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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 582-591, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0162OC

Retrovirally Introduced Prostaglandin D2 Synthase Suppresses Lung Injury Induced by Bleomycin

Miyuki Ando, Yousuke Murakami, Fumiaki Kojima, Hirahito Endo, Hidero Kitasato, Atsushi Hashimoto, Hirosuke Kobayashi, Masataka Majima, Matsuhisa Inoue, Hirobumi Kondo, Shinichi Kawai and Izumi Hayashi

Division of Respiratory Internal Medicine, Department of Internal Medicine; Department of Microbiology; Division of Rheumatology, Department of Internal Medicine; and Department of Pharmacology, Kitasato University School of Medicine, Sagamihara-shi, Kanagawa; and Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki-shi, Kanagawa, Japan

Address correspondence to: Izumi Hayashi, Department of Pharmacology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. E-mail: hayashii{at}med.kitasato-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hematopoietic prostaglandin D synthase (PGDS) is a key enzyme to produce prostaglandin (PG) D and J series. These PGs are involved in inflammation and immune system. The PGDS complementary DNA (cDNA)–expressing retrovirally transfected fibroblasts were introduced in vivo, and effect of the expression on lung injury induced by bleomycin was investigated in mice. Intravenous injection of PGDS cDNA-expressing fibroblasts significantly reduced lung edema, leukocyte infiltration in bronchoalveolar lavage (BAL) fluid, and pulmonary collagen content at 4 wk after instillation of bleomycin. Survival rate in mice instilled with the PGDS-expressing fibroblasts was higher than that in mice that received the mock transfection. Administration of 15-deoxy-{Delta}12,14-PGJ2, which is a nonenzymatic metabolite of PGD2, also attenuated the lung injury, suggesting mediation of PGs produced by PGDS for the attenuation. Introduction of PGDS cDNA-expressing fibroblasts suppressed expression of basic fibroblast growth factor, connective tissue growth factor, and collagen messenger RNAs in the lungs, as well as the levels of total proteins and hemoglobin in BAL fluid. These data suggest that the suppressive effect of PGDS on the lung injury could be partly mediated by edema formation and inhibition of genes involved in the fibrotic change.

Abbreviations: basic fibroblast growth factor, bFGF • bronchoalveolar lavage, BAL • complementary DNA, cDNA • connective tissue growth factor, CTGF • cyclooxygenase, COX • 15-deoxy-{Delta}12,14-prostaglandin J2, 15d-PGJ2 • enzyme-linked immunosorbent assay, ELISA • interleukin-1ß, IL-1ß • messenger RNA, mRNA • phosphate-buffered saline, PBS • prostaglandin, PG • prostaglandin D2 synthase, PGDS • peroxisome proliferator-activated receptor-{gamma}, PPAR{gamma} • reverse transcription-polymerase chain reaction, RT-PCR • transforming growth factor-ß, TGF-ß


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The pathogenesis of lung injury has been widely examined by induction with endotoxin lipopolysaccharide and bleomycin in experimental animals. Inflammatory cell infiltration, fibroblast proliferation, and excess deposition of extracellular matrix proteins (including collagen) in the lung parenchyma have been characterized in bleomycin-induced lung injury. Chronic inflammatory response also leads to loss of the normal alveolar architecture and impaired lung function (1, 2). It not only occurs in infectious and idiopathic interstitial pneumonia, but also in drug-induced interstitial fibrosis by treatment with bleomycin during cancer chemotherapy in humans (3, 4).

Several growth factors and cytokines, such as transforming growth factor-ß (TGF-ß) (5), interleukin-1ß (IL-1ß) (6), and basic fibroblast growth factor (bFGF) (7, 8), are considered to be possible mediators of lung injury and pulmonary fibrosis (9). Various observations also indicate that prostaglandins (PGs) could be involved in the pathogenesis (10, 11). Among them, PGE2 is a potent inhibitor of fibroblast proliferation and collagen synthesis (12, 13), and mice with a deficiency of inducible cyclooxygenase (COX)-2 are more susceptible to bleomycin-induced lung injury (14). Such proinflammatory mediators as TGF-ß, IL-1ß, and tumor necrosis factor-{alpha} induce fibroblasts to synthesize PGE2. On the other hand, TGF-ß stimulates autocrine synthesis of PGE2, resulting a negative feedback mechanism by which PGE2 display antiproliferative effects against TGF-ß (5). Thus, several growth factors and PGs are implicated in the pathogenesis of lung injury or pulmonary fibrosis in a complex manner.

The PGs are a family of structurally related molecules that are produced by cells in response to a variety of stimuli and regulate cellular growth, differentiation, and homeostasis (15). One of the PG derivatives, PGD2, is produced from PGH2, a common precursor of various prostanoids, by catalysis of the generating enzyme PGD2 synthase (PGDS). Two distinct types of PGDS have been characterized: one is the lipocalin-type PGDS that is known as brain-type enzyme, and the other is hematopoietic PGDS known as spleen-type enzyme (16). PGD2 is involved in numerous physiologic and pathophysiologic functions. It is known as a potent somnogen and nociceptive modulator in central nervous system. This event is mediated by lipocalin-type PGDS. On the other hand, hematopoietic PGDS could participate in acute and chronic inflammation. In allergic reaction, PGD2 has been reported to aggravate airway hypersensitivity (17, 18). On the contrary, PGD2 and its metabolite, cyclopentenone 15-deoxy-{Delta}12,14-PGJ2 (15 d-PGJ2), exert an anti-inflammatory effect on carrageenin-induced rat pleurisy by decreasing vascular permeability and plasma extravasation. This effect is mediated via the peroxisome proliferator–activated receptor-{gamma} (PPAR{gamma}) (19). This report prompted us to evaluate the anti-inflammatory effect of the PGs on lung injury. Thus, in the present study, we here examined whether in vivo expression of cDNA for hematopoietic PGDS affected lung injury induced by bleomycin in mice. For this, we used retroviral expression system, because the gene transfer method employing retroviruses can effectively introduce genes into various types of cells. Furthermore, The ex vivo method of retrovirus-mediated gene transfer can induce local and constitutive expression of the target gene in specific tissues in vivo (20).

