demonstrates a switch in the type of Ag-specific immune response to a Th1 type. Thus, another possible mechanism for the attenuation of allergic inflammation is the unexpected induction of Th1 cytokines, particularly IFN-{gamma}. It is well-established that IFN-{gamma} can inhibit several aspects of allergen-induced inflammation, including lung eosinophilia, goblet cell hyperplasia, and CD4+ T cell infiltration and proliferation (29, 30, 31). In our studies, we found IFN-{gamma} levels trended higher in the lung lavage fluid of OVA-treated ROR{gamma}–/– mice compared with similarly treated wt controls. We also observed exaggerated IFN-{gamma} secretion by splenocytes stimulated with OVA in vitro. One possible mechanism for this aberrant immune response in ROR{gamma}–/– mice could be that ROR{gamma} plays a critical role in the functional maturation of APC and their subsequent capacity to direct T cell differentiation. Another possibility is that ROR{gamma} expression by lymphocytes is critical for suppressing transcription of Th1 cytokines.

Recently it has been suggested that IL-17-producing Th (Th17) cells may contribute to the pathogenesis of a number of inflammatory diseases, including allergic asthma (32). In established allergic inflammation, IL-17 has been shown to reduce inflammation by inhibiting the synthesis of Th2 chemokine and cytokine production (33). However, two independent investigations have shown that IL-17 may play a critical role in the induction of allergic inflammation. Mice lacking IL-17 demonstrated attenuated eosinophilic lung inflammation, Th2 cytokine production, and airway hyperresponsive (33, 34). Recent studies showed that Th17 cells are absent in ROR{gamma}–/– mice and indicated that ROR{gamma}t (or ROR{gamma}2), a ROR{gamma} isoform, is responsible for orchestrating the differentiation of Th17 cells both in vitro and in vivo (35). Thus, impaired IL-17 production during the induction phase of allergic inflammation could contribute to the reduced allergic inflammation that we observed in ROR{gamma}–/– mice.

While aberrant cytokine production by ROR{gamma}–/– mice in response to allergen is an attractive mechanism for the reductions in allergic lung inflammation observed in these animals, several other possibilities warrant discussion. ROR{gamma}–/– mice lack lymph nodes and Peyer’s patches, raising the possibility that lack of regional lymph nodes may be responsible for the attenuated response to aerosolized Ag (17, 18). Secondary lymphoid tissue provides an important microenvironment for presentation of Ag and amplification of T cell populations. Therefore, a lack of lymph nodes provides a plausible explanation for the attenuated response to OVA in ROR{gamma}-deficient mice. However, this explanation is not consistent with studies conducted with other mouse lines lacking lymph nodes, where it has been shown that experimental allergic inflammation can develop. Lymphotoxin-{alpha}-deficient mice, similar to ROR{gamma}-deficient mice, are born without detectable lymph nodes or Peyer’s patches. Surprisingly, robust inflammation was observed in the lungs of sensitized animals following repeated airway challenges with OVA (36). Allergic inflammation was completely abrogated in splenectomized lymphotoxin-{alpha}-deficient mice, suggesting that Ag presentation and allergen-specific T cell expansion occur in the spleen of these mice. Thus, it is unlikely that the absence of lymph nodes alone can account for the drastic reduction in allergic lung disease seen in ROR{gamma}–/– mice because their spleens are available to subserve this function.

