【摘要】
Dexamethasone, a synthetic corticosteroid, is widely used as a potent anti-inflammatory drug in various diseases including corneal angiogenesis. However, dexamethasone??s impact on interleukin (IL)-1ß-dependent inflammatory angiogenesis is unknown. Here, we show that dexamethasone inhibits IL-1ß-induced neovascularization and the expression of the angiogenesis-related factors, vascular endothelial growth factor-A, KC, and prostaglandin E2 in the mouse cornea 2 days after IL-1ß implantation. IL-1ß caused IB- phosphorylation in corneal stromal cells but not in infiltrated CD11b+ cells 2 days after IL-1ß implantation. In contrast, both cell types were positive for phosphorylated IB- 4 days after IL-1ß implantation. Dexamethasone significantly inhibited IB- phosphorylation 2 and 4 days after IL-1ß implantation. Furthermore, dexamethasone inhibited IL-1ß-induced expression of vascular endothelial growth factor-A, KC, and prostaglandin E2, and signaling of nuclear factor (NF)-B in corneal fibroblasts in vitro. A selective NF-B inhibitor attenuated IL-1ß-induced corneal angiogenesis. These findings suggest that NF-B activation in the corneal stromal cells is an important early event during IL-1ß-induced corneal angiogenesis and that dexamethasone inhibits IL-1ß-induced angiogenesis partially via blocking NF-B signaling.
--------------------------------------------------------------------------------
Dexamethasone, a synthetic corticosteroid analogue, is a potent anti-inflammatory drug that is used in the treatment of various immune and inflammatory diseases, including those of the eye. Folkman and Ingber1 reported the anti-angiogenic function of a class of steroids, including dexamethasone, which they named the angiostatic steroids. The anti-angiogenic effect of dexamethasone has been confirmed in various animal models.2,3 For instance, Ishibashi and colleagues2 showed that dexamethasone reduces laser-induced subretinal neovascularization in monkey. Furthermore, dexamethasone inhibits cauterization-induced corneal neovascularization.4 Despite the widespread use of dexamethasone and other steroids in the clinical practice, little is known about the detailed mechanisms by which these molecules exert their anti-angiogenic effects in vivo.5
The cornea, a transparent and avascular tissue, encompasses extracellular matrix, keratocytes,6 and leukocytes.7 Corneal neovascularization occurs in a number of corneal disorders and causes significant loss of visual acuity. In corneal diseases, a number of cytokines and growth factors are up-regulated and induce infiltration of neutrophils, macrophages, and lymphocytes.8 Infiltration of inflammatory cells is often accompanied by an angiogenic response. However, the mechanistic role of inflammatory cells in corneal angiogenesis is only beginning to be understood.
Corneal stromal cells, known as keratocytes or corneal fibroblasts, are normally quiescent but can readily respond to injury and transit into activated phenotypes under pathological conditions.6 After corneal injury, corneal stromal cells are activated and migrate to the site of injury.9 In fibroblast growth factor-2-implanted corneas, stromal cells but not leukocytes express vascular endothelial growth factor (VEGF).10 However, it is unknown whether corneal stromal cells contribute to inflammation-induced neovascularization.
Interleukin (IL)-1ß, a multipotent cytokine, is critically involved in the acute inflammatory response, activation of inflammatory and antigen-presenting cells, chemotaxis, up-regulation of adhesion molecules and costimulatory factors on cells, and neovascularization.11 In the eye, IL-1 activity has been correlated with corneal neovascularization.12,13 The expression of IL-1ß in the cornea is increased in various corneal diseases including chemical burns14 and herpetic stromal keratitis.15-17 Previously, we reported that IL-1ß causes corneal angiogenesis by inducing VEGF-A, COX-2/prostanoids, and CXC chemokines (such as KC and MIP-2).18,19 Furthermore, IL-1ß induces infiltration of various inflammatory cells, including neutrophils and macrophages into the cornea.19 We demonstrated that infiltration of the CD11b+ inflammatory cells, a major source of angiogenic factors, is important in IL-1ß-induced corneal neovascularization.19 Dexamethasone blocks the transcription of inflammatory proteins by prohibiting the activity of the transcription factor nuclear factor (NF)-B.20 NF-B plays an important role in IL-1ß-related inflammatory diseases, including various corneal diseases.21,22 Blockade of NF-B reduces corneal epithelial defects during healing in a model of corneal injury.21 However, it is unknown whether dexamethasone has an impact on IL-1ß-dependent angiogenesis. In this work we investigate dexamethasone??s potential in inhibiting IL-1ß-induced angiogenesis and candidate mechanisms both in vivo and in vitro.
【关键词】 dexamethasone inhibits interleukin-ß -induced neovascularization
Materials and Methods
All animal experiments were approved by the Committee on the Ethics of Animal Experiments at the Kyushu University Graduate School of Medical Sciences and the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. Male BALB/c mice, 6 to 10 weeks old, were purchased from Kyudo (Saga, Japan) and Taconic (Hudson, NY).
Corneal Micropocket Assay in Mice
BALB/c mice were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg). Hydron pellets (0.3 µl) containing 30 ng of human IL-1ß (201-LB; R&D Systems, Minneapolis, MN) were prepared and implanted into the corneas. Pellets were positioned at 1 mm to the corneal limbus. Implanted eyes were treated with Levofloxacin eye drops (Santen Pharmaceuticals, Osaka, Japan) to prevent infection. Dexamethasone (5 mg/kg) (D2915; Sigma Chemical Co., St. Louis, MO) was injected intraperitoneally daily, starting 1 day before (C1) and continued until the 5th day after implantation. A peptide inhibitor of NF-B, SN50 (P-600; Biomol International, Plymouth Meeting, PA), or the control peptide SN50M (P-601; Biomol International) was applied topically to IL-1ß-implanted eyes twice a day from days C1 to 5. Two, 4, and 6 days after implantation, digital images of the corneal vessels were obtained and recorded using Viewfinder 3.0 (Pixera, San Jose, CA) or OpenLab software, version 2.2.5 (Improvision Inc., Lexington, MA) with standardized illumination and contrast and were saved to disks. The quantitative analysis of neovascularization in the mouse corneas was performed using Scion Image software (version 4.0.2; Scion Corp., Frederick, MD).
Isolation of the Cornea-Infiltrating Cells
On days 2 and 4 after IL-1ß implantation, five corneas including limbal vessel were harvested, pooled, and dissected with microscissors. The tissues were then treated twice at 37??C for 30 minutes with 0.5 mg/ml collagenase type D (Boehringer-Mannheim, Indianapolis, IN) in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 10% fetal calf serum (Life Technologies, Inc.), 10 mg/ml gentamicin, 50 µmol/L 2-mercaptoethanol, and 5 mg/ml HEPES buffer. The supernatants were collected, passed through a stainless-steel mesh sieve, and washed three times.
Flow Cytometry
Infiltrated cells into the cornea were stained with phycoerythrin-conjugated anti-CD11b mAb (1:50, RM2804; Caltag Laboratory, Burlingame, CA) for 30 minutes on ice. After washing with phosphate-buffered saline (PBS) twice, flow cytometry was performed using the FACSCaliber system (Becton-Dickinson, Mountain View, CA).
Enzyme-Linked Immunosorbent Assay (ELISA) of VEGF-A, KC, MIP-2, and Prostaglandin E2 in Mouse Corneas
Four corneas with or without dexamethasone treatment were harvested at the indicated time points after pellet implantation. The corneas were pooled in 200 µl of media, dissected with scissors, extracted with 200 µl of Triton X-100 buffer (50 mmol/L HEPES, 150 mmol/L NaCl, 1% Triton X-100, and 10% glycerol containing 1 mmol/L phenylmethyl sulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mmol/L sodium orthovanadate), and centrifuged. The supernatants were used in ELISA kits (R&D Systems) for mouse KC (MKC00B), mouse MIP-2 (MM200), mouse VEGF-A (MMV00), and prostaglandin E2 (PGE2; DE0100).
Mice were sacrificed under deep anesthesia with pentobarbital sodium (60 mg/kg i.p.). The eyes were harvested, snap-frozen in optimal cutting temperature (OCT) compound (Sakura Finetechnical, Tokyo, Japan), and 10-µm sections were prepared, air-dried, and fixed in ice-cold acetone for 10 minutes. The sections were blocked with 3% skim milk and stained with anti-phospho-IB- (1:100, no. 9246; Cell Signaling, Beverly, MA) and anti-CD11b mAb (1:100, 550282; BD Pharmingen, San Diego, CA). After an overnight incubation, sections were washed and stained for 20 minutes with secondary antibodies (Abs) (Chemicon International, Temecula, CA), fluorescein isothiocyanate-conjugated goat anti-rat (1:100, AP136F), Cy5-conjugated donkey anti-rabbit (1:100, AP182S), and Cy5-conjugated goat anti-mouse (1:100, AP181S).
Cell Culture
Immortalized keratocytes from corneal stroma of C57BL/6 WT mice (MK/T-1 cells)23 were grown in low-glucose Dulbecco??s minimum essential medium (no. 11885-084; Life Technologies, Inc.), supplemented with 10% fetal bovine serum at 37??C in 5% CO2.
Western Blot Analysis
After culture for 12 hours in serum-free medium with or without the 100 nmol/L dexamethasone, MK/T-1 cells were stimulated with 1 ng/ml IL-1ß for 15 minutes (whole cell lysates) or 30 minutes (nuclear extracts) at 37??C. After rinsing with ice-cold PBS, the cells were lysed in a mammalian cell lysis kit (MCL1; Sigma Chemical Co.). Nuclear extracts were prepared with a nuclear extract kit (no. 40010; Active Motif, Carlsbad, CA). Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon membranes (Millipore, Bedford, MA). Blots were incubated with anti-IB- (1:1000, no. 9242; Cell Signaling), anti-phospho-IB- (1:1000, no. 9241; Cell Signaling), anti-phospho-NF-B p65 (1:1000, no. 3033; Cell Signaling), anti-NF-B p65 (1:1000, no. 3034; Cell Signaling), or anti-ß-tubulin (1:1000, ab11308; Abcam, Cambridge, UK) and visualized with a secondary antibody coupled to horseradish peroxidase (Amersham, Arlington Heights, IL) and enhanced chemiluminescence system.
DNA-Binding Activity of NF-B
After culture for 12 hours in serum-free medium with or without the 100 nmol/L dexamethasone, MK/T-1 cells were stimulated with 1 ng/ml IL-1ß for 30 minutes at 37??C. After preparation of whole cell lysates, the activity of NF-B p65 transcription in MK/T-1 cells was measured using the Transfactor NF-B p65 colorimetric kit (631930; Clontech, Palo Alto, CA), which allows identification of DNA-protein interactions.
Quantification of VEGF, KC, and PGE2
The concentrations of VEGF, KC, and PGE2 in the conditioned media from MK/T-1 cells were measured using ELISA kits as described previously.24 In brief, MK/T-1 cells were seeded in 24-well dishes at 2.5 x 104 cells in a 2-ml volume per well, and when subconfluent, the medium was replaced with serum-free medium for 24 hours, with or without 100 nmol/L dexamethasone, with or without 1 ng/ml IL-1ß at 37??C.
Statistical Analysis
Comparisons were evaluated by the two-tailed unpaired Student??s t-test. N-numbers per group were as indicated. Data are presented as mean ?? SD. The differences between the groups were considered statistically significant for values of P < 0.05.