The present study demonstrates that the retrovirul expression of PGDS cDNA could dramatically reduce bleomycin-induced lung injury, accompanied by reduction of edema formation and attenuated expressions of bFGF and collagen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of Lung Injury by Bleomycin
All of the following procedures conformed to the Guide for the care and use of laboratory animals published by the U.S. National Institutes of Health. Animals were housed in the Experimental Animal Center of the School for 1 wk under a specific pathogen-free environment, and thereafter acclimatized in the laboratory for 1 h before the commencement of each experiment. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (Abbott Laboratories, Chicago, IL). Bleomycin hydrochloride (100 mg/ml, 5 mg/kg; Nippon Kayaku Co., Tokyo, Japan) was sprayed into the trachea (21) of 5- to 6-wk-old male C57BL/6 mice (SLC Japan, Hamamatsu, Shizuoka, Japan) using Microsprayer (model IA-1c; Penn-Century, Pennsylvania, PA). The same volume of saline was used as the control. The mice were killed at 2 and 4 wk after instillation by severing the abdominal aorta.

Production of Cells with Constitutive PGDS Expression and In Vivo Transfer
Complementary DNA for human PGDS was amplified from normal human synovial cells (Cell System-SS Cells; Dainippon Pharmaceutical, Osaka, Japan) by reverse transcription-polymerase chain reaction (RT-PCR) using the following primer; 5'-ttgaattcatgccaaactacaaactcac-3' (sense) and 5'-ttggatccctagagtttggtttggggcc-3' (antisense). A 600-bp of the full coding region of PGDS cDNA was ligated into the EcoRI and BamHI sites of pLXSN (BD Biosciences Clontech, Palo Alto, CA). The plasmid constructed in this way was transfected to the packaging cell line (PT67; BD Biosciences Clontech) for retroviral delivery and expression. The transfected cells were selected by neomycin resistance and expression of PGDS was confirmed by RT-PCR. Subsequently, the culture supernatant was used to infect C57BL/6J-emb cells (RIKEN Cell Bank, Ibaraki, Japan). The infected fibroblasts were also selected by neomycin resistance. For preparation of the mock transfectant, pLXSN without any insert was also used to transfect PT67 cells followed by selection with neomycin resistance. Then the supernatant was used for infection of cells as above. The recombinant fibroblasts were transferred to C57BL/6 mice by injection (2 x 105 cells) into the tail vein. Instillation of bleomycin was done just before in vivo transfer. Uptake of the cells into lung was confirmed by in situ hybridization for cDNA encoding the neomycin resistance gene within the retroviral vector.

Administration of 15d-PGJ2
Mice were subcutaneously injected with 15d-PGJ2 (1.5 µg/ml, 150 ng/kg; Cayman Chemical, Ann Arbor, MI) twice a day from the instillation of bleomycin until the end of the experiment.

Measurement of Water Content in Lung
The wet lung weight was measured after careful excision of extraneous tissues. The lung was ashed at 180°C for 48 h and the dry weight was measured. Water content was calculated by subtracting dry weight from wet weight.

Collection of BAL Fluid
Bronchoalveolar lavage (BAL) fluid was collected for the analysis of leukocytes, total protein, and hemoglobin. The animals were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and killed with a severing the abdominal aorta. BAL was performed by using phosphate-buffered saline (PBS) (0.035 ml/g) to flush the airway five times using a polypropylene 20-gauge intravenous catheter. BAL fluid was placed in conical polypropylene tubes on ice. The recovered amount of BAL fluid was always over 90% of the injected fluid volume. Bleomycin instillation did not significantly decrease the recovery rate. The leukocytes in BAL fluid were immediately stained with trypan blue dye to confirm viability, and the total nucleated cells were counted using a hemocytometer. The leukocyte differential count was determined by examination of cytospin preparations stained with Diff-Quik (Kokusai Shiyaku Co., Kobe, Japan). Cell classifications were examined by counting at least 300 cells on each slide, and the percentage of each leukocyte type was recorded. A 500-µl aliquot of BAL fluid samples from five mice in each group were centrifuged at 1,100 rpm for 5 min at 4°C. The supernatant was stored frozen at -20°C for measurements of hemoglobin and total protein.

Measurement of Total Protein and Hemoglobin Content in BAL Fluid
The amounts of total protein and hemoglobin in BAL fluid were measured as indicators of lung permeability and cellular injury in fibrosis. Total protein was determined by the BCA assay (Pierce Chemical Co., Rockford, IL) according to directions by the manufacturers. After centrifugation of BAL fluid, the cell pellet was suspended in 50 µl of distilled water to lyse erythrocytes. Then the sample (50 µl) was mixed with an equal volume of 0.67 mM phosphate buffer (pH 7.2) containing 3.5 mM sodium lauryl sulfate and the absorbance at 540 nm was measured to determine the hemoglobin concentration. Mouse hemoglobin (Sigma-Aldorich Co., St. Louis, MO) was used as the standard.