Another possibility is an impairment of T cell migration to the lung from secondary lymphoid organs. As discussed above, since ROR{gamma}–/– mice lack regional lymph nodes, the spleen may serve as the major location of Ag presentation and T cell expansion. Previous reports have suggested that lymphocyte trafficking out of the spleen is impaired in ROR{gamma}-deficient mice. Zhang et al. (19) have shown that spleens from ROR{gamma}-deficient mice contain of 2- to 3-fold more cells. This increase in cell number was predominantly due to increased numbers of B lymphocytes. The numbers of CD4+ and CD8+ T lymphocytes were only marginally increased; however, CD4 and CD8 cells in blood were significantly reduced, suggesting that T and B cells accumulate in the spleens of ROR{gamma}-deficient mice. A defect in the splenic microenvironment was confirmed when it was shown by adoptive transfer experiments that both B and T cells from C57BL/6 bone marrow donors accumulated in the spleens of ROR{gamma}–/– recipients following bone marrow transplantation but also that lymphocytes from ROR{gamma}-deficient mice did not accumulate in the spleens of C57BL/6 or RAG-2 mice (19). Thus, if the spleen is functioning as the secondary lymphoid organ for Ag presentation and trafficking out of the spleen is impeded, a reduced cellular response to Ag challenge may be observed, as was the case for our studies. Robust cytokine production by ROR{gamma}–/– splenocytes stimulated in vitro with OVA, but reduced cell numbers and cytokine levels in the lung in vivo, support the possibility of impaired migration of OVA-specific T cells from the spleen to the lungs of ROR{gamma}–/– mice. High IFN-{gamma} levels produced by OVA-stimulated ROR{gamma}–/– splenocytes supports this contention in light of the well-recognized suppressive effects of IFN-{gamma} on CD4+ T cell migration (31).

A reduction in the CD4+ lymphocyte pool is an additional mechanism that could contribute to the attenuated allergic lung inflammation observed ROR{gamma}–/– mice. While we found similar numbers of CD4+ cells in the lung lavage fluid of saline-exposed ROR{gamma}–/– mice compared with wt controls, the number of peripheral blood CD4+ T cells is substantially reduced, a consequence of accelerated apoptosis of CD4+CD8+ thymocytes (17, 19). Taken together, our data and that of other reports discussed above suggest that several mechanisms may work together in concert to inhibit the full expression of allergic lung inflammation when ROR{gamma}–/– is absent.

Interestingly, low-grade spontaneous inflammation was observed in ROR{gamma}–/– controls that were not exposed to OVA. Evidence for spontaneous inflammation included elevated leukocyte numbers in the lung lavage fluid, perivascular aggregates of lymphocytes on histological sections, and elevated Ig levels of the IgM, IgG, and IgE classes. An infectious etiology for these findings is unlikely based on negative screening serologies for common rodent pathogens and low IL-1beta and TNF-{alpha} levels in the lungs of these mice. One possible explanation for increased Ig levels is the observation that ROR{gamma}–/– mice have a 3-fold increase in the numbers of B lymphocytes in the spleen (19). It is possible that, in the presence of reduced numbers of T cells, the B lymphocytes become unregulated. An alternative explanation is that ROR{gamma}–/– plays a more direct role as a negative regulator of B cell Ig synthesis. In support of this notion are similar findings in mice deficient in another nuclear receptor, vitamin D receptor. These animals have normal numbers of T cells in the spleen, thymus, lymph nodes, and peripheral blood but similarly dysregulated IgE production and, interestingly, attenuated allergic lung inflammation in response to Ag as well (37).

A number of nuclear receptors have been implicated in the regulation of immune functions, including allergen-induced inflammation (10, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49). Although nuclear receptors inhibit inflammation by different mechanisms, most of them all appear at least in part related to an inhibition of the NF-{kappa}B signaling pathway, including inhibition of I{kappa}B{alpha}. Preliminary in vitro studies have indicated that ROR{gamma} does not affect NF-{kappa}B signaling (A. M. Jetten, unpublished observations), suggesting that the alterations in cytokine profiles is mediated by a different mechanism. Thus far, the anti-inflammatory role of the GR and its agonists is the best studied. However, adverse physiological effects of systemic and high-dose inhaled corticosteroids have spurred interest in identifying other nuclear receptors with a more favorable therapeutic/toxic ratio. Our study suggests a role for ROR{gamma} in the development of allergic lung inflammation and has uncovered a role for this nuclear receptor in Ig regulation. Recently, several ROR ligands have been identified (50, 51, 52). Cholesterol and cholesterol derivatives have been reported to bind as agonists to ROR, whereas certain retinoids may function as partial antagonists. These studies suggest that it might be possible to design synthetic ROR (ant)agonists that influence the physiological functions of these orphan nuclear receptors, with potential applications to a number of human diseases. Further studies of the role of ROR in immune cell function and host defense are needed to determine whether ROR ligands could provide useful therapeutic strategies for asthma and other inflammatory diseases.


