Inflammatory Cytokines, Angiogenesis, and Fibrosis in the Rat Peritoneum
2002; Elsevier BV; Volume: 160; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61176-5
ISSN1525-2191
AutoresPeter J. Margetts, Martin Kolb, Lisa Yu, Catherine M. Hoff, Clifford J. Holmes, Daniel C. Anthony, Jack Gauldie,
Tópico(s)Aortic aneurysm repair treatments
ResumoPeritonitis, a common complication of peritoneal dialysis, is followed by acute changes in the function of the peritoneum. The role of inflammatory cytokines in these processes is not clearly identified. We used adenoviral-mediated gene transfer to transiently overexpress interleukin (IL)-1β (AdIL-1β) or tumor necrosis factor (TNF)-α (AdTNF-α) in the rat peritoneum then used a modified equilibrium test to study the histological and functional changes. Overexpression of IL-1β or TNF-α led to an acute inflammatory response. Both inflammatory cytokines induced an early expression of the angiogenic cytokine, vascular endothelial growth factor, along with increased expression of the profibrotic cytokine, transforming growth factor-β1, along with fibronectin expression and collagen deposition in peritoneal tissues. Both inflammatory cytokines induced angiogenesis, increased solute permeability, and ultrafiltration dysfunction at earlier time points. Changes in structure and function seen in AdTNF-α-treated animals returned to normal by 21 days after infection, whereas AdIL-1β-treated animals had persistently increased vasculature with submesothelial thickening and fibrosis. This was associated with up-regulation TIMP-1. TNF-α or IL-1β both induce acute changes in the peritoneum that mimic those seen in peritoneal dialysis patients who experience an episode of peritonitis. These functional changes were associated with early angiogenesis that resolved rapidly after exposure to TNF-α. IL-1β exposure, however, led to a different response with sustained vascularization and fibrosis. IL-1β inhibition may be a therapeutic goal in acute peritonitis to prevent peritoneal damage. Peritonitis, a common complication of peritoneal dialysis, is followed by acute changes in the function of the peritoneum. The role of inflammatory cytokines in these processes is not clearly identified. We used adenoviral-mediated gene transfer to transiently overexpress interleukin (IL)-1β (AdIL-1β) or tumor necrosis factor (TNF)-α (AdTNF-α) in the rat peritoneum then used a modified equilibrium test to study the histological and functional changes. Overexpression of IL-1β or TNF-α led to an acute inflammatory response. Both inflammatory cytokines induced an early expression of the angiogenic cytokine, vascular endothelial growth factor, along with increased expression of the profibrotic cytokine, transforming growth factor-β1, along with fibronectin expression and collagen deposition in peritoneal tissues. Both inflammatory cytokines induced angiogenesis, increased solute permeability, and ultrafiltration dysfunction at earlier time points. Changes in structure and function seen in AdTNF-α-treated animals returned to normal by 21 days after infection, whereas AdIL-1β-treated animals had persistently increased vasculature with submesothelial thickening and fibrosis. This was associated with up-regulation TIMP-1. TNF-α or IL-1β both induce acute changes in the peritoneum that mimic those seen in peritoneal dialysis patients who experience an episode of peritonitis. These functional changes were associated with early angiogenesis that resolved rapidly after exposure to TNF-α. IL-1β exposure, however, led to a different response with sustained vascularization and fibrosis. IL-1β inhibition may be a therapeutic goal in acute peritonitis to prevent peritoneal