Determination of Hydroxyproline
The hydroxyproline level in the hydrolysate was measured after conversion of hydroxyproline into pyrrol. Homogenized whole mouse lungs were hydrolyzed with 2 ml of 6 N HCl for 18 h at 110°C. The hydrolysate was evaporated to dryness and was dissolved in 0.4 ml of distilled water. Then the specific activity of hydroxyproline in the hydrolysate was measured after converting it into pyrrol. Aliquots (20 µl) of samples were diluted to 1 ml with distilled water. The diluent was mixed with 5 µl of 1 N NaOH, 0.75 g of KCl, and 0.25 ml of borate buffer (pH 8.7) and let stand for 25 min. Then 250 µl of chloramine-T solution (0.2 M) dissolved in Methycellsolb was added and the mixture was incubated for 25 min at room temperature. An aliquot (0.75 ml) of 3.6 M sodium thiosulfate was mixed and heated at 100°C for 30 min. Modified hydroxyproline in the mixture was extracted by addition of toluene (1.25 ml). The toluene layer was passed through a mini-column packed with 200 mg of sodium sulfate. The eluate (1 ml) was mixed with 0.4 ml of Ehrlich solution and the absorbance of the mixture was measured at 560 nm. Standard curves were generated for each experiment using 4-hydroxyproline. Results were expressed as micrograms of hydroxyproline in whole lung tissues.

In Situ Hybridization
Cryosections were mounted on saline-coated glass slides and fixed with 4% (wt/vol) paraformaldehyde. A digoxigenin-labeled antisense riboprobe resistance gene was for the neomycin prepared by in vitro transcription of the pCRII-TOPO vector containing the appropriate cDNA. The sense riboprobe was prepared by the same procedure. Sections of mouse lungs were treated with 10 µg/ml proteinase K and hybridized with the labeled riboprobes in the hybridization solution (Novagen, Madison, WI) for 18 h at 50°C in a moistened plastic box. The specimens were treated with 20 µg/ml of RNase A followed by intensive washing. Binding of the probes was visualized with an alkaline phosphatase–conjugated anti-digoxigenin antibody, 5-bromo-4-chloro-3 indolyl-phosphate, and 4-nitroblue tetrazolium chloride. All the slides were counterstained with hematoxylin.

Northern Blot Analysis
Total RNA was isolated from lung tissue by lysis in Trizol at 3 d after transfection. Ten micrograms of RNA was separated on 1% agarose gel and blotted onto a Hybond N+ membrane (Amersham). A 600-base EcoRI-BamHI fragment of human PGDS cDNA was used as the probe in the hybridization experiments. PGDS cDNA was labeled with [32P]CTP by random primer labeling. After hybridization with PGDS, the membranes were washed and exposed to Kodak Biomax MS-1 film (Eastman Kodak Co., Rochester, NY) at -70°C.

RT-PCR
Lung tissue samples (weighing {bsim} 50 mg) from three mice in each group were homogenized in guanidine isothiocyanate and phenol (Isogen; Nippon Gene, Toyama, Japan). Total RNA was extracted according to the manufacturer's instruction. Single-stranded cDNA was synthesized from 250 ng of the RNA using 0.8 µg of oligo-p(dT)15 primer and 10 units of AMV reverse transcriptase (Roche Diagnostics, Basel, Switzerland). Expression of IL-1ß, TGF-ß, bFGF, connective tissue growth factor (CTGF), type I collagen, PPAR{gamma}, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was analyzed by RT-PCR using 25 ng of the cDNA as the template. PCR was performed in 20 µL of 20 mM Tris-HCl (pH 8.7) containing 10 mM KCl, 5 mM (NH4)2SO2, 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.5 µM of forward and reverse primers, and 1 unit of HotStarTaq DNA polymerase (QIAGEN GmbH, Hilden, Germany). The following oligonucleotide primers were used: IL-1ß, 5'-GACCTTCCAGGATGAGGA-3' (bases 246–263), 5'-ACTCCACTTTGCTCTTGA-3' (bases 685–668); TGF-ß, 5'-AGACGGAATACAGGGCTTTCGATTCA-3' (985–1010 bp), 5'-CTTGGGCTTGCGACCCACGTAGTA-3' (1477–1454 bp); b-FGF, 5'-ACCAGCCACTTCAAGGAC-3' (63–81 bp), 5'-TATGGCCTTCTGTCCAGGTC-3' (435–416 bp); CTGF, 5'-CCGGATCGAGCTTTCTGGCTGCACC-3' (939–956 bp), 5'-GGCTGCAGTCTCCGTACATCTTCCTG (1175–1157 bp); type I collagen, 5'-ATTTTTCAAGGGTGCCAGTTTCG-3' (835–857 bp), 5'-TGCAGCAGGTTGTCTTGGATGTC-3' (1238–1216 bp); PPAR{gamma}, 5'-ATTTTTCAAGGGTGCCAGTTTCG-3' (835–857 bp), 5'-TGCAGCAGGTTGTCTTGGATGTC-3' (1238–1216 bp); GAPDH, 5'-CCCTTATTGACCTCAACTACATGGT-3' (100–125 bp), 5'-GAGGGGCCATCCACAGTCTTCTG-3' (569–547 bp). The reaction was started at 95°C for 10 min and subsequent conditions were: 94°C, 30 s; 53°C, 45 s; 72°C, 45 s for 20 cycles, followed by a final extension for 10 min at 72°C. PCR was done for 23 cycles (TGF-ß, CTGF, and Collagen), 25 cycles (bFGF), 29 cycles (IL-1ß), or 35 cycles (PPAR{gamma} and GAPDH). PCR products were analyzed on 2% agarose gel electrophoresis. The product sizes were the same as predicted from the sequences. Gels were visualized under UV light after staining with ethidium bromide and digital images were captured using a CCD camera system (FAS-III; Toyobo, Tokyo, Japan).

Immunohistochemistry
The trachea and lungs were fixed by inflation to a pressure of 25 cm H2O using freshly prepared 4% paraformaldehyde in PBS. Then the trachea was ligated. The heart and lungs were removed en bloc and fixed in paraffin. Sections (3 µm) were cut from the blocks and stained with hematoxylin and eosin or immunohistochemically. Immunoperoxidase staining was done using a Vector Stain Elite ABC kit (Vector Labs, Inc., Burlingame, CA). Sections were immersed in methanol containing 3% (vol/vol) H2O2 for 20 min to block endogenous peroxidase activity. The sections were preincubated with 0.3% (vol/vol) bovine serum albumin (Sigma-Aldrich Chemical Co) in PBS for 20 min, and then incubated with diluted goat serum (1:100) for 20 min. This was followed by incubation for 1 h in a humid chamber with a polyclonal rabbit anti-mouse bFGF antibody (1:50 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Then the sections were washed twice with PBS, and incubated for 30 min with biotinylated goat anti-rabbit IgG (1:2,000), followed by washing twice with PBS. Color was developed with 3,3'-diaminobenzidine (Dojindo, Kumamoto, Japan). Random sampling of tissue sections from five mice per each group was performed for the immunohistologic evaluation.