   Acknowledgments
 
We thank the Keck family for their generous support of the Keck Animal Models Facility at the University of North Carolina.


   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 This work is supported by Intramural Research Program of the National Institute on Environmental Health Sciences, National Institutes of Health, and the extramural National Institutes of Health Grant HL071802. Back

2 Address correspondence and reprint requests to Stephen L. Tilley, 8033 Burnett-Womack, CB# 7219, University of North Carolina, Chapel Hill, NC 27599-7219. E-mail address: stephen_tilley{at}med.unc.edu Back

3 Abbreviations used in this paper: GR, glucocorticoid receptor; EOS, eosinophil; PAS, periodic acid-Schiff; PMN, polymorphonuclear neutrophil; Raw, airway resistance; RL, dynamic resistance; ROR, retinoid-related orphan receptor; Th17, IL-17-producing Th. Back

Received for publication November 10, 2005. Accepted for publication December 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kumar, R., E. B. Thompson. 1999. The structure of the nuclear hormone receptors. Steroids 64: 310-319. [Medline]
  2. Staples, J. E., T. A. Gasiewicz, N. C. Fiore, D. B. Lubahn, K. S. Korach, A. E. Silverstone. 1999. Estrogen receptor {alpha} is necessary in thymic development and estradiol-induced thymic alterations. J. Immunol. 163: 4168-4174. [Abstract/Free Full Text]
  3. Alroy, I., T. L. Towers, L. P. Freedman. 1995. Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. Mol. Cell. Biol. 15: 5789-5799. [Abstract]
  4. Bissonnette, R. P., T. Brunner, S. B. Lazarchik, N. J. Yoo, M. F. Boehm, D. R. Green, R. A. Heyman. 1995. 9-Cis retinoic acid inhibition of activation-induced apoptosis is mediated via regulation of fas ligand and requires retinoic acid receptor and retinoid X receptor activation. Mol. Cell. Biol. 15: 5576-5585. [Abstract]
  5. Moore, K. J., M. L. Fitzgerald, M. W. Freeman. 2001. Peroxisome proliferator-activated receptors in macrophage biology: friend or foe?. Curr. Opin. Lipidol. 12: 519-527. [Medline]
  6. Trifilieff, A., A. Bench, M. Hanley, D. Bayley, E. Campbell, P. Whittaker. 2003. 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. [Medline]
  7. Wittke, A., V. Weaver, B. D. Mahon, A. August, M. T. Cantorna. 2004. Vitamin D receptor-deficient mice fail to develop experimental allergic asthma. J. Immunol. 173: 3432-3436. [Abstract/Free Full Text]
  8. Serhan, C. N., P. R. Devchand. 2001. Novel antiinflammatory targets for asthma: a role for PPAR{gamma}?. Am. J. Respir. Cell Mol. Biol. 24: 658-661. [Free Full Text]
  9. Benayoun, L., S. Letuve, A. Druilhe, J. Boczkowski, M. C. Dombret, P. Mechighel, J. Megret, G. Leseche, M. Aubier, M. Pretolani. 2001. Regulation of peroxisome proliferator-activated receptor {gamma} expression in human asthmatic airways: relationship with proliferation, apoptosis, and airway remodeling. Am. J. Respir. Crit. Care Med. 164: 1487-1494. [Abstract/Free Full Text]
  10. Jetten, A. M.. 2004. 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. [Medline]
  11. Stapleton, C. M., M. Jaradat, D. Dixon, H. S. Kang, S. C. Kim, G. Liao, M. A. Carey, J. Cristiano, M. P. Moorman, A. M. Jetten. 2005. Enhanced susceptibility of staggerer (ROR{alpha}sg/sg) mice to lipopolysaccharide-induced lung inflammation. Am. J. Physiol. 289: L144-L152.
