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
2003; Elsevier BV; Volume: 120; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2003.12033.x
ISSN1523-1747
AutoresAshley J. Fowler, Mary Sheu, Matthias Schmuth, Jack Kao, Joachim W. Fluhr, Linda D. Rhein, Jon L. Collins, Timothy M. Willson, David J. Mangelsdorf, Peter M. Elias, Kenneth R. Feingold,
Tópico(s)Food Allergy and Anaphylaxis Research
ResumoActivators of liver X receptors (LXR) stimulate epidermal differentiation and development, but inhibit keratinocyte proliferation. In this study, the anti-inflammatory effects of two oxysterols, 22(R)-hydroxy-cholesterol (22ROH) and 25-hydroxycholesterol (25OH), and a nonsterol activator of LXR, GW3965, were examined utilizing models of irritant and allergic contact dermatitis. Irritant dermatitis was induced by applying phorbol 12-myristate-13-acetate (TPA) to the surface of the ears of CD1 mice, followed by treatment with 22ROH, 25OH, GW3965, or vehicle alone. Whereas TPA treatment alone induced an ≈2-fold increase in ear weight and thickness, 22ROH, 25OH, or GW3965 markedly suppressed the increase (greater than 50% decrease), and to an extent comparable to that observed with 0.05% clobetasol treatment. Histology also revealed a marked decrease in TPA-induced cutaneous inflammation in oxysterol-treated animals. As topical treatment with cholesterol did not reduce the TPA-induced inflammation, and the nonsterol LXR activator (GW3965) inhibited inflammation, the anti-inflammatory effects of oxysterols cannot be ascribed to a nonspecific sterol effect. In addition, 22ROH did not reduce inflammation in LXRβ-/- or LXRαβ-/- animals, indicating that LXRβ is required for this anti-inflammatory effect. 22ROH also caused a partial reduction in ear thickness in LXRα-/- animals, however (≈50% of that observed in wild-type mice), suggesting that this receptor also mediates the anti-inflammatory effects of oxysterols. Both ear thickness and weight increased (≈1.5-fold) in the oxazolone-induced allergic dermatitis model, and 22ROH and GW3965 reduced inflammation by ≈50% and ≈30%, respectively. Finally, immunohistochemistry demonstrated an inhibition in the production of the pro-inflammatory cytokines interleukin-1α and tumor necrosis factor α in the oxysterol-treated sites from both TPA- and oxazolone-treated animals. These studies demonstrate that activators of LXR display potent anti-inflammatory activity in both irritant and allergic contact models of dermatitis, requiring the participation of both LXRα and LXRβ. LXR activators could provide a new class of therapeutic agents for the treatment of cutaneous inflammatory disorders. Activators of liver X receptors (LXR) stimulate epidermal differentiation and development, but inhibit keratinocyte proliferation. In this study, the anti-inflammatory effects of two oxysterols, 22(R)-hydroxy-cholesterol (22ROH) and 25-hydroxycholesterol (25OH), and a nonsterol activator of LXR, GW3965, were examined utilizing models of irritant and allergic contact dermatitis. Irritant dermatitis was induced by applying phorbol 12-myristate-13-acetate (TPA) to the surface of the ears of CD1 mice, followed by treatment with 22ROH, 25OH, GW3965, or vehicle alone. Whereas TPA treatment alone induced an ≈2-fold increase in ear weight and thickness, 22ROH, 25OH, or GW3965 markedly suppressed the increase (greater than 50% decrease), and to an extent comparable to that observed with 0.05% clobetasol treatment. Histology also revealed a marked decrease in TPA-induced cutaneous inflammation in oxysterol-treated animals. As topical treatment with cholesterol did not reduce the TPA-induced inflammation, and the nonsterol LXR activator (GW3965) inhibited inflammation, the anti-inflammatory effects of oxysterols cannot be ascribed to a nonspecific sterol effect. In addition, 22ROH did not reduce inflammation in LXRβ-/- or LXRαβ-/- animals, indicating that LXRβ is required for this anti-inflammatory effect. 