damage. Peritonitis is a common complication of peritoneal dialysis.1Bunke CM Brier ME Golper TA Outcomes of single organism peritonitis in peritoneal dialysis: gram negatives versus gram positives in the Network 9 Peritonitis Study.Kidney Int. 1997; 52: 524-529Crossref PubMed Scopus (142) Google Scholar The response of the peritoneum to infective organisms involves the inflammatory cytokines and the interaction between resident cell populations: macrophages, mesothelial cells, and fibroblasts.2Topley N Liberek T Davenport A Li FK Fear H Williams JD Activation of inflammation and leukocyte recruitment into the peritoneal cavity.Kidney Int. 1996; 56: S17-S21Google Scholar, 3Jorres A Ludat K Sander K Dunkel K Lorenz F Keck H Frei U Gahl GM The peritoneal fibroblast and the control of peritoneal inflammation.Kidney Int. 1996; 56: S22-S27Google Scholar The earliest response involves tumor necrosis factor (TNF)-α and interleukin (IL)−13Jorres A Ludat K Sander K Dunkel K Lorenz F Keck H Frei U Gahl GM The peritoneal fibroblast and the control of peritoneal inflammation.Kidney Int. 1996; 56: S22-S27Google Scholar derived from macrophages in response to bacterial products.4Visser CE Brouwer-Steenbergen JJ Struijk G Krediet RT Beelen RH Production of IL-1 beta and TNF-alpha by peritoneal macrophages depends on the bacterial species and the inoculum.Adv Perit Dial. 1997; 13: 201-204PubMed Google Scholar What follows is a complex interaction between inflammatory cytokines, such as IL-6, chemokines and subsequent leukocyte transmigration, prostaglandins, nitric oxide, and adhesion molecules. There is also an anti-inflammatory response consisting of soluble receptors to TNF-α,5Zemel D Imholz AL de Waart DR Dinkla C Struijk DG Krediet RT Appearance of tumor necrosis factor-alpha and soluble TNF-receptors I and II in peritoneal effluent of CAPD.Kidney Int. 1994; 46: 1422-1430Crossref PubMed Scopus (117) Google Scholar IL-1 receptor antagonist,6Brauner A Hylander B Wretlind B Tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-1 receptor antagonist in dialysate and serum from patients on continuous ambulatory peritoneal dialysis.Am J Kidney Dis. 1996; 27: 402-408Abstract Full Text PDF PubMed Scopus (55) Google Scholar and repair molecules such as hyaluronan.7Yung S Coles GA Davies M IL-1 beta, a major stimulator of hyaluronan synthesis in vitro of human peritoneal mesothelial cells: relevance to peritonitis in CAPD.Kidney Int. 1996; 50: 1337-1343Crossref PubMed Scopus (67) Google Scholar In the first month after peritonitis, patients develop an acute dysfunction of the peritoneum as a dialysis membrane.8Ates K Koc R Nergizoglu G Erturk S Keven K Sen A Karatan O The longitudinal effect of a single peritonitis episode on peritoneal membrane transport in CAPD patients.Perit Dial Int. 2000; 20: 220-226PubMed Google Scholar This is characteristically associated with increased transport of small molecular weight solutes and ultrafiltration failure. The mechanism for this is not well established, but vascular effects of nitric oxide9Combet S Van Landschoot M Moulin P Piech A Verbavatz JM Goffin E Balligand JL Lameire N Devuyst O Regulation of aquaporin-1 and nitric oxide synthase isoforms in a rat model of acute peritonitis.J Am Soc Nephrol. 1999; 10: 2185-2196PubMed Google Scholar or prostaglandins10Steinhauer HB Schollmeyer P Prostaglandin-mediated loss of proteins during peritonitis in continuous ambulatory peritoneal dialysis.Kidney Int. 1986; 29: 584-590Crossref PubMed Scopus (62) Google Scholar have been hypothesized to play a role. Recurrent peritonitis has been shown, in some studies, to be associated with long-term peritoneal membrane changes.11Davies SJ Bryan J Phillips L Russell GI Longitudinal changes in peritoneal kinetics: the effects of peritoneal dialysis and peritonitis.Nephrol Dial Transplant. 