Dexamethasone Inhibits IL-1ß-Induced Angiogenesis in the Mouse Cornea
To investigate whether dexamethasone affects IL-1ß-induced angiogenesis, we implanted IL-1ß-containing pellets into the corneas of BALB/c mice, treated them with different doses of dexamethasone or vehicle, and quantified the amount of angiogenesis 6 days after implantation. Dexamethasone inhibited IL-1ß-induced angiogenesis in a dose-dependent manner (Figure 1, A and B) . Five mg/kg dexamethasone completely blocked IL-1ß-induced angiogenesis on day 6 (Figure 1, A and B) . Furthermore, dexamethasone inhibited IL-1ß-induced corneal angiogenesis by 77.5, 78.2, and 75.7% on day 2 (P < 8 x 10C8, n = 6 and 8), day 4 (P < 9 x 10C6, n = 6 and 8), and day 6 (P < 3 x 10C8, n = 6 and 8), respectively (Figure 1, C and D) , suggesting that dexamethasone has an impact on IL-1ß-induced corneal angiogenesis from an early stage.
Figure 1. Dexamethsone??s effect on IL-1ß-induced corneal neovascularization. A: Hydron pellets containing 30 ng of IL-1ß were implanted into the corneas of male BALB/c mice treated with vehicle or various concentrations of dexamethasone (Dex). B: Quantitative analysis of neovascularization was performed on day 6 after implantation (*P < 0.01 versus untreated using the two-tailed Student??s t-test). C: Photomicrographs from vessels in the proximity of the sites of the pellets on days 2, 4, and 6 after implantation. D: Quantitative analysis of IL-1ß-induced corneal neovascularization in dexamethasone-treated (n = 6) and control mice (n = 8) was performed on days 2, 4, and 6 (77.5, 78.2, and 75.7% reduction, respectively) (*P < 0.01).
Dexamethasone Inhibits IL-1ß-Induced Expression of Angiogenesis-Related Factors
To investigate whether dexamethasone changes the expression of angiogenesis-related factors, such as VEGF-A25 and CXC chemokines26 (KC and MIP-2), we quantified these factors in corneas of mice during IL-1ß-induced angiogenesis. VEGF-A, KC, and MIP-2 levels were significantly increased in IL-1ß-implanted corneas compared with those of controls on day 2 (P < 0.05, n = 3) (Figure 2, ACC) . VEGF-A and KC protein levels were significantly reduced by dexamethasone on day 2 (P = 0.004 and 0.03; n = 3 and 4, respectively) (Figure 2, A and B) . However, dexamethasone did not significantly affect MIP-2 protein levels in IL-1ß-implanted corneas (P = 0.8, n = 3 and 4) (Figure 2C) . Interestingly, dexamethasone increased KC protein levels in IL-1ß-implanted corneas on day 4 (P = 0.03, n = 4). These data show that dexamethasone inhibits IL-1ß-induced VEGF-A and KC but not MIP-2 expression 2 days after implantation.
Figure 2. Kinetics of the levels of angiogenesis-related factors after IL-1ß pellet implantation. Lysates of four corneas with (black) or without (gray) Dex treatment were prepared and individually assayed using VEGF (A), KC (B), MIP-2 (C), or PGE2 (D) ELISA at the indicated times (n = 3 to 8; *P < 0.05, **P < 0.01).
Furthermore, to examine whether dexamethasone impacts IL-1ß-induced PGE2 expression, we quantified its concentration in corneal extracts by ELISA. Dexamethasone treatment showed a significant decrease in PGE2 levels 2 (P = 0.04, n = 3) and 4 days (P = 0.004, n = 7 and 8) after IL-1ß implantation (Figure 2D) .
Dexamethasone Inhibits IL-1ß-Induced Infiltration of CD11b+ Cells
To examine the effect of dexamethasone on IL-1ß-induced infiltration of inflammatory cells, we quantified the number of CD11b+ cells in corneas of implanted animals using flow cytometry and histology. Corneas from mice treated with dexamethasone or vehicle control were harvested, and the percentage of CD11b+ cells were determined by FACScan. The percentage of the infiltrating CD11b+ cells was increased to 55 ?? 10.9% and 28.1 ?? 9.9% on days 2 and 4 after IL-1ß implantation, respectively (Figure 3A) . In comparison, the percentage of infiltrating CD11b+ cells in dexamethasone-treated mice was 51.3 ?? 6.9% and 33.3 ?? 5.8% on days 2 and 4 after IL-1ß implantation, respectively (Figure 3A) . There was no statistical difference between the results in the dexamethasone-treated and untreated IL-1ß-implanted mice (P = 0.6, n = 4 on day 2; and P = 0.2, n = 7 and 8 on day 4). To analyze the effect of dexamethasone on the number of infiltrated CD11b+ cells into corneas, we next performed immunostaining for CD11b. On day 2, in corneas of dexamethasone-treated mice significantly less CD11b+ cells were found when compared with those of control mice (P < 0.05, n = 5), whereas there was no significant difference between the number of CD11b+ cells in dexamethasone-treated and untreated control (P = 0.8, n = 4 and 5). These data indicate that 5 mg/kg dexamethasone inhibits IL-1ß-induced infiltration of CD11b+ into cornea on day 2 but not day 4.
Figure 3. IL-1ß-induced cell infiltration into the cornea. The analysis of infiltrating cells obtained after IL-1ß implantation and treatment with or without dexamethasone (Dex) on days 2 and 4. A: FACS analysis (CD11b) of infiltrating cells obtained from five IL-1ß-implanted corneas treated with Dex or control on day 2 (n = 4, A and B) and day 4 (n = 7 to 8, C and D). B: Representative photomicrographs of CD11b-stained sections from IL-1ß-implanted corneas with or without Dex on days 2 and 4. C and D: Quantification analysis of the number of CD11b+ cells from IL-1ß-implanted corneas treated with Dex or vehicle control on day 2 (C; 39.8% reduction, *P = 0.019) and day 4 (D, P = 0.8). Original magnifications, x200.
Dexamethasone Inhibits NF-B Activation of Stromal Fibroblasts in IL-1ß-Implanted Corneas
To elucidate the molecular events underlying the inhibition of IL-1ß-induced angiogenesis by dexamethasone, we examined whether dexamethasone affects NF-B signaling in IL-1ß-implanted corneas. We performed immunohistochemistry with Abs against CD11b and phosphorylated IB-, a key signaling molecule upstream of NF-B. Surprisingly, IB- phosphorylation was observed mainly in stromal cells (94 ?? 29.3 cells/field at x200) but not in the CD11b+ cells (13 ?? 4.69 cells/field) in IL-1ß-implanted corneas on day 2 (Figure 4, A and B) . On day 4, most (91.9 ?? 6.7%, n = 8) of CD11b+ cells (38.8 ?? 14.0 cells/field) as well as stromal cells (49.5 ?? 10.9 cells/field) were positive for phosphorylated IB- (Figure 4, A and B) . In dexamethasone-treated mice, we observed infiltration of CD11b+ cells in corneas both on day 2 and day 4. On day 2, phosphorylated IB- cells were not observed in dexamethasone-treated mice (Figure 4A) . On day 4, some of CD11b+ cells were stained with Ab against phosphorylated IB-, whereas CD11bC stromal cells were negative for phosphorylated IB- (Figure 4, A and C) . These results show that IL-1ß induces IB- phosphorylation mainly in stromal cells but not infiltrated CD11b+ cells on day 2 (Figure 4B , P < 6 x 10C6, n = 6) and in both CD11b+ cells and stromal cells on day 4 (Figure 4C) and that dexamethasone inhibits NF-B signaling predominantly in the CD11bC stromal cells (Figure 4C) .
Figure 4. Histological detection of cell activation markers. A: Immunohistochemical detection of CD11b (green) and phosphorylated IB- (red) in IL-1ß-implanted corneas of Dex or vehicle control-treated mice on days 2 and 4. B: The number of phospho-IB-+CD11b+ cells (white) and phospho-IB-+CD11bC cells (black) in the stroma of IL-1ß-implanted corneas on day 2 (n = 6, *P < 0.0001) and day 4 (n = 8, P = 0.1). Each value represents the mean number of cells from six to eight randomly selected microscopic fields ?? SD. C: Comparison of the number of phospho-IB-+CD11b+ (white, 52.3% inhibition) or phospho-IB-+CD11bC cells (black, 92.1% inhibition) in IL-1ß-implanted corneas with or without Dex treatment (day 4, n = 8; *P < 0.01). Original magnifications, x200.
Dexamethasone Inhibits IL-1ß-Induced NF-B Activation in Corneal Stromal Fibroblasts
To investigate the impact of dexamethasone on IL-1ß-induced expression of various angiogenesis-related factors and its relation to NF-B signaling, we cultured corneal stromal fibroblasts, MK/T1, and treated them with dexamethasone or control and measured in these cells the concentration of various angiogenic factors and NF-B activity. Dexamethasone significantly inhibited IL-1ß-induced production of VEGF-A, KC, and PGE2 by MK/T1 cells (P = 0.02, 0.008, and 0.04, respectively; n = 6) (Figure 5, ACC) . To understand how dexamethasone modulates IL-1ß-induced angiogenesis, we next examined the effect of dexamethasone on IL-1ß-dependent NF-B-p65 activity in MK/T1 cells. Dexamethasone significantly inhibited IL-1ß-induced DNA-binding activity and nuclear localization of NF-B-p65 in MK/T1 cells (P = 0.03, n = 6) (Figure 5D) . Furthermore, Western blots using nuclear extracts from dexamethasone- and vehicle control-treated MK/T1 cells revealed that dexamethasone inhibited IL-1ß-induced translocation of NF-B-p65 into the nucleus (Figure 5E) . To detect the signaling molecule that is targeted by dexamethasone, we performed immunoblot analysis of whole cell lysates with antibodies against NF-B signaling molecules. Dexamethasone inhibited IL-1ß-induced IB- degradation as well as NF-B phosphorylation (Figure 5E) . These data indicate that dexamethasone inhibits IL-1ß-induced NF-B signaling through blockade of IB- degradation in corneal stromal cells.
Figure 5. Dexamethasone??s effect on IL-1ß-induced NF-B activation and expression of angiogenesis-related factors in corneal fibroblasts. ACC: Detection of VEGF (A), KC (B), and PGE2 (C) expression in MK/T1 cells after a 24-hour treatment with Dex (100 nmol/L), IL-1ß (1 ng/ml), or vehicle by ELISA (n = 6 to 7; *P < 0.05, **P < 0.01). D: MK/T1 cells were incubated with Dex (100 nmol/L) or vehicle for 12 hours and subsequently with IL-1ß (1 ng/ml) for 30 minutes. DNA-binding activity was measured by an ELISA-based assay at 650 nm. Data are representative of three separate experiments and show means ?? SD from experiments performed in duplicate wells. *P < 0.05, **P < 0.01. E: Western blot analysis with anti-IB-, anti-pNF-B, anti-NF-B, or ß-tubulin Abs using whole cell lysates (WCLs) or with anti-NF-B Ab using nuclear extracts (Nuc) of MK/T1 cells treated with Dex (100 nmol/L) or vehicle for 12 hours and subsequently with IL-1ß (1 ng/ml).