22ROH also caused a partial reduction in ear thickness in LXRα-/- animals, however (≈50% of that observed in wild-type mice), suggesting that this receptor also mediates the anti-inflammatory effects of oxysterols. Both ear thickness and weight increased (≈1.5-fold) in the oxazolone-induced allergic dermatitis model, and 22ROH and GW3965 reduced inflammation by ≈50% and ≈30%, respectively. Finally, immunohistochemistry demonstrated an inhibition in the production of the pro-inflammatory cytokines interleukin-1α and tumor necrosis factor α in the oxysterol-treated sites from both TPA- and oxazolone-treated animals. These studies demonstrate that activators of LXR display potent anti-inflammatory activity in both irritant and allergic contact models of dermatitis, requiring the participation of both LXRα and LXRβ. LXR activators could provide a new class of therapeutic agents for the treatment of cutaneous inflammatory disorders. liver X receptor 25-hydroxycholesterol peroxisome-proliferator-activated receptor retinoic-acid-activated receptor 22(R)-hydroxycholesterol transepidermal water loss Nuclear hormone receptors, the largest known family of transcription factors, have been divided into four major subgroups based upon their dimerization and DNA binding properties (Mangelsdorf and Evans, 1995Mangelsdorf D.J. Evans R.M. The RXR heterodimers and orphan receptors.Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2830) Google Scholar). The class II subfamily includes the retinoic-acid-activated receptor (RAR), thyroid hormone receptor, vitamin D receptor, peroxisome-proliferator-activated receptors (PPARs), and liver X receptor (LXR). These receptors recognize small hydrophobic compounds, such as 1,25(OH)2-vitamin D, free fatty acids, retinoids, thyroid hormone, and certain oxysterols. Activation of class II receptors requires heterodimerization with RXR, which allows for optimal regulation of gene expression. In the epidermis, ligand activation of several class II nuclear hormone receptors, including RAR, PPARα, vitamin D receptor, and LXR, regulates keratinocyte proliferation and differentiation in vitro and in vivo (Bikle, 1996Bikle D.D. 1,25 OH2D3-modulated calcium induced keratinocyte differentiation.J Invest Dermatol Symp Proc The. 1996; 1: 22-27PubMed Google Scholar;Eichner et al., 1996Eichner R. Gendimenico G.J. Kahn M. et al.Effects of long-term retinoic acid treatment on epidermal differentiation in vivo, specific modification in the program of terminal differentiation.Br J Dermatol. 1996; 135: 687-695Crossref PubMed Scopus (27) Google Scholar;Fisher and Voorhees, 1996Fisher G.J. Voorhees J.J. 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Topical application of PPARα restores tissue homeostasis.J Invest Dermatol. 2000; 115: 361-367Crossref PubMed Scopus (96) Google Scholar). In addition, ligands of RAR, vitamin D receptor, and PPAR display selected types of anti-inflammatory activity (Devchand et al., 1996Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. The PPAR-α leukotriene B4 pathway to inflammation control.Nature. 1996; 384: 39-43Crossref PubMed Scopus (1205) Google Scholar;Muller and Bendtzen, 1996Muller K. Bendtzen K. 1,25-Dihydroxyvitamin D3 as a natural regulator of human immune functions.J Invest Dermatol Symp Proc The. 1996; 1: 68-71PubMed Google Scholar;Duvic et al., 1997Duvic M. Nagpal S. Asano A.T. Chandraratna R.A. Molecular mechanisms of tazarotene action in psoriasis.J Am Acad Dermatol. 1997; 37: S18-S24Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar;Deluca and Cantorna, 2001Deluca H.F. Cantorna M.T. Vitamin D: its role and uses in immunology.FASEB J. 2001; 15: 2579-2585Crossref PubMed Scopus (715) Google Scholar). Recent studies by our laboratory have shown that activators of PPARα inhibit both irritant and allergic contact dermatitis in murine skin (Sheu et al., 2002Sheu M.Y. Fowler A.J. Kao J.K. et al.Topical PPAR-α activators reduce inflammation in irritant and allergic contact dermatitis models.J Invest Dermatol. 