1996; 11: 498-506Crossref PubMed Google Scholar, 12Lin CY Chen WP Yang LY Chen A Huang TP Persistent transforming growth factor-beta 1 expression may predict peritoneal fibrosis in CAPD patients with frequent peritonitis occurrence.Am J Nephrol. 1998; 18: 513-519Crossref PubMed Scopus (53) Google Scholar As in the acute changes after peritonitis, long-term changes are associated with increased solute transport and decreased ultrafiltration. In this setting, human biopsy studies13Mateijsen MA van der Wal AC Hendriks PM Zweers MM Mulder J Struijk DG Krediet RT Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis.Perit Dial Int. 1999; 19: 517-525PubMed Google Scholar, 14Plum J Hermann S Fussholler A Schoenicke G Donner A Rohrborn A Grabensee B Peritoneal sclerosis in peritoneal dialysis patients related to dialysis settings and peritoneal transport properties.Kidney Int. 2001; 59: S42-S47Abstract Full Text PDF Scopus (105) Google Scholar, 15Combet S Miyata T Moulin P Pouthier D Goffin E Devuyst O Vascular proliferation and enhanced expression of endothelial nitric oxide synthase in human peritoneum exposed to long-term peritoneal dialysis.J Am Soc Nephrol. 2000; 11: 717-728PubMed Google Scholar and animal experiments16Margetts PJ Kolb M Yu L Hoff CM Gauldie J A chronic inflammatory infusion model of peritoneal dialysis in rats.Perit Dial Int. 2001; 21: S319-S323PubMed Google Scholar have identified an increase in the peritoneal-associated vasculature, which seems to be the primary cause of increased solute transport. A fibrogenic response and associated vasculopathy have also been identified in patients on long-term peritoneal dialysis.17Honda K Nitta K Horita S Yumura W Nihei H Morphological changes in the peritoneal vasculature of patients on CAPD with ultrafiltration failure.Nephron. 1996; 72: 171-176Crossref PubMed Scopus (199) Google Scholar The association between peritoneal inflammation, angiogenesis, and fibrosis is not clear. IL-1β has been identified in vivo as having strong fibrogenic properties through up-regulation of transforming growth factor (TGF)-β.18Kolb M Margetts PJ Anthony DC Pitossi F Gauldie J Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis.J Clin Invest. 2001; 107: 1529-1536Crossref PubMed Scopus (615) Google Scholar This has been confirmed in in vitro mesothelial cell culture work.19Yang WS Kim BS Lee SK Park JS Kim SB Interleukin-1beta stimulates the production of extracellular matrix in cultured human peritoneal mesothelial cells.Perit Dial Int. 1999; 19: 211-220PubMed Google Scholar Both IL-1β and TNF-α have angiogenic properties as has been demonstrated in studies using rat mesenteric window assays20Norrby K Interleukin-1-alpha and de novo mammalian angiogenesis.Microvasc Res. 1997; 54: 58-64Crossref PubMed Scopus (21) Google Scholar, 21Norrby K TNF-alpha and de novo mammalian angiogenesis.Microvasc Res. 1996; 52: 79-83Crossref PubMed Scopus (22) Google Scholar and TNF-α has been associated with up-regulation of the angiogenic cytokine vascular endothelial growth factor (VEGF) in cell culture.22Ryuto M Ono M Izumi H Yoshida S Weich HA Kohno K Kuwano M Induction of vascular endothelial growth factor by tumor necrosis factor alpha in human glioma cells. Possible roles of SP-1.J Biol Chem. 1996; 271: 28220-28228Crossref PubMed Scopus (456) Google Scholar We have previously described the use of adenovirus-mediated gene transfer of cytokines and growth factors as an effective tool in elucidating functional and morphological changes in the peritoneum.23Margetts PJ Kolb M Hoff CM Shockley TR Gauldie J Gene transfer of transforming growth factor beta 1 to the rat peritoneum: effects on membrane function.J Am Soc Nephrol. 