Specific Blockade of NF-B Inhibits IL-1ß-Induced Angiogenesis in the Mouse Cornea
To examine the role of NF-B in the IL-1ß-induced angiogenesis in the mouse cornea, we treated the animals with the specific NF-B inhibitor peptide SN50 (n = 6) or the control peptide SN50M (n = 5) and quantified their corneal angiogenesis after IL-1ß implantation. SN50 significantly reduced IL-1ß-induced angiogenesis on day 6, whereas the animals treated with the control SN50M peptide showed regular levels of corneal angiogenesis (P = 0.002) (Figure 6, A and B) .
Figure 6. The effect of NF-B inhibition on IL-1ß-induced corneal neovascularization. A: IL-1ß-implanted corneas of BALB/c mice topically treated (3-µl eye drops) with the NF-B inhibitor peptide (SN50) or the control peptide (SN50M) (day 6). B: Quantitative analysis of IL-1ß-induced corneal neovascularization in mice with SN50 (n = 6) and with SN50M (n = 5) treatment on day 6 (65.7% reduction, *P = 0.002).
Dexamethasone potently suppresses the immunity and is commonly used in the treatment of a wide variety of immune and inflammatory diseases.20 IL-1ß, an inflammatory cytokine, is up-regulated in various corneal diseases.14-17 Recently, we showed that IL-1ß induces corneal neovascularization via induction of various angiogenesis-related factors including VEGF, CXC chemokines, and COX-2/prostanoids.18,19 In this study, we demonstrate that dexamethasone inhibits IL-1ß-dependent corneal neovascularization partly through regulation of NF-B signaling and inhibition of VEGF, CXC chemokines, and PGE2 production in corneal stromal fibroblast (Figure 7) .
Figure 7. Schematic of how dexamethasone impacts IL-1ß-induced corneal angiogenesis. Dexamethasone inhibits IL-1ß-induced corneal angiogenesis by suppression of NF-B activation and the expression of angiogenesis-related factors in stromal cells during the early phase of the injury (day 2).
Corticosteroids inhibit inflammation through various different pathways.20 For instance, corticosteroid-induced MAPK phosphatase 1 dephosphorylates and inactivates Jun N-terminal kinase, thereby inhibiting c-Jun-mediated transcription.20 Corticosteroid-glucocorticoid receptor complex also interacts with NF-B to block its transcription activity.20 Recent work suggests that glucocorticoids also have rapid nongenomic effects on inflammation. Hafezi-Moghadam and colleagues27 reported that high-dose corticosteroids exert cardiovascular protection through nontranscriptional mechanisms involving rapid activation of endothelial nitric oxide synthase. In this study we examined the effect of dexamethasone on NF-B signaling and demonstrated that dexamethasone mainly inhibited NF-B activation. However, whether the angiostatic effects of dexamethasone involves nontranscriptional mechanisms remains to be investigated.
We first demonstrated that IL-1ß induces NF-B signaling in the mouse cornea during neovascularization. In the corneal alkali burns model, NF-B is activated in the corneal epithelial and stromal cells.21 In our corneal angiogenesis model, IB- was mainly phosphorylated in the corneal stromal cells, suggesting that NF-B-activated corneal stromal fibroblasts play an important role in corneal inflammatory conditions, such as angiogenesis and wound healing.
Dexamethasone is widely used in the treatment of corneal inflammation; however, steroid therapy in the management of some corneal diseases remains controversial because of its side effects.28,29 Dexamethasone inhibits NF-B but not AP-1 activity in transfected human corneal fibroblasts.30 We show that IL-1ß-induced NF-B activation is inhibited by dexamethasone and that a selective NF-B inhibitor diminishes inflammatory corneal angiogenesis. These findings indicate that NF-B inhibition may be an effective therapeutic option for inflammatory corneal angiogenesis.31-33 Further studies will be necessary to assess the safety and side effects for the treatment of human corneal diseases.
CXC chemokines containing the ELR motif mediate angiogenesis through G protein-coupled receptor CXCR2 on endothelial cells.26,34 Previously, we showed that CXCR2 blockade partially inhibits IL-1ß-induced corneal angiogenesis.19 NF-B is known to play an important role as a master switch in the transactivation of angiogenic CXC chemokines.26 Members of the CXC chemokines, such as KC and MIP-2, induce corneal angiogenesis34 and have been implicated in the pathogenesis of inflammatory corneal diseases.30,35 We observed that dexamethasone inhibits IL-1ß-induced NF-B signaling and the level of KC protein but not that of MIP-2. These data suggest that the contribution of MIP-2 may be less than that of KC in IL-1ß-induced corneal angiogenesis.
A recent report shows that fibroblasts produce SDF-1 and promote tumor progression via SDF-1-CXCR4-mediated angiogenesis.36 Another report demonstrated that human corneal fibroblasts express SDF-1 mRNA.37 We examined whether SDF-1 protein expression was increased in IL-1ß-implanted corneas during neovascularization using ELISA. Unexpectedly, however, we did not detect SDF-1 overexpression in IL-1ß-implanted corneas (data not shown), suggesting that SDF-1 may not be necessary in our IL-1ß-induced corneal angiogenesis model.
The IL-1 receptor, which binds both IL-1 and IL-1ß, is constitutively expressed in corneal fibroblasts.38 After corneal injury, IL-1 protein is detectable in corneal fibroblasts. IL-1 up-regulates the expression of various cytokines or enzymes by corneal fibroblasts,8,22,39 and it also contributes to wound healing. In our model, IL-1ß binds to the IL-1 receptor on corneal fibroblasts, up-regulates various angiogenesis-related factors, and induces corneal neovascularization.
Cancer cells are known to alter their adjacent stroma to form a permissive and supportive microenvironment by producing various growth factors and cytokines.40 Recent studies reported that experimentally induced genetic alterations in stromal fibroblasts cause epithelial neoplasia and invasive carcinoma.36,41 Various angiogenesis-related factors, including CXC chemokines and growth factors, are produced in IL-1ß-treated corneal stromal fibroblasts. Activation of stromal fibroblasts by IL-1ß plays a central role in tumor angiogenesis as well as in corneal angiogenesis.42
To date, steroid therapy has been the standard anti-inflammatory and angiostatic treatment in the cornea.28,29 Our results suggest that specific molecular or cellular targeting strategies, such as blockade of NF-B or regulation of the activation status of corneal stromal cells, may offer novel approaches in the treatment of inflammatory angiogenesis in the cornea.
We thank K. Watari, T. Furuta, K. Kano, M. Takahara, H. Fujii (Kyushu University) and N. Lara-Castillo (Harvard University) for technical support; and K.L. Thomas (Harvard University) for editorial support.
【参考文献】
Folkman J, Ingber DE: Angiostatic steroids. Method of discovery and mechanism of action. Ann Surg 1987, 206:374-383
Ishibashi T, Miki K, Sorgente N, Patterson R, Ryan SJ: Effects of intravitreal administration of steroids on experimental subretinal neovascularization in the subhuman primate. Arch Ophthalmol 1985, 103:708-711
Gross J, Azizkhan RG, Biswas C, Bruns RR, Hsieh DS, Folkman J: Inhibition of tumor growth, vascularization, and collagenolysis in the rabbit cornea by medroxyprogesterone. Proc Natl Acad Sci USA 1981, 78:1176-1180
Proia AD, Hirakata A, McInnes JS, Scroggs MW, Parikh I: The effect of angiostatic steroids and ß-cyclodextrin tetradecasulfate on corneal neovascularization in the rat. Exp Eye Res 1993, 57:693-698
Longenecker JP, Kilty LA, Johnson LK: Glucocorticoid influence on growth of vascular wall cells in culture. J Cell Physiol 1982, 113:197-202
West-Mays JA, Dwivedi DJ: The keratocyte: corneal stromal cell with variable repair phenotypes. Int J Biochem Cell Biol 2006, 38:1625-1631
Nakamura T, Ishikawa F, Sonoda KH, Hisatomi T, Qiao H, Yamada J, Fukata M, Ishibashi T, Harada M, Kinoshita S: Characterization and distribution of bone marrow-derived cells in mouse cornea. Invest Ophthalmol Vis Sci 2005, 46:497-503
Wilson SE, Mohan RR, Mohan RR, Ambrosio R, Jr, Hong J, Lee J: The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Prog Retin Eye Res 2001, 20:625-637
Fini ME, Stramer BM: How the cornea heals: cornea-specific repair mechanisms affecting surgical outcomes. Cornea 2005, 24:S2-S11
Chang LK, Garcia-Cardena G, Farnebo F, Fannon M, Chen EJ, Butterfield C, Moses MA, Mulligan RC, Folkman J, Kaipainen A: Dose-dependent response of FGF-2 for lymphangiogenesis. Proc Natl Acad Sci USA 2004, 101:11658-11663
Dinarello CA: Biologic basis for interleukin-1 in disease. Blood 1996, 87:2095-2147
Dana MR, Zhu SN, Yamada J: Topical modulation of interleukin-1 activity in corneal neovascularization. Cornea 1998, 17:403-409
BenEzra D, Hemo I, Maftzir G: In vivo angiogenic activity of interleukins. Arch Ophthalmol 1990, 108:573-576
Fukuda M, Mishima H, Otori T: Detection of interleukin-1ß in the tear fluid of patients with corneal disease with or without conjunctival involvement. Jpn J Ophthalmol 1997, 41:63-66
Staats HF, Lausch RN: Cytokine expression in vivo during murine herpetic stromal keratitis. Effect of protective antibody therapy. J Immunol 1993, 151:277-283
Biswas PS, Banerjee K, Kim B, Rouse BT: Mice transgenic for IL-1 receptor antagonist protein are resistant to herpetic stromal keratitis: possible role for IL-1 in herpetic stromal keratitis pathogenesis. J Immunol 2004, 172:3736-3744
Biswas PS, Banerjee K, Kim B, Kinchington PR, Rouse BT: Role of inflammatory cytokine-induced cyclooxygenase 2 in the ocular immunopathologic disease herpetic stromal keratitis. J Virol 2005, 79:10589-10600
Kuwano T, Nakao S, Yamamoto H, Tsuneyoshi M, Yamamoto T, Kuwano M, Ono M: Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced angiogenesis. FASEB J 2004, 18:300-310
Nakao S, Kuwano T, Tsutsumi-Miyahara C, Ueda S, Kimura YN, Hamano S, Sonoda KH, Saijo Y, Nukiwa T, Strieter RM, Ishibashi T, Kuwano M, Ono M: Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1ß-induced neovascularization and tumor growth. J Clin Invest 2005, 115:2979-2991
Rhen T, Cidlowski JA: Antiinflammatory action of glucocorticoids??new mechanisms for old drugs. N Engl J Med 2005, 353:1711-1723
Saika S, Miyamoto T, Yamanaka O, Kato T, Ohnishi Y, Flanders KC, Ikeda K, Nakajima Y, Kao WW, Sato M, Muragaki Y, Ooshima A: Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-B, in treatment of corneal alkali burns in mice. Am J Pathol 2005, 166:1393-1403
Lu Y, Fukuda K, Liu Y, Kumagai N, Nishida T: Dexamethasone inhibition of IL-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture. Invest Ophthalmol Vis Sci 2004, 45:2998-3004
Gendron RL, Liu CY, Paradis H, Adams LC, Kao WW: MK/T-1, an immortalized fibroblast cell line derived using cultures of mouse corneal stroma. Mol Vis 2001, 7:107-113
Ueda S, Basaki Y, Yoshie M, Ogawa K, Sakisaka S, Kuwano M, Ono M: PTEN/Akt signaling through epidermal growth factor receptor is prerequisite for angiogenesis by hepatocellular carcinoma cells that is susceptible to inhibition by gefitinib. Cancer Res 2006, 66:5346-5353