2002; 118: 94-101Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). LXRs were classified initially as orphan members of the nuclear receptor superfamily, due to the unknown nature of their ligands. LXRα and LXRβ are now recognized to bind certain endogenous oxysterols, including 22(R)-hydroxycholesterol (22ROH), 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol (Janowski et al., 1996Janowski B.A. Willy P.J. Rama-Devi T. Falck J.R. Mangelsdorf D.J. An oxysterol signaling pathway mediated by the nuclear receptor LXRα.Nature. 1996; 383: 728-731Crossref PubMed Scopus (1458) Google Scholar;Lehmann et al., 1997Lehmann J.M. Kliewer S.A. Moore L.B. et al.Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway.J Biol Chem. 1997; 272: 3137-3140Crossref PubMed Scopus (1037) Google Scholar;Peet et al., 1998Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cholesterol and bile acid metabolism are impaired in mice lacking nuclear oxysterol receptor LXRα.Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar). Activation of LXR in extracutaneous tissues regulates important steps in cholesterol, fatty acid, and bile acid metabolism (Peet et al., 1998Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cholesterol and bile acid metabolism are impaired in mice lacking nuclear oxysterol receptor LXRα.Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar;Chawla et al., 2001Chawla A. Repa J.J. Evans R.M. Mangelsdorf D.J. Nuclear receptors and lipid physiology: opening the X-files.Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1685) Google Scholar). Two genes, α and β, encode the LXR paralogs. Whereas LXRα is expressed predominately in the liver and to a lesser extent in the kidney, spleen, adrenal gland, and the small intestine (Willy et al., 1995Willy P.J. Umensono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. LXR, a nuclear receptor that defines a distinct retinoid response pathway.Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (916) Google Scholar), LXRβ is ubiquitously expressed (Song et al., 1995Song C. Hiipakka R.A. Kokonitis J.M. Liao S. Ubiquitous receptor structures, immunocytochemical localization, and modulation of gene activation by receptors for retinoic acids and thyroid hormones.Ann NY Acad Sci. 1995; 761: 38-49Crossref PubMed Scopus (45) Google Scholar). Our laboratory has shown that both LXRα and LXRβ are present in cultured human keratinocytes and in fetal rat epidermis (Hanley et al., 1999Hanley K. Komuves L.G. Bass N.M. et al.Fetal epidermal differentiation and barrier development in vivo is accelerated by nuclear hormone receptor activators.J Invest Dermatol. 1999; 113: 788-795Crossref PubMed Scopus (86) Google Scholar;Hanley, 2000Hanley K. et al.Oxysterols induce differentiation in human keratinocytes and increase AP-1-dependent involucrin transcription.J Invest Dermatol. 2000; 114: 545-553Crossref PubMed Scopus (93) Google Scholar). LXRβ is the predominate isoform in adult mouse epidermis, however (Komuves et al., 2002Komuves L.G. Schmuth M. Fowler A.J. et al.Oxysterol stimulation of epidermal differentiation is mediated by liver-X-receptor-α in murine epidermis.J Invest Dermatol. 2002; 118: 25-34Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Recently, we demonstrated that topical application of oxysterols to murine epidermis stimulates keratinocyte differentiation and inhibits proliferation (Komuves et al., 2002Komuves L.G. Schmuth M. Fowler A.J. et al.Oxysterol stimulation of epidermal differentiation is mediated by liver-X-receptor-α in murine epidermis.J Invest Dermatol. 2002; 118: 25-34Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Moreover, these effects still occurred in LXRα-/- mice, but not in LXRβ-/- animals, indicating that the stimulation of keratinocyte differentiation and the inhibition of proliferation induced by oxysterols is mediated predominately by LXRβ (Komuves et al., 2002Komuves L.G. Schmuth M. Fowler A.J. et al.Oxysterol stimulation of epidermal differentiation is mediated by liver-X-receptor-α in murine epidermis.J Invest Dermatol. 2002; 118: 25-34Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In addition, oxysterols accelerate the formation of the epidermal permeability barrier during fetal development (Hanley et al., 1999Hanley K. Komuves L.G. Bass N.M. et al.Fetal epidermal differentiation and barrier development in vivo is accelerated by nuclear hormone receptor activators.J Invest Dermatol. 