2001; 12: 2029-2039PubMed Google Scholar In the following experiments, we compared the effects of transient overexpression of TNF-α or IL-1β on the peritoneum of rats using adenovirus-mediated gene transfer (AdTNF-α or AdIL-1β) and controlled with a null adenovirus (AdDL70). Four days after infection with either AdTNF-α or AdIL-1β we saw increased vascularity of the peritoneum, increased solute transport, and decreased ultrafiltration. By day 21, the animals treated with AdTNF-α returned to normal peritoneal morphology and function, but the AdIL-1β-treated animals remained quite abnormal with peritoneal fibrosis and persistent vascularization. The construction of the adenovirus vectors AdIL-1β,18Kolb M Margetts PJ Anthony DC Pitossi F Gauldie J Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis.J Clin Invest. 2001; 107: 1529-1536Crossref PubMed Scopus (615) Google Scholar AdTNF-α,24Marr RA Addison CL Snider D Muller WJ Gauldie J Graham FL Tumour immunotherapy using an adenoviral vector expressing a membrane-bound mutant of murine TNF alpha.Gene Ther. 1997; 4: 1181-1188Crossref PubMed Scopus (69) Google Scholar and AdDL7025Bett AJ Haddara W Prevec L Graham FL An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3.Proc Natl Acad Sci USA. 1994; 91: 8802-8806Crossref PubMed Scopus (690) Google Scholar have been previously described. Adenovirus preparations were purified by CsCl gradient centrifugation and PD-10 Sephadex chromatography (Amersham Pharmacia, Baie d'Urfe, Canada) and plaque-titered on 293 cells as previously described.26Xing Z Ohkawara Y Jordana M Graham F Gauldie J Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions.J Clin Invest. 1996; 97: 1102-1110Crossref PubMed Scopus (199) Google Scholar All animal studies were performed according to the Canadian Council on Animal Care Guidelines. Three groups of female Sprague-Dawley rats (Harlan, Indianapolis, IN), 200 to 250 g (corresponding to 6 to 8 weeks of age27Aleman CL Mas RM Rodeiro I Noa M Hernandez C Menendez R Gamez R Reference database of the main physiological parameters in Sprague-Dawley rats from 6 to 32 months.Lab Anim. 1998; 32: 457-466Crossref PubMed Scopus (87) Google Scholar) were studied. The first group (n = 27) received an intraperitoneal injection of AdIL-1β. The second group (n = 20) received AdTNF-α and the third group (n = 28) received a null control virus (AdDL70). All were administered adenovirus at a dose of 1.5 to 2.0 × 109Combet S Van Landschoot M Moulin P Piech A Verbavatz JM Goffin E Balligand JL Lameire N Devuyst O Regulation of aquaporin-1 and nitric oxide synthase isoforms in a rat model of acute peritonitis.J Am Soc Nephrol. 1999; 10: 2185-2196PubMed Google Scholar plaque-forming units/ml diluted to 100 μl in phosphate-buffered saline on day 0. Animals did not receive anti-inflammatory agents during the experimental protocol. Animals from each group were sacrificed on days 4, 7, 21, and 28 after adenovirus administration. Before sacrifice, 0.09 ml/g of 2.5% Dianeal (Baxter Health Care, McGaw Park, IL) was administered intraperitoneally. Four hours later, the peritoneum was opened and the entire fluid content removed for accurate ultrafiltration measurement. Net ultrafiltration was the volume of fluid removed after 4 hours minus the volume of fluid administered. Blood samples were drawn and the entire anterior abdominal wall was removed after skin and subcutaneous tissue was removed. The lower portion of this tissue was stored in formalin and the upper portion taken for RNA extraction. Mesenteric tissue was taken and frozen