Determination of PGD2 in BAL Fluid
BAL fluids collected as described above were immediately acidified to pH 3.5 with 1N HCl and added to equal volume of ice-cold acetone. The mixture was allowed to stand 5 min on ice. The precipitated proteins were removed by centrifugation, and the supernatant were evaporated to dryness under nitrogen. PGD2 in the residue was derivatized to PGD2-methoxime. The methoximated samples were subsequently purified by Sep-Pak plus C18 Cartridge (Waters, Milford, MA). PGD2 in the purified samples were quantified by using Prostaglandin D2–MOX enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemicals, Ann Arbor, MI).

Administration of COX Inhibitors
PGDS-expressing fibroblasts or the mock fibroblasts were injected into mice received bleomycin as described above. Mice were fed diet containing 3,000 ppm of aspirin alminium (Sigma-Aldrich) or 1,500 ppm of SC-58635 (4-[5-(4-methylphenyl)-3-trifluoromethyl]-1H-pyrazol-1-yl]benzene-sulfonamide) (kindly supplied by Pharmacia Corporation, St. Louis, MO) starting Day 0 until the end of the experiment.

Statistical Analysis
Results are expressed as the mean ± SEM. Differences between groups were analyzed using ANOVA with Fisher's PLSD test for pairwise comparisons (StatView; Abacus Concepts Inc., Berkaley, CA). A P value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lung-Specific Delivery of PGDS cDNA-Expressing Fibroblasts by In Situ Hybridization and Detection of PGDS mRNA by Northern Blot Analysis
Transfected fibroblasts, which constitutively expressed PGDS cDNA, were injected into the tail vein for the in vivo expression. We expected that the cells would be trapped in the pulmonary microvessels by the injection and that expression of the cDNA would increase especially in the lungs. To confirm delivery of the cells to the tissue, an antisense riboprobe for the neomycin-resistance gene in constructed vector was used for detection of PGDS cDNA-transfected and the mock-transfected fibroblasts by in situ hybridization. The gene was detected at the same level in lungs from mice injected with PGDS cDNA-expressing fibroblasts (Figure 1C) and mice of the mock group (Figure 1B) at 1 wk after the introduction. The cells were also detected at 4 wk (Figure 1D). The sense riboprobe did not show significant staining (Figure 1A). Prominent expression of PGDS mRNA was seen in the lungs of mice injected with the cDNA-expressing fibroblasts, and the size of the mRNA was estimated to be {bsim} 1.8 kb by Northern blot analysis (Figures 1E and F).



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  		Figure 1. In vivo transfer of PGDS cDNA-expressing fibroblasts (PGDS) or fibroblasts transfected with the empty vector (MOCK) to the lungs of mice. Lungs were taken from mice at 1 wk (A–C) and 4 wk (D) after the transfer. Uptake of cells into the lungs was confirmed by in situ hybridization for cDNA encoding the neomycin resistance gene within the retroviral vector (A–D). A digoxigenin-labeled antisense (B–D; anti-sense) and the sense (A; sense) riboprobes for the neomycin resistance gene was prepared by in vitro transcription of the pCRII-TOPO vector containing the appropriate cDNA. Expression of PGDS mRNA in the lungs after administration of PGDS cDNA-expressing fibroblasts (PGDS) or fibroblasts transfected with the empty vector (EV) was analyzed by Northern blot (E and F). A 600-base EcoRI–BamHI fragment of human PGDS cDNA was used as the probe in the hybridization experiments. PGDS cDNA was labeled with [32P]dCTP by random primer labeling.

 
Protection of Bleomycin-Induced Lung Injury by Introduction of PGDS cDNA-Expressed Fibroblasts or Administration of 15d-PGJ2
Lung injury was induced by a single instillation of bleomycin. Acute and subacute injuries were evaluated at 2 and 4 wk after the instillation. The water content in lung as an indicator of edema, number of infiltrating leukocytes in BAL fluid, and hydroxyproline content of lung tissue were measured to assess as the injury. The water content and the number of leukocytes were significantly increased in group that received fibroblasts transfected with empty vector (mock) and saline-injected, vehicle-treated group at 2 and 4 wk after the instillation (Figures 2A–2D ; P < 0.01). The hydroxyproline content was also increased significantly at 4 wk (Figures 2E and 2F; P < 0.01). Lung edema (P < 0.01 at 2 and 4 wk) and leukocyte infiltration (P < 0.05 at 2 wk and P < 0.01 at 4 wk) related to the lung injury were significantly attenuated by the introduction of PGDS cDNA-expressing fibroblasts, in comparison with the mock-transfected group (Figures 2A and 2C). Increment of hydroxyproline content was also reduced in PGDS cDNA-introduced group at 4 wk with a statistically significant difference (Figure 2E; P < 0.01). Lymphocytes, neutrophils, and eosinophils in BAL fluid were all significantly increased in the mock-treated group, with a peak at 2 wk after instillation. Percentage of neutrophils, lymphocytes, and eosinophils in the lungs of mice injected with PGDS cDNA-expressing fibroblasts were significantly lower than those in the mock group at 2 wk after instillation of bleomycin (Table 1; P < 0.01 for neutrophils and eosinophils, and P < 0.05 for lymphocytes). Daily subcutaneous injection of 15d-PGJ2 (300 ng/d) significantly suppressed lung edema (P < 0.01 at 2 and 4 wk), infiltration of leukocytes (P < 0.01 at 2 wk and P < 0.05 at 4 wk), and increase in hydroxyproline (P < 0.05 at 2 wk and P < 0.01 at 4 wk) (Figures 2B, 2D, and 2F).