  12. Delerive, P., D. Monte, G. Dubois, F. Trottein, J. Fruchart-Najib, J. Mariani, J. C. Fruchart, B. Staels. 2001. The orphan nuclear receptor ROR {alpha} is a negative regulator of the inflammatory response. EMBO Rep. 2: 42-48. [Medline]
  13. Hirose, T., R. J. Smith, A. M. Jetten. 1994. ROR {gamma}: the third member of ROR/RZR orphan receptor subfamily that is highly expressed in skeletal muscle. Biochem. Biophys. Res. Commun. 205: 1976-1983. [Medline]
  14. Ortiz, M. A., F. J. Piedrafita, M. Pfahl, R. Maki. 1995. TOR: a new orphan receptor expressed in the thymus that can modulate retinoid and thyroid hormone signals. Mol. Endocrinol. 9: 1679-1691. [Abstract/Free Full Text]
  15. He, Y. W., M. L. Deftos, E. W. Ojala, M. J. Bevan. 1998. ROR{gamma}t, a novel isoform of an orphan receptor, negatively regulates Fas ligand expression and IL-2 production in T cells. Immunity 9: 797-806. [Medline]
  16. Littman, D. R., Z. Sun, D. Unutmaz, M. J. Sunshine, H. T. Petrie, Y. R. Zou. 1999. Role of the nuclear hormone receptor ROR {gamma} in transcriptional regulation, thymocyte survival, and lymphoid organogenesis. Cold Spring Harb. Symp. Quant. Biol. 64: 373-381. [Medline]
  17. Kurebayashi, S., E. Ueda, M. Sakaue, D. D. Patel, A. Medvedev, F. Zhang, A. M. Jetten. 2000. Retinoid-related orphan receptor {gamma} (ROR{gamma}) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis. Proc. Natl. Acad. Sci. USA 97: 10132-10137. [Abstract/Free Full Text]
  18. Sun, Z., D. Unutmaz, Y. R. Zou, M. J. Sunshine, A. Pierani, S. Brenner-Morton, R. E. Mebius, D. R. Littman. 2000. Requirement for ROR{gamma} in thymocyte survival and lymphoid organ development. Science 288: 2369-2373. [Abstract/Free Full Text]
  19. Zhang, N., J. Guo, Y. W. He. 2003. Lymphocyte accumulation in the spleen of retinoic acid receptor-related orphan receptor {gamma}-deficient mice. J. Immunol. 171: 1667-1675. [Abstract/Free Full Text]
  20. Marshall, J. S., I. Leal-Berumen, L. Nielsen, M. Glibetic, M. Jordana. 1996. Interleukin (IL)-10 inhibits long-term IL-6 production but not preformed mediator release from rat peritoneal mast cells. J. Clin. Invest. 97: 1122-1128. [Medline]
  21. Schandene, L., C. Alonso-Vega, F. Willems, C. Gerard, A. Delvaux, T. Velu, R. Devos, M. de Boer, M. Goldman. 1994. B7/CD28-dependent IL-5 production by human resting T cells is inhibited by IL-10. J. Immunol. 152: 4368-4374. [Abstract]
  22. Jinquan, T., C. G. Larsen, B. Gesser, K. Matsushima, K. Thestrup-Pedersen. 1993. Human IL-10 is a chemoattractant for CD8+ T lymphocytes and an inhibitor of IL-8-induced CD4+ T lymphocyte migration. J. Immunol. 151: 4545-4551. [Abstract]
  23. Kasama, T., R. M. Strieter, N. W. Lukacs, M. D. Burdick, S. L. Kunkel. 1994. Regulation of neutrophil-derived chemokine expression by IL-10. J. Immunol. 152: 3559-3569. [Abstract]
  24. Takayama, T., A. E. Morelli, N. Onai, M. Hirao, K. Matsushima, H. Tahara, A. W. Thomson. 2001. Mammalian and viral IL-10 enhance C-C chemokine receptor 5 but down-regulate C-C chemokine receptor 7 expression by myeloid dendritic cells: impact on chemotactic responses and in vivo homing ability. J. Immunol. 166: 7136-7143. [Abstract/Free Full Text]
