Abdominal irradiation increases inflammatory cytokine expression and ...

Am J Physiol Gastrointest Liver Physiol 285: G556–G565, 2003; 10.1152/ajpgi.00094.2003.

Abdominal irradiation increases inflammatory cytokine expression and activates NF-␬B in rat ileal muscularis layer C. Linard, A. Ropenga, M. C. Vozenin-Brotons, A. Chapel, and D. Mathe Institute for Radioprotection and Nuclear Safety, Human Health Protection and Dosimetry Division, Independent Section of Radiobiology Applied to Medecine, F-92262 Fontenay-aux-Roses Cedex, France Submitted 24 February 2003; accepted in final form 28 May 2003

Linard, C., A. Ropenga, M. C. Vozenin-brotons, A. Chapel, and D. Mathe. Abdominal irradiation increases inflammatory cytokine expression and activates NF-␬B in rat ileal muscularis layer. Am J Physiol Gastrointest Liver Physiol 285: G556–G565, 2003; 10.1152/ajpgi.00094.2003.—The small bowel is an important dose-limiting organ in abdominal radiotherapy because irradiation can cause acute enteritis that, in turn, leads to progressively reduced motility and finally, in a later phase, to fibrosis. Because these clinical symptoms may be caused by the early stage of an inflammatory process, we characterized the radiation-induced intestinal inflammation in rats. Abdominal ␥-irradiation (10-Gy) induced a cascade of inflammatory events characterized by an early (6 h after exposure) increase in IL-1␤, TNF-␣, and IL-6 mRNA levels in the rat ileal muscularis layer. IL-8 [a cytokine-induced neutrophil chemoattractant (CINC)] mRNA appeared later (at 3 days). The expression of TGF-␤ (a profibrotic cytokine) was higher in irradiated than control tissue at day 1, whereas IL-10 (an anti-inflammatory cytokine) expression vanished completely. Despite strong IL-1ra expression, the IL-1ra/IL-1␤ ratio, which is an indicator of inflammatory balance, was ⫺41% at day 1 in irradiated compared with control tissue. The nuclear transcription factors NF-␬B and activator protein-1 (AP-1) govern transcription of these genes, directly or indirectly. Although expression of the subunits of NF-␬B (p65, p50) and AP-1 (c-fos, c-jun) did not increase, irradiation caused a rapid and persistent translocation of p65 and p50. An imbalance between proinflammatory and anti-inflammatory mediators may contribute to perpetuating intestinal inflammation, thus making it chronic. intestine; inflammation; p65 and p50; c-jun and c-fos

pelvic and abdominal tumors frequently causes severe complications because the intestine is an important dose-limiting organ. Many patients suffer acute damage to the small intestines from hours to years after treatment (48). The pathological changes can be divided into acute enteritis, characterized by diarrhea and chronic enteropathy, characterized by hemorrhage and ulceration leading to progressively reduced motility, and eventually fibrosis and bowel obstruction (14). These clinical symptoms may be caused by the early stage of an inflammatory ABDOMINAL RADIOTHERAPY FOR

Address for reprint requests and other correspondence: C. Linard, Institut de Radioprotection et de Suˆrete´ Nucleáire, De´partement de Protection de la sante´ de l’Homme et de Dosime´trie, Section Autonome de Radiobiologie Appliqueé a` la Me´decine, IRSN, BP 17, F-92262 Fontenay-aux-Roses Cedex, France (E-mail: [email protected]). G556