1999; 113: 788-795Crossref PubMed Scopus (86) Google Scholar;Hanley, 2000Hanley K. et al.Oxysterols induce differentiation in human keratinocytes and increase AP-1-dependent involucrin transcription.J Invest Dermatol. 2000; 114: 545-553Crossref PubMed Scopus (93) Google Scholar), and topical oxysterols improve barrier homeostasis following barrier disruption in normal mice (Komuves et al., 2002Komuves L.G. Schmuth M. Fowler A.J. et al.Oxysterol stimulation of epidermal differentiation is mediated by liver-X-receptor-α in murine epidermis.J Invest Dermatol. 2002; 118: 25-34Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Furthermore, in an animal model of epidermal hyperplasia (Komuves et al., 2002Komuves L.G. Schmuth M. Fowler A.J. et al.Oxysterol stimulation of epidermal differentiation is mediated by liver-X-receptor-α in murine epidermis.J Invest Dermatol. 2002; 118: 25-34Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), oxysterol treatment largely normalized structure, barrier function, and differentiation, suggesting that LXR activators could provide a new category of therapeutic agents for cutaneous diseases that are associated with hyperproliferation and/or disordered differentiation. Activation of several members of the class II family of nuclear hormone receptors can regulate inflammation. Oxysterols, which activate LXR, inhibit the secretion of proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin 1 (IL-1), by macrophages and inhibit lymphocyte activation (Ohlsson et al., 1996Ohlsson B.G. Englund M.C. Karlsson A.L. et al.Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-κB to DNA and the subsequent expression of tumor necrosis factor-α and interleukin-1β in macrophages.J Clin Invest. 1996; 98: 78-89Crossref PubMed Google Scholar). Although these observations suggest that oxysterols could be anti-inflammatory, other studies have shown that oxysterols stimulate the secretion of IL-8 by macrophages, a proinflammatory event (Liu et al., 1997Liu Y. Hulten L.M. Wiklund O. Macrophages isolated from human atherosclerotic plaques produce IL-8.Arteriocler Thromb Vasc Biol. 1997; 2: 317-323Crossref Scopus (151) Google Scholar). Moreover, whether the effects of oxysterols on macrophages and lymphocytes are mediated by LXR or whether they occur via other pathways is unknown. The primary purpose of this study was to determine whether topical applications of oxysterols and/or GW3965, a nonsterol activator of LXR, attenuate inflammation in two distinct models of cutaneous inflammation, irritant and allergic contact dermatitis. Moreover, we addressed whether LXRα and/or LXRβ mediate this anti-inflammatory effect. The results of this study indicate that agonists of LXR, when applied topically to the skin, display receptor-mediated, via both LXRα and LXRβ, anti-inflammatory behavior. Adult CD1 male and female mice, 6–10 wk of age, were purchased from Charles River Laboratories (Wilmington, MA) for use in this study. Phorbol 12-myristate-13-acetate (TPA) induced irritant contact dermatitis was instituted by the topical application of 10 μl 0.03% (wt/vol in acetone) TPA to both the inner and outer surface (20 μl total) of the left ears. Acetone alone (vehicle) was applied to the right ears. Forty-five minutes and 4 h after TPA application, 20 μl of test compounds, 22ROH (10 mM), 25-hydroxycholesterol (25OH; 10 mM), and GW3965 (10 mM), known LXR agonists, were applied to both surfaces of both the left and right ears (40 μl total per ear). Identical treatments were performed with 20 μl of 0.05% clobetasol (1.1 mM), a topical anti-inflammatory glucocorticoid, which served as a positive control. Cholesterol (1%, 2.5 mM), which neither binds nor activates LXR, was applied in a similar fashion as a negative control. Control animals were treated similarly with acetone alone, serving as a vehicle control. All chemical compounds were purchased from Sigma (St. Louis, MO) and were dissolved in absolute acetone (reagent grade) vehicle. GW3965 was synthesized by GlaxoSmithKline High Throughput Chemistry, as described previously (Collins et al, submitted). Allergic contact dermatitis was induced by sensitization (for 2 d) on the shaved backs of CD1 female mice with 20 μl of 15% (wt/vol in acetone) oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one) once a day, followed by challenge on day 7 with a single topical application of 10 μl oxazolone (2%) to the inner and outer surface of the left ears. Acetone alone (10 μl) was applied to the right ears. This challenge was followed by treatment with 22ROH (10 mM), GW3965 (10 mM), clobetasol (0.05%), or acetone at 45 min and 4 h, as described previously. Eighteen hours after the inflammatory insult induced by either TPA or oxazolone, inflammation was assessed as the percentage increase in ear thickness and/or ear weight in the treated left ear versus the vehicle-treated right ear. Ear thickness was measured with a digital caliper (Mitutoyo, Tokyo, Japan), followed by a 6 mm punch biopsy to ascertain changes in ear weights. The extent of inflammation was quantitated according to the following equation: ear swelling (%)=100×(a-b)/b, where a is the thickness/weight of the left (treated) ear and b is the thickness/weight of the right (untreated control) ear. After samples were obtained for assessment of ear thickness/weight, biopsies were obtained from adjacent sites for routine histopathology (fixation in 4% freshly prepared paraformaldehyde in phosphate-buffered saline), or for immunohistochemical analysis (directly frozen in liquid nitrogen). Age- and sex-matched controls from the same genetic background and LXRα-/-, LXRβ-/-, and LXRα/β-/- mice, produced as described previously (Peet et al., 1998Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cholesterol and bile acid metabolism are impaired in mice lacking nuclear oxysterol receptor LXRα.Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar), were used in this study. The irritant contact dermatitis model (TPA) was performed in an identical fashion in these mice, as described above for CD1 mice. Each group of animals was divided randomly, half receiving 22ROH and the other half receiving vehicle alone, at 45 min and 4 h post TPA-induced inflammation. Ear thickness was measured at 18 h as described above, but weight determinations were not performed due to the small numbers of knockout animals available. Biopsies then were taken for hematoxylin and eosin staining and immunohistochemistry. Basal cutaneous permeability barrier function was determined by measuring transepidermal water loss (TEWL) with an electronic water analyzer (MEECO, Warrington, PA). The kinetics of barrier recovery were then determined after acute disruption by sequential applications of cellophane tape (Scotch tape, 3M) (TEWL≥ 6–8 mg per cm2 per h), at 3 and 6 h postdisruption, as described previously (Komuves et al., 2000Komuves L.G. Hanley K. Man M.Q. Elias P.M. Williams M.L. Feingold K.R. Keratinocyte differentiation in hyperprolferative epidermis. Topical application of PPARα restores tissue homeostasis.J Invest Dermatol. 2000; 115: 361-367Crossref PubMed Scopus (96) Google Scholar). Stratum corneum integrity was defined as the number of tape strips required to produce a predetermined elevation in TEWL (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar). Stratum corneum cohesion was defined as the amount of protein removed per stripping, measured with a BioRAD Assay Kit (Hercules, CA), using bovine plasma gamma globulin as the standard, as described previously (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar). Changes in surface pH were measured with a flat glass electrode (Mettler-Toledo, Giessen, Germany), using a pH meter (Skin pH Meter PH 900, Courage and Khazaka, Cologne, Germany). Both paraffin sections (6 μm) and cryosections (5 μm) were used to detect TNF-α and IL-1α in this study. Cryosections alone were assessed in the LXR-deficient animals. Paraffin-embedded, paraformaldehyde-fixed sections were processed as described previously (Sheu et al., 2002Sheu M.Y. Fowler A.J. Kao J.K. et al.Topical PPAR-α activators reduce inflammation in irritant and allergic contact dermatitis models.J Invest Dermatol. 