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  		Figure 2. Effects of PGDS cDNA-expressing fibroblasts and 15d-PGJ2 on lung injury induced by bleomycin in mice. Mice received a single intratracheal dose of bleomycin hydrochloride (5 mg/kg). PGDS cDNA-expressing fibroblasts (2 x 105 cells suspended in 0.2 ml of PBS; closed triangles) or fibroblasts transfected with the empty vector (open triangles) were injected intravenously on the day of bleomycin instillation (A, C, and E). 15d-PGJ2 (150 ng/kg; closed circles) or sterile saline vehicle (100 µl; open circles) was subcutaneously injected into the backs of mice twice a day after instillation of bleomycin until the end of the experimental period (B, D, and F). Mice were killed at 2 and 4 wk after the treatment. The water content in lung (A and B), the number of leukocytes in the BAL fluid (C and D), and the hydroxyproline content of lung tissue (E and F) were assessed as indicators of pulmonary inflammation and fibrotic change. Data are expressed as the mean ± SEM for seven mice per group. *P < 0.05, **P < 0.01, PGDS cDNA-expressing fibroblasts-introduced group versus the mock group or 15d-PGJ2–administered group versus the vehicle group.

 

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  		TABLE 1 Influence of PGDS cDNA-expressing fibroblasts on classification of leukocytes in BAL fluid from mice treated with bleomycin

 
Systemic Improvement in Bleomycin-Instillated Mice by Expression of PGDS cDNA or Administration of 15d-PGJ2
Compared with the saline-treated group, instillation of bleomycin resulted in significant weight loss at 2 and 4 wk after treatment with bleomycin (Figures 3A and 3B ; P < 0.01). Considerable improvement of weight loss was observed in mice injected with PGDS cDNA-expressing fibroblasts or treated with 15d-PGJ2 (P < 0.01). Observation was continued until 4 wk after instillation of bleomycin. When the survival rate was compared, the mice died every week in the vehicle-treated and the mock-transfected groups (n = 61) after instillation of bleomycin (Figure 3C). In contrast, 24 of 25 mice in PGDS cDNA-introduced group and 36 of 38 mice in 15d-PGJ2–treated group survived at 4 wk. The survival rate of the groups was significantly higher than that of the vehicle-treated and the mock-transfected groups (P < 0.001).



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  		Figure 3. Effects of PGDS cDNA-expressing fibroblasts and 15d-PGJ2 on body weight and survival rate in bleomycin-instilled mice. (A) Changes in body weight of PGDS cDNA-expressing fibroblasts-introduced group (closed triangles), the mock group (open triangles), and the control group without bleomycin instillation (open squares). (B) Changes in body weight of 15d-PGJ2–administered group (closed circles), the vehicle-administered group (open circles), and the control group without bleomycin instillation (open squares). Data are expressed as the mean ± SEM. **P < 0.01, PGDS cDNA-expressing fibroblasts-introduced group versus the mock group or 15d-PGJ2–administered group versus the vehicle group. (C) Survival rate of PGDS cDNA-expressing fibroblast-introduced group (closed triangles), 15d-PGJ2-administered group (closed circles), and the mock and the vehicle-treated groups (open circles). Observation was done until 28 d after instillation of bleomycin. The survival rate was evaluated with a log-rank test over the period (Kaplan-Meier method, P < 0.001).

 
Production of PGD2 in BAL Fluid from Mice Treated with PGDS cDNA-Expressing Fibroblast
Amounts of PGD2 in BAL fluid were measured by ELISA. Mice were instilled with bleomycin and subsequently injected with PGDS cDNA-expressing fibroblasts or the mock fibroblasts. The amounts in PGDS cDNA-introduced group were 94.5 + 21.3 pg (n = 7) and 97.8 + 9.8 pg (n = 7) at 2 and 4 wk after the transfection, respectively. On the other hands, the amounts in the mock-transfected group were 49.5 + 8.8 pg (n = 7) and 19.4 + 5.1 pg (n = 7) at 2 and 4 wk after the transfection, respectively. Significant differences of the amounts of PGD2 were observed between PGDS cDNA-introduced group and the mock group at both 2 wk (P = 0.02) and 4 wk (P = 0.0004) after the introduction.

Suppression of Plasma Extravasation by PGDS and 15d-PGJ2 in Bleomycin-Induced Lung Injury
The levels of total protein and hemoglobin in BAL fluid were compared between PGDS cDNA-expressing fibroblast-injected and the mock-treated groups or 15d-PGJ2–administered and the vehicle-groups, respectively. Bleomycin resulted in marked elevation of both the total protein and hemoglobin levels in BAL fluid 2 wk after the instillation (Figure 4 ; P < 0.01). The increase was significantly reduced by intravenously bolus injection of PGDS cDNA-expressing fibroblasts at 2 and 4 wk (Figure 4A; P < 0.01) or by daily subcutaneous injection of 15d-PGJ2 at 4 wk (Figure 4B; P < 0.05). The hemoglobin level of the BAL fluid was also significantly decreased in both PGDS cDNA-expressing fibroblasts-introduced and 15d-PGJ2–treated groups (Figures 4C and 4D; P < 0.05).