process. Some studies report that inflammatory cytokines, such as IL-1␤, TNF-␣, and IL-6, all induced by ionizing radiation, significantly contribute to the disorders associated with radiotherapy in the blood (18), peripheral lymphoid tissues, and lungs (20, 41). In animal models, increased cytokine expression after irradiation has been reported in hematopoietic, lung, spleen, and other tissues (11, 23, 52). The expression of these cytokines changes in a time- and tissue-specific manner. Nonetheless, the radiation-induced inflammatory state of the intestines has never been characterized clearly. Increased levels of IL-1␤, IL-2, IL-6, and IL-8 have been reported only in patients with radiation-induced proctitis (22). Cytokines are glycoproteins produced by a wide variety of cells. They are functionally grouped into proinflammatory cytokines (mainly IL-1␤, IL-6, IL-8, and TNF-␣) and anti-inflammatory cytokines [mainly IL-4, IL-10, IL-1 receptor antagonist (IL-1ra), and TGF-␤]. Investigation of the balance between pro- and antiinflammatory events in the gut may provide important insights into the pathogenic mechanisms of radiation. An imbalance between IL-1␤ and IL-1ra is reported to be an important factor in the pathogenesis of inflammatory bowel disease (IBD) and may explain why the acute inflammatory response develops into chronic persistent inflammation in some patients (9). Cytokines probably play a role in initiating and perpetuating these uncontrolled disease processes. There is, however, a remarkable paucity of information on cellular interactions in complex gut inflammatory diseases such as Crohn’s disease and ulcerative colitis, and animal models of IBD have not provided substantial additional data. Some reports suggest that the intestinal muscle layer, including mesenchymal tissue, fibroblasts, myofibroblasts, and muscle cells, may be the source of the inflammatory mediators that account for acute inflammation-induced changes in motor function and later for intestinal fibrosis (24, 40). Intestinal motility dysfunctions with modifications of transit and contractility have been reported after irradiation (16, 46). Many inflammatory responses, particularly in the gut, are mediated by the activation of transcription factors such as NF-␬B and activator protein-1 (AP-1) The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

0193-1857/03 $5.00 Copyright © 2003 the American Physiological Society

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ABDOMINAL IRRADIATION AND RAT ILEAL MUSCULARIS LAYER

(40, 43). NF-␬B is an inducible transcriptional factor defined as a heterodimeric complex of two subunits, p65 (Rel A) and p50/p105 (2, 3). In unstimulated cells, NF-␬B is sequestered in the cytoplasm as an inactive complex bound to inhibitor ␬B (I␬B), an inhibitory protein. Activation of NF-␬B by inflammatory cytokines, such as IL-1␤ and TNF-␣, induces a cascade of reactions leading to I␬B phosphorylation and degradation by proteasomes (2). Activated NF-␬B then translocates into the nucleus, thereby activating the transcription of a variety of genes (35). Besides modulating genes that directly influence cell proliferation and death, NF-␬B regulates the expression of several cytokines, including IL-1␤ and TNF-␣ (3, 45). It establishes a positive feedback loop that amplifies the inflammatory response and increases chronic inflammation (43). The number of NF-␬B positive cells has been correlated to the degree of inflammation in human IBD (39). Until now, ionizing radiation has been shown to activate NF-␬B only in vitro in fibroblasts (6), endothelial cells (17), HeLa cells (28), and astrocytes (37), and in vivo in peripheral lymphoid tissues (52). Another nuclear target for cytokines is transcription factor AP-1, a homo- or heterodimeric transcription factor composed of members of the Jun and Fos families of DNA-binding proteins (7, 21). AP-1 binding sites are activated during inflammation in various types of cells and tissues (21). Radiation-induced activation of NF-␬B has recently been related to AP-1 activation. Cooperative interaction between these factors seems necessary to obtain inducible expression of proinflammatory cytokines during the prodromal stage of radiation (4). The in vivo activation of irradiation-induced transcriptional factors has not yet been characterized in the intestines. In the present study, we investigated in vivo the acute effect of ionizing radiation on the balance between some proinflammatory [IL-1␤, TNF-␣, IL-6, and IL-8, a cytokine-induced neutrophil chemoattractant (CINC)] and anti-inflammatory (IL-1ra, TGF-␤, and IL-10) cytokines by quantifying the changes in mRNA levels in the muscularis layer of rat ileum and monitoring the time course of the activation of NF-␬B and AP-1 transcriptional factors. MATERIALS AND METHODS