in liquid nitrogen. Whole blood was centrifuged at 5000 rpm for 10 minutes and the serum removed. Peritoneal fluid samples were centrifuged at 1500 rpm for 5 minutes. Samples were analyzed on a Hitachi 917 automated chemistry analyzer (Roche Diagnostics, Laval, Canada) for glucose and albumin. Mass transport of glucose out of the peritoneum was calculated as (initial dialysate glucose × initial volume) − (final dialysate glucose × final volume). Albumin clearance was calculated as mass transport divided by the serum solute concentration. All values were corrected for animal weight at sacrifice. Cell number in the peritoneal fluid was counted with a hemocytometer. The fluid was plated to a glass slide by cytospin (Shandon Inc., Pittsburgh, PA) and stained (Biochemical Science Inc., Swedesboro, NJ). Cell differential was counted on at least 300 cells. Tissue samples at sacrifice from the lower anterior abdominal wall or omentum were taken and fixed in a sufficient amount of 4% phosphate-buffered formaldehyde for 24 hours. The tissue samples were then paraffin-processed, embedded, and 5-μm sections cut. Cut sections were then stained for Masson's trichrome and immunohistochemistry was performed with antibodies to Factor VIII-von Willebrand factor (vWF) antigen (DAKO Corp., Carpinteria CA). Negative control sections were run in parallel with nonimmune mouse or rabbit serum. All sections were deparaffinized in xylene followed by 100% ethanol and then placed in a methanol H2O2 solution for 30 minutes to block endogenous peroxidase activity. After hydration to water with graded alcohols, the sections were placed in 0.05 mol/L Tris-buffered saline, pH 7.6. The sections were digested with 0.05% Pronase (Sigma Chemical Co., St. Louis, MO) in Tris-buffered saline with calcium chloride for 17 minutes at room temperature then blocked in 5% normal goat serum followed by a 1-hour incubation in the 1:500 rabbit anti-human factor VIII in 1% normal goat serum. Sections were then incubated in a prediluted kit of a biotinylated goat anti-rabbit followed by a streptavidin/peroxidase conjugate (Zymed Labs, San Francisco, CA) as per the manufacturer's instructions. Immunohistochemistry incubations were performed at room temperature and sections were washed in between incubations 3 × 5 minutes with 0.05 mol/L Tris-buffered saline, pH 7.6, except before the addition of primary antibody. Sections were rinsed in 0.05 mol/L acetate buffer, pH 5.0, before development in an 3-amino-9-ethylcarbazole (AEC) chromogen substrate for 15 minutes. Sections were counterstained in Mayer's hematoxylin for 2 minutes before mounting with glycerin gelatin. Low-power fields from factor VIII-immunostained sections of the anterior abdominal wall were digitized using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). All sections were analyzed by the same image-processing algorithm using Leica Qwin Image Processing Software (Leica Imaging Systems, Cambridge, England). Results are reported as number of vessels/mm2 of peritoneal tissue. We were able to estimate the total vessel cross-section area in each digitized image and we could therefore calculate an average cross-sectional area per vessel for each slide analyzed. A portion of mesentery was taken and frozen for a hydroxyproline assay, modified from Woessner's method.28Woessner Jr, JF The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3388) Google Scholar Tissues were weighed, homogenized in water, centrifuged at 1000 rpm for 5 minutes, and the superficial fatty material removed by vacuum suction. Solid material was precipitated with trichloroacetic acid with centrifugation at 1500 rpm for 15 minutes at 4°C. Samples were hydrolyzed overnight