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  		Figure 4. Effects of PGDS cDNA-expressing fibroblasts and 15d-PGJ2 on the total protein and hemoglobin levels in BAL fluid in bleomycin-induced lung injury. Bleomycin-instilled mice were treated with PGDS cDNA-expressing fibroblasts (A and C, closed columns), fibroblasts transfected with the empty vector (A and C, right-diagonally hatched columns), 15d-PGJ2 (B and D, left-diagonally hatched columns), or the vehicle (B and D, open columns) as described in Figure 2. Columns indicated by broken lines show a group received with intratracheal saline instead of bleomycin. The amounts of total protein (A and B) and hemoglobin (C and D) in BAL fluid were measured at 2 and 4 wk after instillation of bleomycin. Data are expressed as the mean ± SEM of five mice per group. Ordinate indicate the amounts as mg per ml of BAL fluid. *P < 0.05, **P < 0.01, PGDS cDNA-expressing fibroblasts introduced group versus the mock group or 15d-PGJ2–administered group versus the vehicle group.

 
Attenuation of bFGF, TGF-ß, IL-1ß, CTGF, and Collagen mRNAs in Lung by Introduction of PGDS cDNA-Expressed Fibroblasts or Administration of 15d-PGJ2
Expression of mRNAs for several growth factors and collagen in whole lung tissue was analyzed in bleomycin-treated mice by RT-PCR (Figure 5) . Compared with lung from saline-injected, control mice without instillation of bleomycin, considerable expressions of bFGF, TGF-ß, IL-1ß, CTGF, and collagen mRNAs were observed in the lungs of bleomycin-treated, vehicle mice and mock-transfected mice at 2 and 4 wk. These inductions were dramatically decreased to the level of the control mice at 2 and 4 wk after the instillation of bleomycin by introduction of PGDS cDNA-expressing fibroblasts. Marked differences were also observed between the lungs of 15d-PGJ2–administered mice and the vehicle-treated mice. Constitutive expression of PPAR{gamma} was detected in all of the lungs tested.



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  		Figure 5. Expression of mRNAs for bFGF, TGF-ß, IL-1ß, CTGF, type I collagen, and PPAR{gamma} in the lungs of mice with lung injury induced by bleomycin. Bleomycin-instillated mice were treated with PGDS cDNA-expressing fibroblasts (PGDS), fibroblasts transfected with the empty vector (MOCK), 15d-PGJ2 (15d-PGJ2), or the vehicle (vehicle) as described in Figure 2. Control mice (control) received intratracheal saline instead of bleomycin. Lungs were isolated 2 and 4 wk after the treatment and total RNA was extracted for RT-PCR. Numbers in parentheses indicate the sizes of PCR products.

 
Immunohistochemical Detection of bFGF in Mice with PGDS cDNA Expression or 15d-PGJ2 Treatment
In the vehicle-treated group with bleomycin-instillation, infiltration of inflammatory cells into the alveolar and interstitial regions, as well as thickened and edematous alveolar walls, were observed at Week 2, when evaluated by hematoxylin–eosin staining of sections. Inflammatory infiltrates affected large regions of the lungs. Multiple areas of fibrotic change, located mainly in the subpleural regions, were observed at Week 4 (data not shown). When mice were treated with PGDS cDNA-expressing fibroblasts or with 15d-PGJ2, fibrotic changes of the interalveolar septa were less severe (data not shown). Expression of bFGF protein in the lungs of the mock-transfected group (Figures 6A and 6B) or the vehicle-treated group (Figures 6E and 6F) increased 2 and 4 wk after instillation of bleomycin. The tissues were immunohistochemically stained intensively by antibody against bFGF. In contrast, lung sections from PGDS cDNA-expressing fibroblast-treated mice (Figures 6C and 6D) or 15d-PGJ2–treated mice (Figures 6G and 6H) showed less staining against the antibody than those from the mock-transfected mice or the vehicle-treated mice, respectively.



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  		Figure 6. Immnunohistochemical observation of bFGF expression in lungs of bleomycin-instilled mice. Bleomycin-instilled mice were treated with PGDS cDNA-expressing fibroblasts (C and D), fibroblasts transfected with the empty vector (A and B), 15d-PGJ2 (G and H), or the vehicle (E and F) as described in Figure 2. Sections of lung tissues were prepared 2 wk (A, C, E, and G) and 4 wk (B, D, F, and H) after instillation of bleomycin. Control mice (I) received intratracheal saline instead of bleomycin. Original magnification: x200; bar: 200 µm.

 
Effects of COX Inhibitors on Attenuated Lung Injury by Introduction of PGDS cDNA
Mice were treated with bleomycin and PGDS cDNA-expressing fibroblasts. A selective COX-2 inhibitor, SC-58635, and aspirin to inhibit both COX-1 and COX-2, were administered orally, and effects of the inhibitors on attenuated lung injury by introduction of PGDS cDNA were examined (Figure 7) . Decreased number of leukocytes, total protein, and hemoglobin in BAL fluid, lung water content, and hydroxyproline content in lung by treatment with PGDS cDNA-expressing fibroblasts were aggravated by administration of aspirin. SC-58635 also reversed the attenuated number of leukocytes, total protein in BAL fluid, water content, and hydroxyproline content in lung significantly.



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  		Figure 7. Reversible effects of COX inhibitor on attenuated lung injury by introduction of PGDS cDNA. Bleomycin-instilled mice were treated with PGDS cDNA-expressing fibroblasts (closed, horizontally hatched, and vertically hatched columns) or the mock fibroblasts (open columns). Mice were fed diet containing 3,000 ppm of aspirin aluminum (horizontally hatched columns) or 1,500 ppm of SC-58635 and (vertically hatched columns) starting Day 0 until the end of the experiment. Mice were killed at 2 wk (A, B, and C) or 4 wk (D and E) after the instillation. Numbers of leukocytes (A), total protein (B), hemoglobin (C) in BAL fluid, lung water content (D), and hydroxyproline content in lung (E) were estimated as described in Figures 2 and 4. Data are expressed as the mean ± SEM of four to eight mice per group. *P < 0.05, **P < 0.01, PGDS cDNA-expressing fibroblasts introduced group versus the mock group. {ddagger}P < 0.01, PGDS cDNA-expressing fibroblasts introduced group versus aspirin-administered or SC-58635–administered group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To clarify whether PGDS-catalyzed products, PGD2 and/or 15d-PGJ2, were suppressors or enhancers of lung injury and the fibrotic change, we focused on constitutive expression of human hematopoietic PGDS by retroviral transfection of fibroblasts with the cDNA, and examined the effect of in vivo transfer of the fibroblasts on bleomycin-induced lung injury in mice. The result demonstrated that in vivo introduction of retrovirally transfected PGDS cDNA could effectively suppress the lung injury, and that PGs catalyzed by PGDS could be a suppressor against the injury.