Animals and treatment. Male Wistar rats (Elevage Janvier, Le Genest, France), weighing 200–250 g, were allowed water and food ad libitum. All experiments were conducted in accordance with the French Ministry of Agriculture regulations for animal experimentation (87–848, October 19, 1987). Anesthetized rats received a single abdominal dose of 10Gy-␥ (60Co; ICO-4000) at a dose rate of 0.96-Gy/min. Control rats were submitted to the same conditions but were not exposed to the radiation source. Tissues were collected 6 h and 1 and 3 days after exposure. Each ileum was dissected on ice and separated into muscularis and mucosa layers by scraping. The muscularis layers were frozen in small aliquots at ⫺80°C after collection. RNA extraction and RT-PCR. The mRNA levels of the cytokines, NF-kB, and AP-1 subunits and of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase AJP-Gastrointest Liver Physiol • VOL

(HPRT) were measured by real-time PCR. Total RNA was prepared with the RNeasy total RNA isolation kit (Qiagen, France) according to the manufacturer’s instructions. The cDNA was produced from 1 ␮g of total RNA by reverse transcription with 200 U of Superscript reverse transcriptase (GIBCO) in a 20-␮l reaction containing 1 ⫻ Superscript buffer (GIBCO), 1 mM 2-deoxynucleotide 5⬘-triphosphate, 20 ng random hexamer, 10 mM DTT, and 20 U RNase inhibitor. After incubation for 50 min at 42°C, the reaction was terminated by a denaturing enzyme for 10 min at 70°C. RNA integrity was confirmed by denaturing agarose gel electrophoresis and ethidium bromide staining. For IL-1␤, TNF-␣, and IL-6, we used primers from the manufacturer to amplify first-strand cDNA through 36 PCR cycles with TaqMan (Applied Biosystems). PCR amplification of the other cytokines and NF-kB and AP-1 subunits used Syber PCR master mix; the primer sequences, which are listed in Table 1, were designed with Primer Express software (Applied Biosystems). Optimized PCR used the Abi Prism 7700 Sequence detection system (Qiagen). PCR fluorescent signals were normalized to the fluorescent signal obtained from the housekeeping gene HPRT for each sample. Preparation of protein extracts. Cytoplasmic and nuclear protein extracts were prepared according to methods described previously (51). Briefly, small aliquots (⬍0.1 g) were immersed in 1 ml ice-cold lysis buffer (in mM): 10 HEPES, pH 7.9, 10 KCl, 1.5 MgCl2, 1 DTT, 0.5 PMSF, and 5 ␮l/ml protease inhibitor cocktail (Sigma). They were homogenized on ice with a Dounce homogenizer, kept on ice for 15 min, and then 1% IGEPAL was added to the homogenate. After a brief vortexing, they were incubated on ice for 20 min and then centrifuged at 4°C (12,500 rpm) for 30 s. The supernatant corresponding to this cytoplasmic extract was collected into a new tube. The pelleted nuclei were resuspended into 50–200 ␮l of extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 5% glycerol, 1 mM DTT, 0.5 mM PMSF, and 5 ␮l/ml protease inhibitor cocktail) and kept on ice for 30 min. The nuclear suspension was centrifuged at 12,500 rpm for 15 min at 4°C to collect the supernatants containing nuclear protein extracts. Protein concen-

Table 1. Specifications of the primer sets used to analyse cytokine mRNA expression Cytokine