in 6 N HCl at 110°C. Hydroxyproline content is quantified by Erlich's reagent (Sigma) and assayed by measuring the optical density at 557 nm. A hydroxyproline standard sample (Sigma) was used to create a standard curve. Peritoneal fluid taken at sacrifice was analyzed for the following cytokines using enzyme-linked immunosorbent assay (ELISA) (all R&D Systems, Minneapolis, MN) as directed by the manufacturer: human TGF-β (cross-reactive with rat), murine VEGF (cross-reactive with rat), rat IL-6, rat TNF-α (cross-reactive with murine), human IL-1β (not cross-reactive with rat), and murine TNF-soluble receptor II (TNFsrII, cross-reactive with rat). To measure total TGF-β, samples were first activated using 1 mol/L HCl for 10 minutes then normalized with 1 N NaOH to dissociate TGF-β from its latency-associated binding protein. Frozen omental tissue was taken and homogenized in Trizol reagent (Life Technologies, Burlington, Ontario, Canada). Protein was extracted from the phenol layer after centrifugation according to the manufacturer's protocol. Total protein in this extract was assayed using a standard protein assay method (BioRad DC Protein Assay; BioRad Laboratories, Mississauga, Canada). Equal quantities of protein were then assayed using murine VEGF ELISA (R&D Systems). The peritoneal surfaces of the anterior abdominal wall sections were immersed for 15 minutes in Trizol reagent. The parietal peritoneum was gently scraped and the Trizol collected and processed according to the manufacturer's instruction for isolation of RNA. The concentration of RNA resuspended in RNase-free water was measured by optical density at 260 nm. RNA (8 μg) was then hybridized overnight with a custom probe set (Pharmingen, Mississauga, ON) labeled with α-32P-UTP (New England Nuclear, Boston, MA). The custom probe set contains different length probes for fibronectin, TGF-β1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and housekeeping genes GAPDH and L32 for loading control. The hybridized samples were extracted using phenol/chloroform and acetate precipitation and then washed with ethanol. The extracted bound RNA was then run on a 5% polyacrylamide gel, transferred to blotting paper, dried, and exposed for 4 days to film (Eastman Kodak, Rochester, NY). The images obtained were digitized and analyzed for band density using Scion Image software (Scion Corp, Frederick, MD). Densities were standardized to L32. Data are presented ± SEM unless otherwise noted. Comparison between groups was made by t-test. We used regression analysis to compare the cytokine concentration on day 4 in the peritoneal fluid and subsequent vascularization of the peritoneal membrane and combined all three treatment groups (AdIL-1β, AdTNF-α, AdDL70) in these results. In previous work, we have identified an early (48 hour) inflammatory response to AdDL70 in similar dosage used in these experiments.29Hoff CM Piscopo D Inman KL Shockley TR Adenovirus-mediated gene transfer to the peritoneal cavity.Perit Dial Int. 2000; 20: 128Google Scholar There were no adenoviral effects identified by 4 days,23Margetts PJ Kolb M Hoff CM Shockley TR Gauldie J Gene transfer of transforming growth factor beta 1 to the rat peritoneum: effects on membrane function.J Am Soc Nephrol. 2001; 12: 2029-2039PubMed Google Scholar the earliest time point used in our experiments. We therefore did not include a nonadenoviral control group in these experiments. We have previously shown that adenovirus is effectively taken up, and the transgene product expressed, by mesothelial cells in the peritoneum.23Margetts PJ Kolb M Hoff CM Shockley TR Gauldie J Gene transfer of transforming growth factor beta 1 to the rat peritoneum: effects on membrane function.J Am Soc Nephrol. 