Techniques involving retroviruses are capable of promoting constitutive gene expression and production of the gene product by target cells (20). Bolus injection of fibroblasts transfected with cDNA for the PGDS attenuated edema, leukocyte migration, and elevation of hydroxyproline in lung induced by instillation of bleomycin. Weight loss and survival were also improved by the treatment (Figure 2). With administration of PGDS-expressing fibroblasts, cells were injected via the tail vein. These fibroblasts may have lodged in the microvessels of the lungs where PGDS would be expressed. The lodging of fibroblasts in lung tissues was suggested by in situ hybridization, because DNA encoding the neomycin resistance gene of the vector was detected by the antisense probe in tissue sections from mice injected with PGDS-expressing fibroblasts as well as the mock fibroblasts. The cells remained in lung at 4 wk after the transfer. This suggests that expression of PGDS by the cells could last during the time course. Intensive expression of PGDS mRNA was also detected by Northern blotting in the lungs of mice injected with PGDS-expressing fibroblasts, but not in mice treated with mock fibroblasts (Figure 1). Furthermore, it was confirmed that a significant amount of PGD2 could be detected in BAL fluid from mice instilled with PGDS cDNA-expressing fibroblasts. This indicates that the fibroblasts produce PGD2 in vivo. Therefore, the effect of the PGDS cDNA-expressing fibroblasts was specific for expression of the gene, and any influence of the transfer of fibroblasts per se was negligible. Histologic observation also confirmed the suppression of lung injury by the treatments (data not shown).

The production of PGs begins with the release of arachidonic acid from membrane phospholipids by phospholipase A2 in response to inflammatory stimuli. Arachidonic acid is converted to PGH2 by the enzymes COX-1 and COX-2. It is thought that COX-1 is expressed constitutively in most tissues of the body and maintains homeostatic processes, such as mucus secretion. In contrast, COX-2 is mainly an inducible enzyme and is primarily involved in the regulation of inflammation. Cell-specific prostaglandin synthases convert PGH2 into a series of PGs, including PGI2, PGH2, PGE2, PGD2, and thromboxane A2. PGDS catalyzes the isomerization of PGH2 to produce PGD2 with 9-hydroxyl and 11-keto groups in the presence of sulfhydryl compounds. PGD2 appears to inhibit migration of human fetal lung fibroblasts in vitro (22). A cyclopentenone, 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2), is recognized as a potent apoptotic and growth-inhibitory factor (23, 24). 15d-PGJ2 is the terminal metabolite of PGD2 and is formed by dehydration of the cyclopentenone ring of endogenous PGD2. It is produced by a variety of cells, including mast cells, T cells, platelets, and alveolar macrophages (25). In contrast to classical PGs, which bind to cell surface G protein–coupled receptors, 15d-PGJ2 is a natural ligand for a nuclear receptor, the peroxisome proliferator–activated receptor-{gamma} (PPAR{gamma}) (26). Many in vivo studies support a role of 15d-PGJ2 as an anti-inflammatory agent (19, 27, 28). 15d-PGJ2 and PPAR{gamma} ligands inhibit inflammation in models of ischemia-reperfusion injury, colitis, and adjuvant-induced arthritis (22).

Daily administration of 15d-PGJ2 was examined whether the PG improved lung injury induced by bleomycin as PGDS-expressing fibroblasts did. The treatment also showed attenuation of all of the inflammatory parameters (i.e., lung edema, leukocyte infiltration, and hydroxyproline content in lung), indicating 15d-PGJ2 to be effective for suppression of the lung injury. Injected 15d-PGJ2 would be absorbed into the blood from the subcutaneous tissues and delivered to the microcirculation in the lungs where it would act on fibroblasts and other types of cells. Therefore, the suppression by 15d-PGJ2 may support protective effects of PGDS-expressing fibroblasts on the lung injury. These results strongly indicate that stable expression of PGDS or administration of 15d-PGJ2 could suppress bleomycin-induced lung injury mediated by complex inflammatory reactions.

Previous studies have shown that the imbalance of various chemical mediators is involved in acute lung injury and fibrosis (9, 29). Fibroblast proliferation and collagen synthesis are partly regulated by complex interactions between stimulatory and inhibitory mediators. Several stimulatory mediators, including TGF-ß, platelet-derived growth factor, IL-1ß, and PGE2, have been suggested to play a role in the pathogenesis of pulmonary fibrosis. Among the various PGs, PGE2 is known to downregulate fibroblast functions, including proliferation (13) and collagen production (30). Further support for the notion that PGE2 has an important downregulatory effect on the evolution of pulmonary fibrosis comes from recent preliminary work showing that COX-2-/- mice have an enhanced fibrotic response to bleomycin (14).