Loureiro RM, D??Amore PA: Transcriptional regulation of vascular endothelial growth factor in cancer. Cytokine Growth Factor Rev 2005, 16:77-89
Strieter RM, Burdick MD, Mestas J, Gomperts B, Keane MP, Belperio JA: Cancer CXC chemokine networks and tumour angiogenesis. Eur J Cancer 2006, 42:768-778
Hafezi-Moghadam A, Simoncini T, Yang Z, Limbourg FP, Plumier JC, Rebsamen MC, Hsieh CM, Chui DS, Thomas KL, Prorock AJ, Laubach VE, Moskowitz MA, French BA, Ley K, Liao JK: Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nat Med 2002, 8:473-479
Leibowitz HM, Kupferman A, Stewart RH, Kimbrough RL: Evaluation of dexamethasone acetate as a topical ophthalmic formulation. Am J Ophthalmol 1978, 86:418-423
Raizman M: Corticosteroid therapy of eye disease. Fifty years later. Arch Ophthalmol 1996, 114:1000-1001
Lu Y, Fukuda K, Nakamura Y, Kimura K, Kumagai N, Nishida T: Inhibitory effect of triptolide on chemokine expression induced by proinflammatory cytokines in human corneal fibroblasts. Invest Ophthalmol Vis Sci 2005, 46:2346-2352
Olivier S, Robe P, Bours V: Can NF-B be a target for novel and efficient anti-cancer agents? Biochem Pharmacol 2006, 72:1054-1068
Miller JW, Stinson WG, Folkman J: Regression of experimental iris neovascularization with systemic -interferon. Ophthalmology 1993, 100:9-14
Manna SK, Mukhopadhyay A, Aggarwal BB: IFN- suppresses activation of nuclear transcription factors NF-B and activator protein 1 and potentiates TNF-induced apoptosis. J Immunol 2000, 165:4927-4934
Addison CL, Daniel TO, Burdick MD, Liu H, Ehlert JE, Xue YY, Buechi L, Walz A, Richmond A, Strieter RM: The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity. J Immunol 2000, 165:5269-5277
Yamagami S, Miyazaki D, Ono SJ, Dana MR: Differential chemokine gene expression in corneal transplant rejection. Invest Ophthalmol Vis Sci 1999, 40:2892-2897
Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA: Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 2005, 121:335-348
Bourcier T, Berbar T, Paquet S, Rondeau N, Thomas F, Borderie V, Laroche L, Rostene W, Haour F, Lombet A: Characterization and functionality of CXCR4 chemokine receptor and SDF-1 in human corneal fibroblasts. Mol Vis 2003, 9:96-102
Wilson SE, Lloyd SA, He YG: Glucocorticoid receptor and interleukin-1 receptor messenger RNA expression in corneal cells. Cornea 1994, 13:4-8
Hong JW, Liu JJ, Lee JS, Mohan RR, Mohan RR, Woods DJ, He YG, Wilson SE: Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Invest Ophthalmol Vis Sci 2001, 42:2795-2803
Mueller MM, Fusenig NE: Friends or foes??bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 2004, 4:839-849
Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD, Cunha GR: Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res 1999, 59:5002-5011
Saijo Y, Tanaka M, Miki M, Usui K, Suzuki T, Maemondo M, Hong X, Tazawa R, Kikuchi T, Matsushima K, Nukiwa T: Proinflammatory cytokine IL-1ß promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction. J Immunol 2002, 169:469-475
作者单位：Shintaro Nakao*, Yasuaki Hata*, Muneki Miura*, Kousuke Noda, Yusuke N. Kimura, Shuhei Kawahara*, Takeshi Kita*, Toshio Hisatomi*, Toru Nakazawa, Yiping Jin¶, M. Reza Dana¶, Michihiko Kuwano, Mayumi Ono||, Tatsuro Ishibashi* and Ali Hafezi-MoghadamFrom the Departments of Ophthalmology,* and
【摘要】
Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor-ß1, mediates mesangial cell/fibroblast proliferation and extracellular matrix production by renal cells. Here, we show that renal tubular epithelial cells from patients with minimal change nephritic syndrome produced CTGF after glucocorticoid treatment. In addition, the glucocorticoid dexamethasone (DEX) increased CTGF mRNA levels in the kidneys of C57B6 but not SJL mice and produced intermediate CTGF mRNA levels in the kidneys of F1 (C57B6 x SJL) mice, midway between the levels found for parental strains. DEX also increased CTGF mRNA levels in cultured tubular epithelial cells derived from C57B6 (mProx24) but not SJL (MCT) mice via transcriptional up-regulation of CTGF mRNA. Transient transfection experiments using luciferase reporter constructs bearing CTGF promoter fragments revealed that the C897- to C628-bp fragment contained DEX-responsive positive regulatory elements, which were active in mProx24 but not MCT cells. Long-term DEX treatment resulted in fibronectin deposition in the kidneys of C57B6 but not SJL mice, and this effect was inhibited by co-administration of CTGF anti-sense oligodeoxynucleotides. Thus, glucocorticoid-induced renal fibrogenesis seems to be influenced by genetic background, with the critical DEX-responsive elements in the C897- to C628-bp region of the CTGF promoter.
--------------------------------------------------------------------------------
Connective tissue growth factor (CTGF) is a member of the CTGF, cyr 61/cef 10, nov (CCN) protein family, which participates in a wide variety of biological processes such as embryonic development and tissue repair.1,2 CTGF itself is a potent and ubiquitously expressed growth factor for fibroblasts, chondrocytes and vascular endothelial cells.2 CTGF is also a downstream mediator of transforming growth factor (TGF)-ß1, a key cytokine involved in renal fibrogenesis, ie, glomerulosclerosis and interstitial fibrosis. As such CTGF plays a unique role in mediating mesangial cell/fibroblast proliferation and extracellular matrix production by renal cells.3,4 Glomerular visceral/parietal epithelial cells, mesangial cells, fibroblasts, and endothelial cells have been shown to produce CTGF in fibrosing kidneys.4-6 We previously showed that tubular epithelial cells are potent producers of CTGF and that CTGF itself probably plays a role in renal fibrogenesis, as determined using the remnant kidney model.7,8
Minimal change nephrotic syndrome is a benign disease that rarely leads to renal failure.9 Although its frequency remains to be clarified, however, minimal change nephrotic syndrome follows a steroid-dependent course, and relapsing patients sometimes develop renal functional impairment as a result of glomerulosclerosis and/or interstitial fibrosis that is thought to be attributable to long-term protein overload.10 As an alternative hypothesis, we proposed that these changes might be attributable to long-term exposure to glucocorticoids, which have been reported to be potent inducers of CTGF in the kidney.11 In this study, we immunohistochemically analyzed human renal biopsy material and mouse kidneys to determine which kidney cells expressed CTGF in response to glucocorticoid treatment. Our results showed that glucocorticoid induced CTGF expression in tubular epithelial cells. We then examined the molecular mechanisms responsible for this effect using cultured mouse tubular epithelial cells. Finally, we determined whether glucocorticoid treatment itself induced renal fibrogenesis.
【关键词】 dexamethasone connective expression epithelial strain-specific
Materials and Methods
Cell Culture
Cultured mouse proximal tubular epithelial cell lines, mProx2412 and MCT,13 derived from C57B6 and SJL mice, respectively, were maintained in Dulbecco??s modified Eagle??s medium containing 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. For experimental purposes, the cells were seeded in six-well plates (1 x 105 cells/well) and incubated overnight in growth medium, after which the medium was replaced with modified K-1 medium (50:50 Ham??s F-12/Dulbecco??s modified Eagle??s medium with 5 µg/ml of transferrin and 5 µg/ml of insulin). After 48 hours, various doses of dexamethasone (DEX; Sigma, St. Louis, MO) or rhTGF-ß1 (R&D Systems, Minneapolis, MN) were added to the wells, and the cells were harvested for mRNA extraction as described below after various incubation times. To examine the mechanism of DEX-induced CTGF expression in these cells, neutralizing anti-TGF-ß (Genzyme Corp., Cambridge, MA) and anti-CTGF7 antibodies, as well as rmTNF- (10 ng/ml; R&D Systems), the AP-1 inhibitor curcumin (4.0 µmol/L; Sigma), the PKC inhibitor H7 (1.0 µg/ml; Sigma), the MEK inhibitor U0126 (10 µmol/L; Sigma), or the tyrosine kinase inhibitor genistein (10 µmol/L; Sigma) were added to the cultures 30 minutes before DEX (1000 nmol/L). After an incubation of 3 hours, the cells were harvested. To determine the CTGF mRNA stability of these DEX-treated cultured cells, those that had been incubated in the presence or absence of DEX (1000 nmol/L) for 3 hours were treated with the transcriptional inhibitor actinomycin-D (5 µg/ml; Sigma) for varying periods of time, after which they were harvested for RNA extraction.
In a separate experiment, cells were harvested from collagenase-digested fragments of proximal tubules obtained from the cortices of both C57B6 and SJL mice using a modification of a previously described procedure.14 Briefly, cortices were minced and incubated with 0.5 mg/ml of collagenase (type IV-S; Sigma) and 0.5 mg/ml of soybean trypsin inhibitor (Sigma) in Hanks?? salt solution for 30 minutes at 37??C. After the large undigested fragments were removed by gravitation, the suspension was mixed with an equal volume of Hanks?? solution containing 10% horse serum and centrifuged at 500 rpm for 7 minutes at room temperature. The pellets were washed once with Dulbecco??s modified Eagle??s medium by centrifugation, after which they were resuspended in the modified K-1 medium. Cells isolated using this technique were previously shown to be predominantly proximal tubular cells.14 The response of these cells to DEX and rhTGF-ß1 was determined as described above for the mProx24 and MCT cell lines.
Treatment of Mice with DEX
Animal care and treatment were provided in con-formity with institutional guidelines. Male C57B6, SJL, F1(C57B6xSJL), Balb/C, and 129 mice (5 to 7 weeks of age) were injected intraperitoneally with 1 mg of DEX in phosphate-buffered saline (PBS) per kg body weight; control mice were injected with PBS alone. These animals were then serially sacrificed after 1, 3, 12, 24, or 48 hours, and their kidneys, liver, and lungs were harvested and processed for RNA isolation and immunohistochemistry.
Separate cohorts of C57B6 and SJL mice were injected intraperitoneally daily for 2 weeks with 1 mg/kg of DEX. We previously reported that when a given anti-sense oligodeoxynucleotide (ODN) was injected intravenously into rodents, it was absorbed into the proximal tubular epithelium where it was retained for nearly 48 hours and during which time it could block transcription of a target gene.7,15 Thus, these mice were similarly injected intravenously with the CTGF anti-sense (5'-TGCGACGGAGGCGAGCAT-3')7 or mutated anti-sense ODNs (5'-TGCAGTGGAAATGAGTGC-3') at a concentration of 1.0 mg/kg every 2 days from day 0 to day 14. The experimental groups consisted of C57B6 mice that were treated with DEX and anti-sense ODN (C57-AS, n = 8), C57B6 mice treated with DEX and mutated anti-sense ODN (C57-mAS, n = 8), SJL mice treated with DEX and anti-sense ODN (SJL-AS, n = 8), SJL mice treated with DEX and mutated anti-sense ODN (SJL-mAS, n = 8), and negative control mice of both strains (C57-NC, n = 8; SJL-NC, n = 8). After 12 hours or 2 weeks of treatment, the renal tissues from these animals were harvested for RNA extraction and immunohistochemistry.