  25. Tournoy, K. G., J. C. Kips, R. A. Pauwels. 2000. Endogenous interleukin-10 suppresses allergen-induced airway inflammation and nonspecific airway responsiveness. Clin. Exp. Allergy 30: 775-783. [Medline]
  26. Berg, D. J., M. W. Leach, R. Kuhn, K. Rajewsky, W. Muller, N. J. Davidson, D. Rennick. 1995. Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inflammatory responses. J. Exp. Med. 182: 99-108. [Abstract/Free Full Text]
  27. Grunig, G., D. B. Corry, M. W. Leach, B. W. Seymour, V. P. Kurup, D. M. Rennick. 1997. Interleukin-10 is a natural suppressor of cytokine production and inflammation in a murine model of allergic bronchopulmonary aspergillosis. J. Exp. Med. 185: 1089-1099. [Abstract/Free Full Text]
  28. Enk, A. H., J. Saloga, D. Becker, M. Mohamadzadeh, J. Knop. 1994. Induction of hapten-specific tolerance by interleukin 10 in vivo. J. Exp. Med. 179: 1397-1402. [Abstract/Free Full Text]
  29. Cohn, L., R. J. Homer, N. Niu, K. Bottomly. 1999. T helper 1 cells and interferon {gamma} regulate allergic airway inflammation and mucus production. J. Exp. Med. 190: 1309-1318. [Abstract/Free Full Text]
  30. Iwamoto, I., H. Nakajima, H. Endo, S. Yoshida. 1993. Interferon {gamma} regulates antigen-induced eosinophil recruitment into the mouse airways by inhibiting the infiltration of CD4+ T cells. J. Exp. Med. 177: 573-576. [Abstract/Free Full Text]
  31. Gajewski, T. F., F. W. Fitch. 1988. Anti-proliferative effect of IFN-{gamma} in immune regulation. I. IFN-{gamma} inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol. 140: 4245-4252. [Abstract]
  32. Weaver, C. T., L. E. Harrington, P. R. Mangan, M. Gavrieli, K. M. Murphy. 2006. Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 24: 677-688. [Medline]
  33. Schnyder-Candrian, S., D. Togbe, I. Couillin, I. Mercier, F. Brombacher, V. Quesniaux, F. Fossiez, B. Ryffel, B. Schnyder. 2006. Interleukin-17 is a negative regulator of established allergic asthma. J. Exp. Med. 203: 2715-2725. [Abstract/Free Full Text]
  34. Nakae, S., Y. Komiyama, A. Nambu, K. Sudo, M. Iwase, I. Homma, K. Sekikawa, M. Asano, Y. Iwakura. 2002. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity 17: 375-387. [Medline]
  35. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, D. J. Cua, D. R. Littman. 2006. The orphan nuclear receptor ROR{gamma}t directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126: 1121-1133. [Medline]
  36. Gajewska, B. U., D. Alvarez, M. Vidric, S. Goncharova, M. R. Stampfli, A. J. Coyle, J. C. Gutierrez-Ramos, M. Jordana. 2001. Generation of experimental allergic airways inflammation in the absence of draining lymph nodes. J. Clin. Invest. 108: 577-583. [Medline]
  37. O’Kelly, J., J. Hisatake, Y. Hisatake, J. Bishop, A. Norman, H. P. Koeffler. 2002. Normal myelopoiesis but abnormal T lymphocyte responses in vitamin D receptor knockout mice. J. Clin. Invest. 109: 1091-1099. [Medline]
  38. Chinetti, G., J. C. Fruchart, B. Staels. 2003. Peroxisome proliferator-activated receptors and inflammation: from basic science to clinical applications. Int. J. Obes. Relat. Metab. Disord. 3: (27 Suppl.):S41-S45.