2001; 12: 2029-2039PubMed Google Scholar The peak of expression is 4 to 7 days after infection, and the total duration of expression is 10 to 14 days. In these experiments, we confirmed the high levels of expression of the transgene product by analysis of the peritoneal dialysis fluid with the appropriate ELISA. Four days after infection peritoneal fluid was taken after a 4-hour dwell and analyzed by ELISA. Animals treated with AdIL-1β had 326 ± 89 pg/ml of human IL-1β and animals treated with AdTNF-α had 2076 ± 1131 pg/ml of rodent TNF-α measured in the peritoneal fluid (Table 1).Table 1Cytokine Concentration in the Peritoneal Fluid and Tissue 4, 7, and 21 Days after Infection with Control Adenovirus, AdTNF-α, or AdIL-1βDay 4Day 7Day 21AdDL70AdTNF-αAdIL-1βAdDL70AdTNF-αAdIL-1βAdDL70AdTNF-αAdIL-1βHuman IL-1β (pg/ml)<10<10326 ± 89*P < 0.05 compared with AdDL70 control.†P < 0.05 compared with AdTNF-α.<10<1077 ± 105<10<10<10Rat IL1β (pg/ml)32 ± 10302 ± 255*P < 0.05 compared with AdDL70 control.310 ± 186*P < 0.05 compared with AdDL70 control.31 ± 10114 ± 92638 ± 80046 ± 2128 ± 2251 ± 301TNF-α (pg/ml)69 ± 82076 ± 1131*P < 0.05 compared with AdDL70 control.69 ± 7†P < 0.05 compared with AdTNF-α.58 ± 14365 ± 482*P < 0.05 compared with AdDL70 control.83 ± 12†P < 0.05 compared with AdTNF-α.nd66 ± 1169 ± 9Total TGF-β (pg/ml)131 ± 80186 ± 82756 ± 441*P < 0.05 compared with AdDL70 control.†P < 0.05 compared with AdTNF-α.88 ± 50306 ± 137*P < 0.05 compared with AdDL70 control.1056 ± 610*P < 0.05 compared with AdDL70 control.†P < 0.05 compared with AdTNF-α.63 ± 264 ± 18166 ± 101VEGF (pg/ml)75 ± 44172 ± 130*P < 0.05 compared with AdDL70 control.210 ± 173*P < 0.05 compared with AdDL70 control.85 ± 49205 ± 133*P < 0.05 compared with AdDL70 control.262 ± 157*P < 0.05 compared with AdDL70 control.63 ± 2430 ± 447 ± 17Tissue VEGF (pg/ml)43 ± 1072 ± 4076 ± 1647 ± 1445 ± 2178 ± 8*P < 0.05 compared with AdDL70 control.ndndndData shown ± SD.nd, No data.* P < 0.05 compared with AdDL70 control.† P < 0.05 compared with AdTNF-α. Open table in a new tab Data shown ± SD. nd, No data. The inflammatory response after intraperitoneal administration of AdTNF-α, AdIL-1β, or control adenovirus was measured from various markers in the peritoneal fluid and the results are shown in Table 2. The cellular response to overexpression of IL-1β and TNF-α was dramatic with a significant increase in the total number of cells and a disproportionate increase in neutrophils measured in the peritoneal fluid. There was also an associated weight loss that was more dramatic in the AdIL-1β-treated animals compared to control adenovirus-treated animals. Rodent IL-6 was measured by ELISA in peritoneal fluid and was elevated after exposure to IL-1β or TNF-α (Table 1). Finally, we measured rodent TNFsrII by ELISA in peritoneal fluid and this was significantly elevated 4 and 7 days after infection with AdIL-1β and AdTNF-α (Table 1).Table 2Inflammatory Changes Measured 4 and 7 Days after Infection with Control Adenovirus, AdTNF-α, AdIL-1βDay 4Day 7AdDL70AdTNF-αAdIL-1βAdDL70AdTNF-αAdIL-1βWhite blood cells (104/ml)77 ± 69158 ± 81*P < 0.05 compared with AdDL70 control.952 ± 275*P < 0.05 compared with AdDL70 control.†P < 0.05 compared with AdTNF-α.132 ± 164262 ± 148763 ± 541*P < 0.05 compared with AdDL70 control.†P < 0.05 compared with AdTNF-α. Monocyte, %19.415.38.0*P < 0.05 compared with AdDL70 control.28.920.313.2 Macrophage, %46.241.430.750.549.427.1 Neutrophil, %4.231.7*P < 0.05 compared with AdDL70 control.48.3*P < 0.05 compared with AdDL70 control.3.525.9*P < 0.05 compared with AdDL70 control.58.3*P < 0.05 compared with AdDL70 control.