Twelve renal biopsy specimens from patients with minimal change nephrotic syndrome, six of whom had just received steroid pulse therapy (500 mg/24 hours of methyl-prednisolone for 3 days) and six who did not, were examined for the presence of CTGF. The specimens, all of which were confirmed cases of minimal change nephrotic syndrome, came from patients who underwent renal biopsy at our hospital between 1996 and 2002 for the evaluation of nephrotic range proteinuria. The protocol for the use of human materials was approved by the ethics committee of the institution. The specimens from these patients, as well as renal samples from the mice described above, were fixed in 4% paraformaldehyde overnight and processed for paraffin embedding. Sections (4 µm) were cut from these blocks, after which they were deparaffinized, rehydrated, and treated with proteinase K. The sections were then boiled in citrate buffer in a microwave to unmask antigenic sites. Endogenous biotin was blocked by using a biotin blocking system (X0590; DAKO Corp., Carpinteria, CA). The sections were then immersed in 3% H2O2 in methanol to inhibit endogenous peroxidase and incubated with 1% nonfat milk in PBS to block nonspecific binding. Rabbit polyclonal anti-CTGF (1:200) or anti-fibronectin (anti-FN; Life Technologies, Inc., Grand Island, NY) (1:400) primary antibodies were then applied to the sections, after which the sections were washed and then incubated with a biotin-conjugated anti-rabbit IgG secondary antibody. A catalyzed signal amplification system peroxidase (K1500; DAKO) was used to visualize the antibody reactions.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Real-Time RT-PCR
Total RNA was extracted from the homogenates of kidney tissues and cultured cells using TRIzol (Life Technologies, Inc.) according to the manufacturer??s instructions. All RNA samples were pretreated with RNase-free DNase I (Qiagen, Basel, Switzerland). The RT-PCR assay was performed to detect the expression of the glucocorticoid receptor gene using glucocorticoid receptor primers, forward 5'-CAAAGCCGTTTCACTGTCC-3' and reverse 5'-ACAATTTCACACTGCCACC-3'.16 The PCR protocol was as follows: 94??C for 2 minutes, followed by 30 cycles at 94??C for 1 minute, 56??C for 1 minute, and 72??C for 1 minute, and ending with 72??C for 7 minutes. Real-time quantitative one-step RT-PCR assay was performed to quantify CTGF, TGF-ß1, fibronectin EIIIA isoform (FN-EIIIA), 1 chain of type I procollagen (1COLI), angiotensinogen, and GAPDH mRNAs using QuantiTect SYBR Green RT-PCR (Qiagen) and an ABI Prism 7700 sequence detection system (Applied Biosystems, Tokyo, Japan). The primers used for real-time RT-PCR were as follows: CTGF primers, forward 5'-GTGGAATATTGCCGGTGCA-3', reverse 5'-CCATTGAAGCATCTTGGTTCG-3'; TGF-ß1 primers, forward 5'-TCGTGGAACTGCCCTACCAG-3', reverse 5'-ATGTTGGTGAGGGCGGAGAG-3'; FN-EIIIA primers, forward 5'-ATCCGGGAGCTTTTCCCTG-3', reverse 5'-TGCAAGGCAACCACACTGAC-3'; 1COLI primers, forward 5'-TGTAAACTCCCTCCACCCCA-3', reverse 5'-TCGTCTGTTTCCAGGGTTGG-3'; angiotensinogen primers, forward 5'-GAGGCAAATCTGAGCAACATTG-3', reverse 5'-GAGTTCGAGGAGGATGCTATTGA-3'; and GAPDH primers, forward 5'-TGCAGTGGCAAAGTGGAGATT-3', reverse 5'-TTGAATTTGCCGTGAGTGGA-3'. All of these oligonucleotides were designed by using Primer Express software (Perkin Elmer, Foster City, CA). Preliminary RT-PCR experiments in which these primer sets were used yielded appropriately sized, single products.
Luciferase Reporter Constructs and Transient Transfection Assay
A series of firefly luciferase reporter minigenes were constructed bearing various 5' fragments of the CTGF gene. The plasmids, pCT-897.L, pCT-628.L, pCT-483.L, and pCT-202.L contained genomic DNA C897, C628, C483, and C202 bp upstream of the transcription start site, respectively. In these plasmids, genomic fragments were placed 5' of the firefly luciferase cDNA in pGL3b (Promega, Madison, WI). The accuracy of all constructed plasmids was verified by sequencing.
Transient transfections were performed using TransFast (Promega) according to the manufacturer??s instructions. The Dual Luciferase system (Promega) was used for the sequential measurement of firefly and Renilla luciferase activity using the specific substrates of beetle luciferin and coelenterazine, respectively. One µg of pCT-897.L or isomolar amounts of other firefly luciferase constructs were co-transfected with 0.5 µg of Renilla luciferase control plasmid (pRL-TK; Promega) into 1.0 x 105 cells plated in each well of a six-well plate. The medium was changed 12 hours later, and the cells were incubated with or without 1000 nmol/L DEX in K-1 medium for another 36 hours. Quantification of luciferase activity and the calculation of relative ratios were performed manually using a luminometer (TD-20/20; Turner Designs, Sunnyvale, CA). The genomic sequences from C897 to C628 bp upstream of the transcription start site of the CTGF gene in C57B6 and SJL mice were determined using capillary sequence methods.
Statistical Analysis
Values are presented as the means ?? SE. Statistical differences between groups were evaluated using a Bonferroni/Dunnett??s test; P values that were 0.05 were considered to be statistically significant.
Demographic data for the 12 patients whose biopsies were examined in this study are shown in Table 1 . Although no CTGF protein expression was detected in the kidneys from the six untreated minimal change nephrotic syndrome patients (Figure 1A) , CTGF protein localized in the tubular epithelial cells in four of the six kidney tissues from minimal change nephrotic syndrome patients who had been treated or who had just finished their treatment with steroid pulse therapy (Figure 1B) . There were no differences in the demographic data between the CTGF-positive and -negative minimal change nephrotic syndrome patients (data not shown). In light of these findings, we next examined the effects of DEX on the expression of CTGF mRNA in the kidneys of different mouse strains. DEX treatment significantly increased CTGF mRNA levels in the kidneys of C57B6 mice (Figure 2A) , had no effect in the kidneys of SJL mice (Figure 2B) , and slightly increased CTGF mRNA levels in the kidneys of Balb/C and 129 mice (data not shown). Interestingly, DEX significantly increased CTGF mRNA levels in the lungs of C57B6 (4.3 ?? 0.5-fold, P < 0.05) but not SJL mice after 3 hours. Regardless of DEX treatment, however, CTGF mRNA levels in the liver were significantly lower than those in the kidneys and lungs in both strains of mice. DEX slightly, but not significantly, increased CTGF mRNA levels in the kidneys of F1 (C57B6 x SJL) mice to levels that were approximately midway between the levels found in C57B6 and SJL mice (Figure 2C) . Immunohistochemistry using an anti-CTGF antibody localized CTGF protein exclusively in the renal tubular epithelial cells of DEX-treated, but not untreated, C57B6 mice (Figure 2, D and E) .
Table 1. Demographic Data of Patients with Minimal Change Nephrotic Syndrome at Biopsy
Figure 1. Localization of CTGF protein in the kidneys of patients with minimal change nephrotic syndrome. A: An untreated patient. No CTGF expression was detected. B: A patient who just completed steroid pulse therapy. CTGF protein was found exclusively in tubular epithelial cells (arrows). Figures are representative sections from six samples each. DAB stain. Original magnifications, x200.
Figure 2. DEX induction of CTGF expression in the mouse kidney. A: CTGF mRNA levels in DEX-treated C57B6 mice. B: CTGF mRNA levels in DEX-treated SJL mice. C: CTGF mRNA levels in DEX-treated F1 (C57B6 x SJL) mice. CTGF protein localization in the kidneys of DEX-treated C57B6 mice 24 hours after treatment (arrows) (D) and in the kidneys of untreated C57B6 mice (E). Each of the data in A, B, and C were obtained from four mice and data in D and E are representative of the sections from four mice in each case. DAB stain. Original magnifications, x100.
The mProx24 and MCT tubular epithelial cell lines derived from C57B6 and SJL mice, respectively, were used to examine the effects of DEX on the induction of CTGF. RT-PCR revealed that both cell lines were positive for glucocorticoid receptor mRNA (Figure 3A) . As expected from our in vivo findings, DEX increased CTGF mRNA levels in mProx24 cells in a dose-dependent manner (Figure 3B) , with levels at 1 and 3 hours after DEX treatment being significantly greater than controls (Figure 3D) . Because DEX facilitated CTGF mRNA degradation in mProx24 cells (Figure 3F) , it was likely that this increase was attributable to the up-regulation of CTGF gene transcription by DEX. In contrast, DEX did not increase CTGF mRNA levels in MCT cells (Figure 3, C and E) . Because DEX facilitated CTGF mRNA degradation in MCT cells to the same degree as in mProx24 cells (Figure 3G) , it seems reasonable that DEX does not up-regulate CTGF gene transcription in MCT cells. These findings were unlikely to have been attributable to the spontaneous transformation of both cell lines in culture because DEX also significantly increased CTGF mRNA levels in primary cultured cells derived from C57B6, but not SJL, mice (Figure 3H) . CTGF mRNA levels increased in both cell lines as well as both primary cultured cells in response to rhTGF-ß1 (Figure 4) , suggesting that they possessed the capacity to synthesize CTGF. DEX was reported to induce the expression of angiotensinogen and FN mRNA in certain cultured tubular epithelial cells.17,18 We also found that DEX significantly increased angiotensinogen but not FN-EIIIA mRNA levels in both mProx24 and MCT cells (Figure 5, ACD) , indicating that the glucocorticoid receptor binds to DEX and transduces signals into the nucleus to promote target transcription in both cell lines.
Figure 3. DEX induction of CTGF mRNA expression in C57B6-derived mProx24 and SJL-derived MCT renal tubular epithelial cells. A: Glucocorticoid receptor mRNA expression in mProx24 and MCT cells. Dose dependency of DEX induction of CTGF mRNA expression at 3 hours in mProx24 (B) and MCT cells (C). Time course of DEX (1000 nmol/L) induction of CTGF mRNA expression in mProx24 (D) and MCT cells (E). Effect of DEX treatment (1000 nmol/L, 3 hours) on CTGF mRNA stability in mProx24 (F) and MCT cells (G). CTGF mRNA levels were normalized against GAPDH levels and expressed relative to the initial values. In both the mProx24 (F) and MCT cells (G), the rate of degradation of CTGF mRNA was significantly and similarly enhanced by DEX treatment. H: Effect of DEX treatment (1000 nmol/L, 3 hours) on CTGF mRNA levels in the primary cultured cells derived from C57B6 and SJL mice. The data in B to H were each obtained from four independent experiments.