  39. Woerly, G., K. Honda, M. Loyens, J. P. Papin, J. Auwerx, B. Staels, M. Capron, D. Dombrowicz. 2003. Peroxisome proliferator-activated receptors {alpha} and {gamma} down-regulate allergic inflammation and eosinophil activation. J. Exp. Med. 198: 411-421. [Abstract/Free Full Text]
  40. Mueller, C., A. August. 2003. Attenuation of immunological symptoms of allergic asthma in mice lacking the tyrosine kinase ITK. J. Immunol. 170: 5056-5063. [Abstract/Free Full Text]
  41. Mueller, C., V. Weaver, J. P. Vanden Heuvel, A. August, M. T. Cantorna. 2003. Peroxisome proliferator-activated receptor {gamma} ligands attenuate immunological symptoms of experimental allergic asthma. Arch. Biochem. Biophys. 418: 186-196. [Medline]
  42. Fowler, A. J., M. Y. Sheu, M. Schmuth, J. Kao, J. W. Fluhr, L. Rhein, J. L. Collins, T. M. Willson, D. J. Mangelsdorf, P. M. Elias, K. R. Feingold. 2003. Liver X receptor activators display anti-inflammatory activity in irritant and allergic contact dermatitis models: liver-X-receptor-specific inhibition of inflammation and primary cytokine production. J. Invest. Dermatol. 120: 246-255. [Medline]
  43. Schoonjans, K., L. Dubuquoy, J. Mebis, E. Fayard, O. Wendling, C. Haby, K. Geboes, J. Auwerx. 2005. Liver receptor homolog 1 contributes to intestinal tumor formation through effects on cell cycle and inflammation. Proc. Natl. Acad. Sci. USA 102: 2058-2062. [Abstract/Free Full Text]
  44. Ueki, S., Y. Matsuwaki, H. Kayaba, H. Oyamada, A. Kanda, A. Usami, N. Saito, J. Chihara. 2004. Peroxisome proliferator-activated receptor {gamma} regulates eosinophil functions: a new therapeutic target for allergic airway inflammation. Int. Arch. Allergy Immunol. 1: (134 Suppl.):30-36.
  45. Barish, G. D., R. M. Evans. 2004. A nuclear strike against Listeria—the evolving life of LXR. Cell 119: 149-151. [Medline]
  46. Valledor, A. F., M. Ricote. 2004. Nuclear receptor signaling in macrophages. Biochem. Pharmacol. 67: 201-212. [Medline]
  47. Winoto, A., D. R. Littman. 2002. Nuclear hormone receptors in T lymphocytes. Cell 109: (Suppl.):S57-S66. [Medline]
  48. He, Y. W.. 2000. The role of orphan nuclear receptor in thymocyte differentiation and lymphoid organ development. Immunol. Res. 22: 71-82. [Medline]
  49. Joseph, S. B., M. N. Bradley, A. Castrillo, K. W. Bruhn, P. A. Mak, L. Pei, J. Hogenesch, M. O’Connell, R. G. Cheng, E. Saez, et al 2004. LXR-dependent gene expression is important for macrophage survival and the innate immune response. Cell 119: 299-309. [Medline]
  50. Kallen, J. A., J. M. Schlaeppi, F. Bitsch, S. Geisse, M. Geiser, I. Delhon, B. Fournier. 2002. X-ray structure of the hROR{alpha} LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of ROR{alpha}. Structure 10: 1697-1707. [Medline]
  51. Stehlin-Gaon, C., D. Willmann, D. Zeyer, S. Sanglier, A. Van Dorsselaer, J. P. Renaud, D. Moras, R. Schule. 2003. All-trans retinoic acid is a ligand for the orphan nuclear receptor ROR beta. Nat. Struct. Biol. 10: 820-825. [Medline]
  52. Kurebayashi, S., T. Nakajima, S. C. Kim, C. Y. Chang, D. P. McDonnell, J. P. Renaud, A. M. Jetten. 2004. Selective LXXLL peptides antagonize transcriptional activation by the retinoid-related orphan receptor ROR{gamma}. Biochem. Biophys. Res. Commun. 315: 919-927. [Medline]



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