2002; 118: 94-101Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), using polyclonal rabbit antimouse primary antibodies specific for IL-1α (R&D Systems, Minneapolis, MN) and polyclonal goat antimouse specific for TNF-α (Santa Cruz Biotechnology, Santa Cruz, CA). Peroxidase activity was revealed with 3,3′-diaminobenzidine substrate (Vector Laboratories, Burlingame, CA), and methyl green was used as a counterstain. The same antibodies were used for immunohistochemical localization of IL-1α and TNF-α in cryosections. After washing in Tris-HCl buffer, the sections were incubated with blocking buffer for 30 min at room temperature, followed by incubation with primary antibody (1 : 500) in blocking buffer for 2 h. Before incubation with the secondary antibody (1 : 200) for 45 min, the sections were washed in Tris-HCl buffer and incubated for 10 min with levamisole (0.05%) to inhibit endogenous alkaline phosphatase activity. Localization was achieved by the avidin-biotin peroxidase technique, using alkaline phosphatase as the chromagen (reaction time, 13 min and 45 min for IL-1α and TNF-α, respectively). The sections were rinsed with ddH2O, dehydrated, and mounted (Cytoseal 60, Stephens Scientific, Kalamazoo, MI). Nonspecific binding of secondary IgG was not seen when the primary antibody was omitted. The sections were examined with a Zeiss (Axioplan 2) microscope (Jena, Germany) using bright-field optics. Digital images were captured with AxioVision software 2.05 (Carl Zeiss Vision, Munich, Germany). Photos were prepared using Adobe Illustrator (Adobe Systems, Mountain View, CA). All statistical analyses were performed using Prism 3 software (Graph Pad Software, San Diego, CA). Results were compared between multiple groups, using ANOVA, and expressed as mean ± SEM. When results between pairs were analyzed, the Student's t test was used. We initially examined the anti-inflammatory effect of LXR activators in a TPA-induced, irritant contact dermatitis murine model. Both ear thickness (Fig 1A) and ear weight (Fig 1B) markedly increased (1.85-fold and 1.91-fold, respectively) after treatment with TPA. Application of vehicle (acetone) alone did not significantly affect TPA-induced inflammation (1.60-fold and 1.78-fold, for ear thickness and weight, respectively). In contrast, treatment with either 22ROH or 25OH markedly attenuated the TPA-induced increase in ear thickness and ear weight. Because the LXR activators were applied topically, high concentrations (10 mM) were used in order to ensure activation of the target cells. Treatment with 22ROH decreased both ear thickness and ear weight by 51% and 56%, respectively, versus the vehicle control, whereas 25OH decreased thickness and weight by 57% and 69%, respectively (Fig 1A, B). These anti-inflammatory effects were comparable to those achieved by topical clobetasol, a potent glucocorticoid (ear thickness and weight were decreased by 68% and 61%, respectively). Treatment with cholesterol, however, which does not activate LXR, did not alter the extent of TPA-induced inflammation insult (Fig 1A, B). Finally, because oxysterols have biologic effects other than activating LXR, we next employed a nonsterol activator of LXR, GW3965. As shown in Fig 1, GW3965 reduced ear thickness and ear weight by 40% and 38%, respectively, following TPA treatment. These results strongly suggest that LXR activation and not other, nonspecific effects of oxysterols account for the inhibition of TPA-induced inflammation. Hematoxylin and eosin stained sections from the same TPA-treated mice are shown in Fig 2. TPA application alone (Fig 2B) produced a marked increase in ear thickness and an abundance of inflammatory cells infiltrating the epidermis and dermis (Fig 2A vs 2B). In contrast, 22ROH treatment markedly reduced both ear thickness and the extent of the inflammatory infiltrate in the epidermis and