Figure 4. TGF-ß1 induction of CTGF mRNA expression in mProx24 and MCT cells. Dose dependency of TGF-ß1 induction of CTGF mRNA expression at 3 hours in mProx24 (A) and MCT cells (B). Time course of TGF-ß1 (3 ng/ml) induction of CTGF mRNA expression in mProx24 (C) and MCT cells (D). E: Effect of TGF-ß1 (3 ng/ml, 3 hours) on CTGF mRNA levels in the primary cultured cells derived from C57B6 and SLJ mice. The data in A to E were each obtained from four independent experiments.
Figure 5. DEX induction of mRNA expression of angiotensinogen and FN-EIIIA in mProx24 and MCT cells. DEX (1000 nmol/L) induction of angiotensinogen mRNA in mProx24 (A) and MCT cells (B). DEX (1000 nmol/L) induction of FN-EIIIA mRNA in mProx24 (C) and MCT cells (D). The data in A to D were each obtained from four independent experiments.
The induction of CTGF mRNA expression by TGF-ß1 was attenuated by tumor necrosis factor (TNF)- in fibroblasts, an effect that was blocked by the PKC inhibitor Go6983, the MEK inhibitor U0126, and the tyrosine kinase inhibitor genistein in mesangial cells. DEX induction of CTGF mRNA expression was not affected by TNF- or U0126, or by neutralizing anti-TGF-ß or anti-CTGF antibodies (Figure 6) . However, the PKC inhibitor H7 or genistein significantly attenuated DEX induction of CTGF mRNA expression in mProx24 cells (Figure 6) , suggesting that PKC and tyrosine kinase pathways played a role in this process.
Figure 6. Effects of various substances on DEX induction of CTGF mRNA expression in mProx24 cells. Both the PKC inhibitor H7 and the tyrosine kinase inhibitor genistein significantly inhibited DEX induction of CTGF mRNA expression in mProx24 cells, suggesting the involvement of PKC and tyrosine kinase. However, treatment with TNF-, neutralizing anti-TGF-ß, and anti-CTGF antibodies, curcumin and U-0126, was without effects. The data were obtained from four independent experiments in each case.
GenBank data analysis revealed a number of consensus motifs in the promoter sequence of the mouse CTGF gene (representatives are shown in Figure 7A ), although it was found to lack consensus glucocorticoid responsive cis-elements (GRE). Transient transfection experiments with firefly luciferase reporter constructs bearing CTGF promoter fragments revealed that there were universal positive and negative regulatory elements in the C483- to C202-bp and C628- to C483-bp fragments, respectively, elements that were not reactive to DEX (Figure 7, B and C) . In contrast, the C897- to C628-bp fragment was found to contain positive regulatory elements that were responsive to DEX but were active only in mProx24, but not MCT, cells (Figure 7, B and C) . We also identified two polymorphisms in the genomic DNA sequence of the C897- to C628-bp fragment between C57B6 and SJL mice (at C858 and C782 bp; Figure 7D ) that were outside of the consensus motifs (ie, STAT3RE at C740 to C736 bp, and C/EBP at C688 to C676 bp).
Figure 7. DEX-responsive elements in the CTGF promoter. A: Consensus motifs in the mouse CTGF promoter and firefly luciferase reporter constructs. Promoter activity of CTGF promoter fragments with or without DEX treatment in mProx24 (B) and MCT cells (C). D: The genomic DNA sequence at the C897 to C628 bp of CTGF promoter. Polymorphisms between C57B6 and SJL mice are shown in bold (at C858 and C782 bp), and consensus motifs (STAT3RE and C/EBP) are shown in italic. The data in B and C were each obtained from four independent experiments.
To determine the pathophysiological role of glucocorticoid in CTGF-mediated renal fibrogenesis, we treated glucocorticoid-susceptible (C57B6) and nonsusceptible (SJL) mice with DEX. Twelve hours after DEX treatment, both FN-EIIIA and CTGF mRNA levels were elevated in the kidneys of C57B6 mice (Figure 8B) . Treatment of C57B6 mice with CTGF anti-sense ODN at the same time that they received DEX suppressed these elevations in both CTGF and FN-EIIIA mRNA levels (Figure 8A) , supporting the notion that DEX-induced increases in CTGF mRNA levels indirectly resulted in elevations in FN-EIIIA mRNA levels. On the other hand, DEX treatment reduced CTGF and FN-EIIIA mRNA levels in SJL mice (Figure 8B) . DEX treatment resulted in a significant induction in FN protein deposition in the kidneys of C57B6, but not SJL, mice 2 weeks after treatment (Figure 8D) .
Figure 8. Fibronectin deposition in the kidneys of DEX-treated animals is mediated by CTGF. DEX increased the mRNA expression of CTGF and FN-EIIIA in the kidneys of C57B6 (A) and SJL mice (B) after 12 hours. A: Co-treatment with DEX and CTGF-AS significantly inhibited DEX-induced CTGF and FN-EIIIA mRNA expression in the kidneys of C57B6 mice, but there were no significant effects by co-treatment with CTGF-mAS. B: DEX decreased CTGF and FN-EIIIA mRNA levels in the kidneys of SJL mice regardless of co-treatments. C: Minimal deposition of fibronectin protein (arrows) was seen in control C57B6 mouse kidneys (C57-NC). D: C57B6 mice that were treated for 2 weeks with DEX and CTGF-mAS (C57-mAS) displayed fibronectin protein deposition in their kidneys (arrows). E: In contrast, co-treatment of C57B6 mice with CTGF-AS (C57-AS) suppressed DEX-induced fibronectin protein deposition (arrows). Each of the data in A and B was obtained from four mice, and data in C to E are representative of the sections obtained from four mice in each case. DAB stain. Original magnifications, x100.
DEX induction of CTGF expression in the mouse kidney was previously reported by Dammeier.11 In our study, we found that tubular epithelial cells produced CTGF in response to DEX in vivo and in vitro. We also found the DEX-induced CTGF mRNA expression in the kidney is mouse strain-specific, with C57B6 mice responding to treatment in this way whereas SJL mice did not. A biopsy sample from a kidney of a minimal change nephrotic syndrome patient who had been treated with high doses of glucocorticoids revealed the presence of CTGF in tubular epithelial cells. This finding was not disease-specific because similar staining was demonstrated in the kidneys of glucocorticoid-treated lupus nephritis patients (data not shown). TGF-ß1 is a strong inducer of CTGF in tubular epithelial cells8,19 as well as in a variety of cells such as fibroblasts, mesangial cells, endothelial cells, and chondrocytes3 ; TGF-ß1/Smad-responsive regulatory elements were found in the CTGF promoter regions of some of these cells.20-24 In contrast, although CTGF induction by glucocorticoid has similarly been detected in fibroblasts, osteoblasts, and chondrocytes,11,25,26 the molecular mechanisms that control this process have not as yet been elucidated.
We were surprised to discover that the mouse strain-specific responsiveness to DEX that we uncovered was seemingly attributable to relatively simple genetics because F1 (C57B6 x SJL) mice showed responsiveness that was midway between the levels seen in responsive C57B6 mice and unresponsive SJL mice; DEX responsiveness was observed not only in renal cells but also in lung cells of C57B6 mice. It has been reported that glucocorticoid facilitates mRNA degradation and down-regulates the expression of some genes via the 3'-UTR.27 Similarly, DEX was shown to inhibit TNF- gene expression via adenosine/uridine-rich elements in its 3'-UTR.28 Interestingly, the CTGF gene bears adenosine/uridine-rich elements in its 3'-UTR, and DEX shortened the half-life of CTGF mRNA to the same degree in both cell lines, suggesting that the observed differences in DEX-induced CTGF expression were regulated at the transcriptional level. Our luciferase reporter assay results suggested the presence of strain-specific regulatory elements at position C897 to C628 bp in the mouse CTGF gene promoter that were active in mProx24 (C57B6-derived) but not in MCT (SJL-derived) cells. Because both mProx24 and MCT cell lines had glucocorticoid receptors and increased their angiotensinogen and CTGF mRNA levels in response to DEX and rhTGF-ß1, respectively, the presence of these regulatory elements likely accounted for the strain-specific CTGF gene responsiveness to DEX.
Regulatory elements that were constitutively active and responsive to TGF-ß1 were found at C483 to +1 bp in the CTGF gene promoter,20-24 which was recognized as the prototypical CTGF promoter. The prototypical promoter in NIH3T3 fibroblasts reportedly responded to DEX, with the relevant DEX-responsive elements likely being present at C292 to +22 bp in the human CTGF gene promoter.26 The activity found at C897 to C628 bp of the CTGF gene promoter in response to DEX in mProx24 cells in our study has not been previously reported but is consistent with data collected in human chondrocytes, which demonstrated DEX-responsive elements at C802 to C292 bp in their CTGF gene promoter.26 The glucocorticoid receptor generally has three functional domains, ie, a DNA-binding domain, a transcriptional activity domain, and a ligand (glucocorticoid)-binding domain. The C-terminal ligand-binding domain undergoes a conformational change after its binding to glucocorticoid in the cytoplasm, enabling the glucocorticoid-glucocorticoid receptor complex to bind to a series of co-activator proteins, after which the glucocorticoid-glucocorticoid receptor complex enters the nucleus and binds to GRE to regulate target genes.27 In our study, DEX increased angiotensinogen mRNA expression in both cell lines, possibly via the GRE in its 5' promoter sequence.18 However, 5' and 3' sequences of the mouse CTGF gene do not contain consensus GRE (half-) site sequences (GGTACAnnnTGTTCT).27 Instead, a CCAAT/enhancing binding protein site (C/EBP site; C688 to C676 bp) found in the C897- to C628-bp fragment is thought to tether GRE, to which the glucocorticoid-glucocorticoid receptor complex indirectly binds other transcriptional factors in a larger regulatory complex.27 Transcriptional cooperation with the glucocorticoid-glucocorticoid receptor complex was demonstrated to be specific for C/EBP for CYP3A1,29 C/EBPß for phosphoenolpyruvate carboxykinase,30 and ß-casein.31 Induction of the transcription of these genes by glucocorticoid does not require binding of the glucocorticoid-glucocorticoid receptor complex to the consensus GRE. In addition, a STAT3-responsive element (C740 to C736 bp) exists in the C897 to C628 bp fragment of the mouse CTGF promoter, which may also be involved in the tethering of GRE because phosphorylated STAT3 associates with the glucocorticoid-glucocorticoid receptor complex to form a transactivating/signaling complex. This complex was reported to increase the transcription of 2-macroglobulin and fibrinogen genes via STAT3-responsive elements in their 5' promoter sequences.32,33 PKC inhibitors and tyrosine kinase inhibitors universally inhibited the activity of the prototypical CTGF promoter in fibroblasts and mesangial cells20,21 as well as DEX induction of CTGF in the tubular epithelial cells in this study. If phosphorylated STAT3 is truly involved in this signaling process, then a tyrosine kinase most likely plays a direct role because it phosphorylates STAT3.34
The observation has often been made that sclerotic and fibrotic reactions occur at the same time that inflammatory and immune reactions subside in the diseased kidney after glucocorticoid treatment35 ; our above findings may provide a possible explanation for this. Such processes are likely mediated by high concentrations of glucocorticoids because patients with sustained exposure to low levels of glucocorticoids, such as those with Cushing??s syndrome and asthmatics treated with glucocorticoids, have not been reported to develop renal fibrosis. It is well established that organ fibrogenesis is significantly affected by genetic background, and the C57B6 mouse is recognized as a fibrosis-prone strain that is sensitive not only to DEX-induced renal fibrosis but also to TGF-ß1-induced lung fibrosis.36 The fact that four of our six patients with minimal change nephrotic syndrome expressed CTGF in their kidneys after steroid pulse therapy suggests that, at the very least, Japanese individuals may be high responders to glucocorticoids. We are thus conducting experiments to find some differences in the postreceptive pathway after DEX binding between C57B6 and SJL mice and to locate C57B6 strain-specific DEX-responsive elements at C897 to C628 bp in the mouse CTGF promoter. Such information should contribute to our understanding of the genetics of fibrosis susceptibility.