Further experiments were performed to elucidate the mechanism by which PGDS or 15d-PGJ2 suppressed the lung injury. Fibroblasts play an important role in the repair and remodeling processes following injury. Several observations suggest that bFGF may play an important role in lung injury. Basic FGF is a potent mitogenic, angiogenic, differentiation, and chemotactic factor for many types of cells, and has an important role in the repair of tissue injury (8). It upregulates the expression of several cellular factors involved in pulmonary fibrosis, including CTGF and collagen. In the lungs, bFGF has been localized to isolated alveolar type II cells and basement membranes, including those of the alveoli (2). In primary cultured lung fibroblasts, bFGF induced the expression of collagen and CTGF mRNA. An increase in immunoreactive bFGF has been found in mast cells in the extracellular matrix after exposure to bleomycin (8). In the present study, both immunohistochemistry and RT-PCR showed that increased expression of bFGF in lung tissues after instillation of bleomycin was weakened by treatment of mice with PGDS-expressing fibroblasts or 15d-PGJ2 (Figures 5 and 6). Concomitantly, expression of CTGF and collagen mRNA was suppressed in the lung of mice subjected to those treatments. These results suggest that PGDS or 15d-PGJ2 could reduce the expression of bFGF, subsequently resulting downregulation of CTGF and collagen synthesis. TGF-ß–induced procollagen synthesis by lung fibroblasts is modulated by production of PGE2 (12). Moreover, overexpression of COX-2 inhibits fibroblast proliferation in vitro and increases PGE2 levels in the lungs (11). These reports indicate that PGE2 protects fibrosis mediated by TGF-ß. A number of studies have revealed that IL-1ß shows proinflammatory effects. It promotes leukocyte recruitment, as well as expression of procollagen and other extracellular matrix proteins to induce fibrosis (6). Lower expression of TGF-ß and IL-1ß was also observed after introduction of PGDS-expressing fibroblasts or administration of 15d-PGJ2 in the present study. Thus, it is possible that PGDS and the products modulate multiple growth factors, probably at the transcriptional level, providing antifibrotic influence.

An increase in water content is one of the characteristics of lung injury and fibrosis, suggesting the involvement of plasma extravasation in the development and the progression (31). It is generally thought that a decrease of vascular permeability could prevent the release of plasma components into the tissue microenvironment, and these components are an important source of several growth factors and biologically active substances. Introduction of PGDS cDNA or administration of 15d-PGJ2 improved lung edema in the present study. In addition, the protein and hemoglobin levels in BAL fluid were also decreased by treatment with PGDS-expressing fibroblasts or 15d-PGJ2 in the present model (Figure 4). This suggests that PGDS and 15d-PGJ2 could reduce the extravasation from lung microvessels and protect against vascular damage. 15d-PGJ2 is reported to attenuate the development of carrageenin-induced pleurisy and reduces both acute and chronic inflammation (19, 27). Exudation of plasma proteins and leakage of erythrocytes into BAL fluid reached a peak at 2 wk after the instillation of bleomycin, so these events seem to occur in the relatively acute phase of the lung injury. The persistence of chronic inflammation is important in the development of pulmonary fibrosis. Extravasation of plasma through damaged alveolar walls is a common occurrence in inflammatory lung diseases (32). Because suppression of the initial event, such as an increase in extravasation, by PGDS-derived PGs may inhibit the supply of growth factors and signals from the bloodstream, subsequent progression of injury may be effectively attenuated. The early expression of proinflammatory factors and their persistence may be important in mediating pulmonary fibrosis. Thus, emphasis would be placed on the multiple anti-inflammatory effects of PGDS and 15d-PGJ2 in relation to not only acute inflammation, but also chronic pathogenesis such as fibrosis.

Treatment with COX inhibitor completely reversed suppression of bleomycin-induced lung injury by introduction of PGDS-expressing fibroblasts. COX inhibitor blocks production of PG endoperoxide H. Therefore, PGD2 could not be produced even though PGD2 synthase was expressed. As the result, COX inhibitor might offset the protective effects of PGDS on the lung injury. Furthermore COX-2 could be suggested to play a role in production of the PG in part. COX-2–deficient mice have been also reported to be susceptible to pulmonary fibrogenesis (33).

Although PGD2 was detected in BAL fluid, the present study did not determine whether PGD2 or 15d-PGJ2 had a dominantly protective role in lung injury. A major peripheral source of PGDS is mast cells outside the central nervous system. PGD2 is released by mast cells, suggesting a role in immunologic responses (34). A considerable increase of mast cells infiltration was also observed in the present model (data not shown), suggesting the intrinsic production of PGDS-derived products. We could not distinguish the effect of PGDS-expressing fibroblasts from that of 15d-PGJ2 on bleomycin-induced lung injury. Therefore, it cannot be concluded that both 15d-PGJ2 and PGD2 derivatives produced by expression of PGDS bind to the same receptor (i.e., PPAR{gamma}) to mediate their anti-inflammatory effects on fibrosis. 15d-PGJ2 binds directly to PPAR{gamma} and promotes differentiation of C3H10T1/2 fibroblasts into adipocytes (35). Otherwise, PGD2 produced by PGDS may bind to the DP receptor before conversion to 15d-PGJ2 by hydration in the tissues. Further investigations to identify the receptor(s) and signal transduction mechanisms are needed in the future.

In this article, we provide observation showing that in vivo introduction of retrovirally transfected PGDS cDNA could suppress lung injury by bleomycin in mice. The protective mechanism may be diverse, including attenuation of plasma extravasation to be responsible for edema formation and inhibitory expressions of bFGF, TGF-ß, CTGF, and collagen to mediate fibrotic change of lungs. Thus, PGDS-derived PGs could be a downregulator in lung injury as well as PGE2.


   Acknowledgments
 
Part of this work was supported by a research grant from The Japanese Ministry of Education, Culture, Sports, Science and Technology (to S.K. and I.H.), Parent's Association Grant of Kitasato University School of Medicine (to M.A.), and a Research Project grant from the Graduate School of Medical Sciences, Kitasato University (to I.H.). The authors thank Ms. Terumi Mizuno, Sachiko Kurihara, and Masumi Tanaka for their expert technical assistance, Mr. Nobumasa Mitsuoka and Ms Misako Uemura for animal care, and The Radioisotope Research Center at Kitasato University School of Medicine for radioactive experiment. The authors thank Prof. Fumihiko Sakai and Prof. Noriyuki Masuda for his encouragement.

Received in original form August 22, 2002

Received in final form October 26, 2002


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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