dermis (Fig 2B vs 2C). Again, the reduction in inflammation was similar to that seen with clobetasol treatment (Fig 2c vs 2D). These results further demonstrate the anti-inflammatory properties of oxysterols in the TPA-irritant contact dermatitis model. Previous studies with LXRβ–/– and LXRα/β–/– mice demonstrated a slight decrease in the expression of epidermal differentiation markers, with thinning of the normal epidermis, whereas such changes did not occur in LXRα–/– mice (Komuves et al., 2002Komuves L.G. Schmuth M. Fowler A.J. et al.Oxysterol stimulation of epidermal differentiation is mediated by liver-X-receptor-α in murine epidermis.J Invest Dermatol. 2002; 118: 25-34Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). We therefore next determined whether the modest morphologic alterations that occur in animals deficient in LXRβ result in functional abnormalities. In all three strains of LXR-deficient mice (LXRα, LXRβ, and LXRα/β–/–), basal TEWL and the kinetics of barrier recovery following acute barrier disruption were similar to rates observed in wild-type littermates (data not shown). In addition, stratum corneum cohesion and integrity were similar in LXR-deficient mice and control mice (data not shown). Lastly, surface pH was not altered by the absence of either LXRα or LXRβ (data not shown). Thus, LXR deficiency does not produce significant functional cutaneous abnormalities. In order to determine definitively whether the anti-inflammatory effects of oxysterols are mediated by LXR, we next compared the anti-inflammatory effects of oxysterols on TPA-induced inflammation in LXRα–/–, LXRβ–/–, and LXRα/β–/–versus wild-type mice. Treatment with TPA increased ear thickness to a similar extent in LXRα- and LXRβ-deficient mice compared to wild-type mice. Yet, TPA-induced ear thickness was blunted in the LXRα/β–/– double knockout mice (not shown). Treatment with 22ROH following TPA resulted in a similar reduction in ear thickness in the LXR wild-type control mice as seen in the CD1 mice (Fig 1vsFig 3). In contrast, 22ROH treatment did not significantly reduce ear thickness in the LXRβ–/– or LXRα/β–/– animals (Fig 3). Clobetasol treatment resulted in a reduction in ear thickness in LXRβ–/– mice, however, to a degree virtually identical to that seen in wild-type mice (LXRβ–/– 29% decrease versus wild-type 34%, not significant, n=3–4). These results indicate that the failure of 22ROH to inhibit TPA-induced inflammation in LXRβ–/– animals is not due to a general impairment of these animals to elicit an anti-inflammatory reaction. Finally, whereas treatment with 22ROH partially reduced the ear thickness in the LXRα–/– animals, the extent of anti-inflammatory effects was less than those observed in the wild-type animals (32%vs 58%, respectively). These findings indicate that the anti-inflammatory effects produced by oxysterol treatment are inhibited by LXR, and primarily require LXRβ, but that LXRα is also of importance. We next examined the effects of the LXR agonist 22ROH in a model of allergic contact dermatitis. Fig 4(A), (B) demonstrates the marked increase in both ear thickness and weight induced by topical oxazolone applications to sensitized animals (≈1.5-fold). Treatment with 22ROH reduced the oxazolone-induced inflammation, as shown by both decreased ear thickness (Fig 4A) and decreased ear weight (Fig 4B) (58% and 55%, respectively). Moreover, treatment with a nonsterol activator of LXR, GW3965, also significantly decreased ear thickness and ear weight, by 28% and 26%, respectively. Clobetasol (0.05%) treatment produced a stronger anti-inflammatory effect than that observed with the LXR activators at the concentrations employed here (over a 90% reduction in ear thickness and weight). Finally, hematoxylin and eosin stained sections demonstrated a marked increase in ear thickness with an abundance of inflammatory cells in both epidermis and dermis in oxazolone-treated animals, changes that were markedly reduced by 22ROH treatment (Fig 5). Thus, oxysterols reduce inflam
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