We thank M. Otobe for her technical assistance.
【参考文献】
Moussad EE, Brigstock DR: Connective tissue growth factor: what??s in a name? Mol Genet Metab 2000, 71:276-292
Takigawa M: CTGF/Hcs24 as a multifunctional growth factor for fibroblasts, chondrocytes and vascular endothelial cells. Drug News Perspect 2003, 16:11-21
Blom IE, Goldschmeding R, Leask A: Gene regulation of connective growth factor: new targets for antifibrotic therapy? Matrix Biol 2002, 21:473-482
Gupta S, Clarkson MR, Duggan J, Brady HR: Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis. Kidney Int 2000, 58:1389-1399
Riser BL, Denichilo M, Cortes P, Baker C, Grondini JM, Yee J, Narins RG: Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 2000, 11:25-38
Yokoi H, Mukoyama M, Nagae T, Mori K, Suganami T, Sawai K, Yoshioka T, Koshikawa M, Nishida T, Takigawa M, Sugawara A, Nakao K: Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. J Am Soc Nephrol 2004, 15:1430-1440
Okada H, Kikuta T, Kobayashi T, Inoue T, Kanno Y, Takigawa M, Sugaya T, Kopp JB, Suzuki H: Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis. J Am Soc Nephrol 2005, 16:133-143
Inoue T, Okada H, Kobayashi T, Watanabe Y, Kanno Y, Kopp JB, Nishida T, Takigawa M, Ueno M, Nakamura T, Suzuki H: Hepatocyte growth factor counteracts transforming growth factor-ß1, through attenuation of connective tissue growth factor induction, and prevents renal fibrogenesis in 5/6 nephrectomized mice. FASEB J 2003, 17:268-270
Eddy AA, Symons JM: Nephrotic syndrome in childhood. Lancet 2003, 362:629-639
Remuzzi G, Ruggenenti P, Perico N: Chronic renal diseases: renoprotective benefits of renin-angiotensin system inhibition. Ann Intern Med 2002, 136:604-615
Dammeier J, Beer HD, Brauchle M, Werner S: Dexamethasone is a novel potent inducer of connective tissue growth factor expression. J Biol Chem 1998, 273:18185-18190
Takaya K, Koya D, Isono M, Sugimoto T, Sugaya T, Kashiwagi A, Haneda M: Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells. Am J Physiol 2003, 284:F1037-F1045
Okada H, Danoff TM, Kalluri R, Neilson EG: Early role of Fsp1 in epithelial-mesenchymal transformation. Am J Physiol 1997, 273:F563-F574
Sheridan A, Schwartz J, Kroshian V, Tercyak A, Laraia J, Masino S, Lieberthal W: Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia. Am J Physiol 1993, 265:F323-F350
Okada H, Moriwaki K, Kalluri R, Imai H, Ban S, Takahama M, Suzuki H: Inhibition of monocyte chemoattractant protein-1 expression in tubular epithelium attenuates tubulointerstitial alteration in rat Goodpasture syndrome. Kidney Int 2000, 57:927-936
Gupta V, Wagner BJ: Expression of the functional glucocorticoid receptor in mouse and human lens epithelial cells. Invest Ophthalmol Vis Sci 2003, 44:2041-2046
Viedt C, Bruger A, Hansch GM: Fibronectin synthesis in tubular epithelial cells: up-regulation of the EDA splice variant by transforming growth factor ß. Kidney Int 1995, 48:1810-1817
De Haij S, Adcock IM, Bakker AC, Gobin SJ, Daha MR, van Kooten C: Steroid responsiveness of renal epithelial cells. Dissociation of trans-repression and transactivation. J Biol Chem 2003, 278:5091-5098
Inoue T, Okada H, Kobayashi T, Watanabe Y, Kikuta T, Kanno Y, Takigawa M, Suzuki H: TGF-ß1 and HGF coordinately facilitate collagen turnover in subepithelial mesenchyme. Biochem Biophys Res Commun 2002, 297:255-260
Leask A, Holmes A, Black CM, Abraham DJ: Connective tissue growth factor gene regulation. J Biol Chem 2003, 278:13008-13015
Chen Y, Blom IE, Sa S, Goldschmeding R, Abraham DJ, Leask A: CTGF expression in mesangial cells: involvement of SMADs, MAP kinase, and PKC. Kidney Int 2002, 62:1149-1159
Grotendorst GR, Okochi H, Hayashi N: A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ 1996, 7:469-480
Holmes A, Abraham DJ, Sa S, Shiwen X, Black CM, Leask A: CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling. J Biol Chem 2001, 276:10594-10601
Nakanishi T, Kimura Y, Tamura T, Ichikawa H, Yamaai Y, Sugimoto T, Takigawa M: Cloning of a mRNA preferentially expressed in chondrocytes by differential display-PCR from a human chondrocytic cell line that is identical with connective tissue growth factor mRNA. Biochem Biophys Res Commun 1997, 234:206-210
Pereira RC, Durant D, Canalis E: Transcriptional regulation of connective tissue growth factor by cortisol in osteoblasts. Am J Physiol 2000, 279:E570-E576
Kubota S, Moritani NH, Kawaki H, Mimura H, Minato M, Takigawa M: Transcriptional induction of connective tissue growth factor/hypertrophic chondrocyte-specific 24 gene by dexamethasone in human chondrocytic cells. Bone 2003, 33:694-702
Schoneveld OJLM, Gaemers IC, Lamers WH: Mechanisms of glucocorticoid signalling. Biochim Biophys Acta 2004, 1680:114-128
Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias G: Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity 1999, 10:387-398
Rodrigues E, Vilarem MJ, Ribeiro V, Maurel P, Lechner MC: Two CCAAT/enhancer binding protein sites in the cytochrome P4503A1 locus. Eur J Biochem 2003, 270:556-564
Yamada K, Duong DT, Scott DK, Wang JC, Granner DK: CCAAT/enhancer-binding protein b is an accessory factor for the glucocorticoid response from the cAMP response element in the rat phosphoenolpyruvate carboxykinase gene promoter. J Biol Chem 1999, 274:5880-5887
Wyszomierski SL, Rosen JM: Cooperative effects of STAT5 and C/EBPß on ß-casein gene transcription are mediated by the glucocorticoid receptor. Mol Endocrinol 2005, 15:228-240
Lerner L, Henriksen MA, Zhang X, Darnell JE, Jr: STAT3-dependent enhancesome assembly and disassembly: synergy with GR for full transcriptional increase of the 2-macroglobulin gene. Gene Dev 2003, 17:2564-2577
Fuller GM, Zhang Z: Transcriptional control mechanism of fibrinogen gene expression. Ann NY Acad Sci 2001, 936:469-479
Takeda T, Kurachi H, Yamamoto T, Nishio Y, Nakatsuji Y, Morishige K, Miyake A, Murata Y: Crosstalk between the interleukin-6 (IL-6)-JAK-STAT and the glucocorticoid-nuclear receptor pathway: synergistic activation of IL-6 response element by IL-6 and glucocorticoid. J Endocrinol 1998, 159:323-330
Balow JE, Austin HAI, Muenz LR, Joyce KM, Antonovych TT, Klippel JH, Steinberg AD, Plotz PH, Decker JL: Effect of treatment on the evolution of renal abnormalities in lupus nephritis. N Engl J Med 1984, 311:491-495
Kolb M, Bonniaud P, Galt T, Sime PJ, Kelly MM, Margetts PJ, Gauldie J: Differences in the fibrogenic response after transfer of active transforming growth factor-ß1 gene to lungs of "fibrosis-prone" and "fibrosis-resistant" mouse strains. Am J Respir Cell Mol Biol 2002, 27:141-150
作者单位：From the Departments of Nephrology* and Pathology, Saitama Medical School, Saitama; the Center of Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Ibaraki; and the Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of M
| ABSTRACT |
|---|
| TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
|---|
Key Words: alveolar macrophage • cigarette smoke • chronic obstructive pulmonary disease • corticosteroid • dexamethasone
| INTRODUCTION |
|---|
| TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
|---|
Macrophages are suggested to be the orchestrators of the chronic inflammatory response and tissue destruction associated with COPD (7). For example, macrophages contribute to airway inflammation in smokers and patients with COPD by secreting neutrophil and macrophage chemotactic factors and related chemokines such as interleukin (IL)-8 (8, 9), and by the generation of reactive oxygen species (10). Bronchoalveolar lavage (BAL) from asymptomatic smokers and patients with COPD yields higher numbers of macrophages than BAL from nonsmokers (11). Cigarette smoking affects the cellular composition of BAL, and markers of inflammation (12), and macrophages recovered from smokers secrete increased levels of chemotactic factors (13), cytokines (14), and proteases (15) compared with nonsmokers.
Cigarette smoke medium (CSM), produced by bubbling smoke through cell culture medium (16), induces IL-8 release from cultured human bronchial epithelial cells (9). CSM constituents in vitro also increase cytokine mRNA expression (17) and reduce surfactant secretion by alveolar type II cells (16). However, the effects of CSM on cytokine secretion by alveolar macrophages have not been evaluated.
Glucocorticosteroids inhibit cytokine release from inflammatory and other cell types mainly by suppressing the expression of inflammatory genes (18). Dexamethasone inhibits in vitro release of tumor necrosis factor- from human airway smooth muscle cells (19) and granulocyte macrophage-colony stimulating factor (GM-CSF) from monocytes (20). Dexamethasone also inhibits IL-8 release by human airway epithelial cells (21), U937 monocytic cells (22), and porcine alveolar macrophages (23). The inhaled corticosteroids fluticasone propionate and budesonide inhibit tumor necrosis factor-, IL-6, and IL-8 release by alveolar macrophages from nonsmokers (24). GM-CSF release from alveolar macrophages is reduced in subjects with asthma who are treated with inhaled steroids (25), and oral prednisolone reduces leukotriene B4 release by macrophages in subjects with nocturnal asthma (26). The effect of corticosteroids on macrophage function in COPD is not reported. Although corticosteroids are an effective treatment in asthma (27), their clinical efficacy in COPD is controversial (28, 29). Neither high-dose inhaled nor oral corticosteroids reduce the inflammatory response, concentrations of IL-8, nor proteases in induced sputum of patients with COPD (30–32). Consequently, reduced corticosteroid efficacy in COPD could be due to a decreased effect on macrophage function.