†P < 0.05 compared with AdTNF-α. Eosinophil, %27.49.8*P < 0.05 compared with AdDL70 control.11.3*P < 0.05 compared with AdDL70 control.19.43.81.1Change in weight from day 0 (g)1.3 ± 5.3−2.1 ± 2.8−5.6 ± 4.12.8 ± 6.12.2 ± 3.9−5.9 ± 4.2IL-6 (pg/ml)59.1 ± 0.3988 ± 8571451 ± 49766 ± 274 ± 81788 ± 746TNFsrII (pg/ml)34 ± 16604 ± 420*P < 0.05 compared with AdDL70 control.751 ± 364*P < 0.05 compared with AdDL70 control.63 ± 111156 ± 275*P < 0.05 compared with AdDL70 control.917 ± 388*P < 0.05 compared with AdDL70 control.Data shown ± SD.* P < 0.05 compared with AdDL70 control.† P < 0.05 compared with AdTNF-α. Open table in a new tab Data shown ± SD. We also noted a quantitative difference between the inflammatory response to AdIL-1β or AdTNF-α. Specifically the response to transient overexpression of IL-1β was more intense, as measured by total cell count and percentage of neutrophils, especially 7 days after infection. Also, two animals in the AdIL-1β group became very ill in the first week after treatment and required euthanasia. The response to IL-1β was more prolonged, with levels of TNFsrII remaining significantly elevated in AdIL-1β-treated animals 21 days after infection. We analyzed the peritoneal fluid after a 4-hour dwell for rat IL-1β, TNF-α, TGF-β, and VEGF using ELISA (Table 1). Both AdTNF-α and AdIL-1β treatment induced the production of endogenous IL-1β measured using a rodent-specific assay. Interestingly, AdIL-1β-treated animals did not show an increase in peritoneal concentration of TNF-α. Overexpression of both TNF-α and IL-1β induced a significant increase in the peritoneal fluid concentration of total TGF-β1 and VEGF compared to AdDL70-treated animals 7 days after infection. We identified a threefold increase in peritoneal concentration of TGF-β1 7 days after AdIL-1β treatment compared to AdTNF-α. Increases in peritoneal VEGF concentrations were similar after exposure to IL-1β and TNF-α. To confirm that VEGF was present in the tissues after adenovirus infection, we isolated protein from omental homogenates and assayed for VEGF. We noted an increase in tissue VEGF that was significantly elevated 7 days after treatment with AdIL-1β (Table 1). We isolated RNA from parietal peritoneal tissue of animals 4, 7, and 21 days after receiving adenovirus and analyzed it using an RNase protection assay (Figure 1). Quantitative density analysis indexed to L32 and referenced to AdDL70-treated animals revealed that at 4 and 7 days after infection, both TNF-α and IL-1β led to an increase in mRNA signal for fibronectin and TGF-β1. These changes declined to baseline by day 21 except for a small persistent elevation in fibronectin mRNA in AdIL-1β-treated animals at day 21. Most striking was the progressive increase in TIMP-1 mRNA expression seen in AdIL-1β-treated animals at day 21 (Figure 1). Transient overexpression of both IL-1β and TNF-α led to early, significant histological changes in the parietal peritoneum compared to animals treated with the control adenovirus, AdDL70 (Figure 2). The mesothelial cells, which normally are flattened against the basement membrane, became rounded up with enlarged nuclei. The submesothelial zone became substantially thickened with inflammatory cells and edematous changes. These changes were evident in both parietal and visceral peritoneal tissue as seen in omental tissue 7 days after infection (Figure 3).Figure 3Histology of the visceral peritoneum (omentum) 7 days after infection with AdDL70 (A), AdIL-1β (B), or ADTNF-α (C). AdIL-1β and AdTNF-α-treated animals demonstrate increased cellular infiltration in omental tissue with collagen deposition (arrows). Scale bars, 200 μm. Masson's trichrome.View Large Image
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