The aim of this study was to determine the effects of a corticosteroid on cytokine release by alveolar macrophages from patients with COPD. Consequently, we examined the effects of dexamethasone on IL-8 and GM-CSF release by alveolar macrophages from smokers and patients with COPD under basal conditions and after stimulation with IL-1ß or CSM. We chose to evaluate IL-8 because its concentration is elevated in BAL fluid of smokers and patients with COPD (12, 33–35). Similarly, we chose to evaluate GM-CSF because it is elevated in BAL fluid of patients with chronic bronchitis (36). GM-CSF also enhances neutrophil survival (37), and in patients with chronic bronchitis, it localizes to monocytes–macrophages in sputum (38). We also evaluated the effect of lipopolysaccharide (LPS) on macrophage function to account for possible LPS contamination of CSM.
| METHODS |
|---|
| TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
|---|
|
Isolation and Culture of Alveolar Macrophages
BAL was filtered and centrifuged, and the washed cells were resuspended in culture medium (RPMI-1640 containing 10% vol/vol fetal calf serum, 2 mM glutamine, 100 i.u./ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphoteracin) at a concentration of a million cells per milliliter (15). For consistency, alveolar macrophages were seeded for all experiments in 24-well Falcon cell culture plates (Becton Dickinson, Cowley, UK) at a density of 250,000 cells/well and were incubated (37°C, 5% CO2, humidified air) for 2 hours to allow the macrophages to adhere, after which the medium was replaced, removing nonadherent cells. After 24-hour culture, the medium was replaced, and cells were cultured for a further 24 hours under experimental conditions. Cell viability was assessed using Trypan Blue dye exclusion.
CSM
CSM was produced using the method of Wirtz and Schmidt (16). Briefly, smoke from two cigarettes (12-mg tar, 0.9-mg nicotine) was bubbled through a 20-ml culture medium. Absorbance was measured spectrophotometrically, after which the media were diluted approximately 12-fold to give an absorbance of 0.15 at 320 nm. This concentration (nominally one) was serially diluted with untreated media (0.001-fold to 1-fold) and applied to cells. Freshly prepared CSM was used in all experiments.
Cytokine Measurements
GM-CSF and IL-8 were measured in cell-free macrophage culture supernatants using paired antibody quantitative enzyme-linked immunosorbent assays and appropriate blanks (R&D Systems, Abingdon, UK) (31, 41). The lower limit of detection was 15.6 pg/ml for both assays.
Statistical Analysis
Data are presented as means ± SEM. Changes in macrophage secretory products were compared with control subjects using analysis of variance. Comparisons between experimental groups were performed using the Mann-Whitney U test. The concentration of inhibitor causing 50% inhibition of stimulated cytokine release was calculated using GraphPad Prism software (GraphPad Software Inc., San Diego, CA).
| RESULTS |
|---|
| TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
|---|
|
|
Concentrations of IL-8 were significantly higher in macrophages from patients with COPD at all concentrations of IL-1ß and dilutions of CSM . For GM-CSF, there was no significant difference in basal concentrations (discussed earlier here), and this was maintained during stimulation with IL-1ß. In contrast, stimulation with CSM led to a divergence between smokers and patients with COPD in GM-CSF release, with patients with COPD producing significantly more .
LPS (Escherichia coli 055:B5) did not significantly change the release of either IL-8 or GM-CSF by macrophages from either smokers or patients with COPD .
Effect of Dexamethasone on Basal Cytokine Release
Dexamethasone inhibited basal IL-8 release by macrophages from smokers in a concentration-dependent manner, with a maximal inhibition of 27% at 10 µM . In contrast, dexamethasone had no effect on basal release of IL-8 by macrophages from patients with COPD . Dexamethasone also inhibited basal GM-CSF release by macrophages from smokers in a concentration-dependent manner, with a maximal inhibition of 57% at 10 µM, and in contrast to its lack of effect on IL-8 release, also inhibited GM-CSF release (by 44%) from patients with COPD . The concentration causing 50% inhibition (IC50) for inhibition by dexamethasone of GM-CSF release by macrophages from smokers was significantly different to that for release by patients with COPD , with the curve shifted significantly to the left .
|
|
|
|
| DISCUSSION |
|---|
| TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
|---|
In this study, IL-1ß stimulated both IL-8 and GM-CSF release by alveolar macrophages from smokers and patients with COPD. We used IL-1ß as an inflammatory stimulus because its levels are elevated in the BAL fluid of cigarette smokers compared with nonsmokers (46). Although IL-1ß stimulates IL-8 release by BAL macrophages from cigarette smokers (47), IL-1ß–stimulated IL-8 release by macrophages from patients with COPD has not been reported previously. Similarly, although IL-1ß stimulates GM-CSF release by macrophages from patients with asthma and control subjects (25), this study is the first to compare IL-1ß–stimulated GM-CSF release between smokers and patients with COPD. In this study, CSM also stimulated the release of IL-8 and GM-CSF. Stimulation by CSM is unlikely to be due to contamination by LPS because herein LPS alone did not stimulate cytokine release in these cells. The latter observation is in contrast to a number of studies showing that LPS stimulates cytokine release from human alveolar macrophages (48, 49). The reason for this discrepancy is unclear but may be related to a number of factors. For example, responses to LPS can be variable between patients with the same diagnosis (50). In addition, the response by macrophages from smokers is less than that in nonsmokers (51). In a number of cases, the concentrations of LPS exceed that used in this study (48, 49, 52). Also, LPS elicits an increased secretion of cytokines that is inverse to the basal secretion (53). In this study, we observed a marked basal secretion of IL-8 and GM-CSF. Finally, there is inconsistency in the serotype of LPS used between published studies, which hinders a comparison with our present observations. We do not know the serotype of the LPS that may be present in our samples of CSM. Therefore, we cannot exclude a contribution of LPS to our CSM data.
Stimulation by CSM is consistent with release of tumor necrosis factor- and IL-6 by alveolar macrophages from normal subjects after exposure to tobacco smoke (54). The mechanism(s) of CSM-mediated cytokine release by macrophages is not investigated in this study. Cigarette smoke contains 4,700 compounds, including radicals, hydrogen peroxide, peroxynitrite, and acrolein (55). A number of these are found in aqueous solutions of smoke, including hydrogen peroxide (56) and semiquinone radicals that can react with oxygen to produce O2.- (57). Reactive oxygen species activate transcription factors, including nuclear factor-B (NF-B) and activator protein-1, which regulate expression of inflammatory genes such as IL-8 and GM-CSF (58). However, the reactive oxygen species content of our CSM was not determined, and in addition, soluble CS particulates may also have contributed to the increased macrophage activity observed herein. There is scant literature on the validity of comparison between CSM and in vivo exposure to cigarette smoke. In this study, the relationship between CSM and exposure of macrophages to cigarette smoke in vivo is not known. However, in rats, cigarette smoke condensates in vitro and cigarette smoke in vivo induce similar patterns of DNA damage (59).
The profile of release of IL-8 by IL-1ß and CSM was similar for both subject groups. The profile of IL-1ß–stimulated GM-CSF release was also similar. In contrast, CSM-stimulated release of GM-CSF was elevated in macrophages from patients with COPD compared with smokers. The reason for selective elevation of CSM-induced GM-CSF release is unknown but may be due to increased oxidant sensitivity of macrophages from patients with COPD.
In this study, dexamethasone had different inhibitory effects on cytokine release by alveolar macrophages from cigarette smokers and patients with COPD. The most striking difference was the lack of inhibitory effect of dexamethasone on IL-8 release by macrophages from patients with COPD compared with the inhibition by macrophages from smokers. In macrophages from normal volunteers, fluticasone proprionate or budesonide inhibit LPS-induced IL-8 release by alveolar macrophages by approximately 33% and approximately 60%, respectively (24). In addition, dexamethasone inhibits IL-1ß–induced IL-8 release by 64% in macrophages from normal subjects but only by 29% in cigarette smokers (47). Our present observation extends these findings and demonstrates a trend to increased resistance to steroids by macrophages from normal subjects to smokers to patients with COPD. The mechanisms underlying the relative steroid insensitivity of macrophages from patients with COPD in this study are not investigated but include altered glucocorticoid receptor function and apoptosis. There is no difference in glucocorticoid receptor expression in mononuclear cells in bronchial biopsies from patients with chronic bronchitis compared with nonsmoking control subjects (60). In contrast, there are more apoptotic macrophages in bronchial biopsies from patients with chronic bronchitis than from patients with asthma or healthy control subjects (61). Specific studies are required to determine the functional significance of these observations to macrophage corticosteroid insensitivity in COPD.
In contrast to its lack of inhibitory effect on IL-8 release by macrophages from patients with COPD, dexamethasone inhibited basal and IL-1ß–stimulated GM-CSF release and. However, macrophages from patients with COPD were less responsive than those from smokers, and the concentration–response curve was shifted to the right. These observations indicate a differential cytokine-specific effect of dexamethasone. This suggestion is consistent with the observation that dexamethasone inhibits IL-8 release by only approximately 50% compared with complete inhibition of GM-CSF release from human primary airway epithelial cells (41), which indicates differential corticosteroid sensitivity of inflammatory genes. In this study, in contrast to IL-1ß stimulation, GM-CSF release after CSM exposure was steroid insensitive . Similarly, dexamethasone did not inhibit IL-1ß–stimulated tumor necrosis factor- release by alveolar macrophages from cigarette smokers compared with nonsmokers (47). This lack of inhibitory effect of dexamethasone on cytokine release was mimicked by hydrogen peroxide treatment of a macrophage-like cell line (47). These combined observations suggest that oxidative mechanisms contribute, at least in part, to CSM stimulation of cytokine release by alveolar macrophages. This further indicates that steroid responsiveness of cytokine release by macrophages is both stimulus and cytokine-dependent. This proposal is consistent with the observation that human rhinovirus-induced respiratory epithelial cell expression of IL-8 and IL-6 is via an NF-B–independent pathway, whereas induction of GM-CSF is partially dependent on NF-B activation (62). Because glucocorticoids inhibit NF-B activity (63), the greater inhibition seen herein by dexamethasone on GM-CSF production compared with IL-8 production may reflect a greater relative contribution of NF-B activity to GM-CSF gene transcription rather than IL-8 gene transcription. However, this is not likely to be a general rule and may be an oversimplification, as IL-8 responses can also be mediated via NF-B (64).
The clinical efficacy of corticosteroids in COPD is controversial (28, 29). However, neither high doses of inhaled nor oral corticosteroid treatment reduces markers of airway inflammation, including IL-8, in induced sputum in patients with COPD (30, 31, 44). Any lack of efficacy could be due to a reduced steroid sensitivity by macrophages, the predominant inflammatory cell in COPD (7). In this study, we show that alveolar macrophages from patients with COPD display reduced sensitivity to dexamethasone compared with macrophages from smokers. Specifically, inhibition of the neutrophil chemotactic factor, IL-8, and the cell survival cytokine, GM-CSF, was reduced. It should be noted that the difference in inhibition by dexamethasone of cytokine release between smokers and patients with COPD in this study was relatively small. However, when combined with the comparative lack of effect of corticosteroids on other aspects of COPD pathophysiology, for example, reduced inhibition of neutrophil apoptosis (1), small reductions in efficacy could be additive and become clinically significant. Thus, any lack of efficacy of steroids on macrophage activity in COPD could lead to reduced inhibition of neutrophil chemoattractants and increased survival, with perpetuation of pulmonary neutrophilic inflammation. It is difficult to predict at this stage of understanding of the pathophysiology of COPD the relative impact on disease progression of individual experimental observations.
| REFERENCES |
|---|
| TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES
|
|---|