Primer Set

IL-8 (CINC) 5⬘-GACTGTTGTGGCCCGTGAG-3⬘ 5⬘-CCGTCAAGCTCTGGATGTTCT-3⬘ IL-10 5⬘-GTTGCCAAGCCTTGTCAGAAA-3⬘ 5⬘-TTTCTGGGCCATGGTTCTCT-3⬘ IL-1ra 5⬘-GCGCTTTACCTTCATCCGC-3⬘ 5⬘-CTGGACAGGCAAGTGATTCGA-3⬘ TGF-␤1 5⬘-TCCCAAACGTCGAGGTGAC-3⬘ 5⬘-CAGGTGTTGAGCCCTTTCCA-3⬘ p105 5⬘-AGCACCAAGACCGAAGCAA-3⬘ 5⬘-TCTCCCGTAACCGCGTAGTC-3⬘ p65 5⬘-CCACGATCTGTTTCCCCTCAT-3⬘ 5⬘-TGATCTCCACATATGGCCCAG-3⬘ c-Fos 5⬘-TGACTTCTTGTTTCCGGCATC-3⬘ 5⬘-CACATCTGGCACAGAGCGG-3⬘ c-Jun 5⬘-CACCTGACTGAGGCGCTGA-3⬘ 5⬘-CCGAAGCTGCACAAAGTTCAT-3⬘ HPRT 5⬘-GCTCGAGATGTCATGAAGGAGA-3⬘ 5⬘-TCAGCGCTTTAATGTAATCCAGC-3⬘

PCR Product Size

83 78 65 94 149 68 67 78 109

Values are base pairs. HPRT, hypoxanthine-guanine phosphoribosyltransferase; CINC, cytokine-induced neutrophil chemoattractant; IL-1ra, IL-1 receptor antagonist. 285 • SEPTEMBER 2003 •

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trations of cytoplasmic and nuclear extracts were measured with a modified Bradford method from Bio-Rad (Hercules, CA). The samples were then stored at ⫺80°C. Western blot analysis. We separated 20 ␮g of proteins from each of the samples described above on 12% SDS-polyacrylamide gel and transferred them onto nitrocellulose membranes. Membranes were blocked for 1 h with 5% nonfat dry milk. Enhanced chemiluminescence was used to detect specific proteins with the appropriate antibodies: anti-p65 (F-6, dilution of 1:500; Santa Cruz), anti-p50 (E-10, dilution of 1:500; Santa Cruz) and anti-I␬B␣ (C-21, dilution of 1:200; Santa Cruz). Densitometric analyses used the Biocom analyzer (Les Ulis, France). NF-␬B transcription factor activation. The DNA binding activity of NF-␬B in the nuclear extract was determined by EMSA. An aliquot of 2.5 ␮g nuclear proteins was incubated with a reaction buffer [in mM: 25 Tris (pH 7.5), 50 KCl, 6.25 MgCl2, 0.5 EDTA, and 0.5 dithiothreitol; and 10% glycerol and 1 ␮g poly(dI-dC)]. Then 105 counts/min of [32P]-endlabeled double-stranded DNA nucleotides containing the consensus ␬B motif 5⬘-AGTGAGGGGACTTTCCCAGGC-3⬘ and 5⬘-GCCTGGGAAAGTCCCCTCACT-3⬘ were added to the reaction and incubated 30 min at room temperature. Specificity of the DNA/protein binding was determined by competition reactions by using a 10-fold molar excess of unlabeled NF-␬B oligonucleotides. For supershift analysis, nuclear extracts were incubated with 2 ␮g of the polyclonal antibodies against the NF-␬B subunit of p65 and p50 (Santa Cruz Biotechnology, Santa Cruz, CA). Two microliters of 0.1% bromophenol blue dye were then added to each sample. After electrophoresis (an aliquot of 20 ␮l through a 6% nondenaturing polyacrylamide gel for 2 h at 150 volts), gel was dried and the protein-DNA complexes were visualized by a PhosphoImager. Quantitation of NF-␬B activation was assayed with Trans-AM NF-␬B kits (Active Motif; Rixensart, Belgium) that included a 96-well plate with an immobilized oligonucleotide containing the NF-␬B consensus-binding site (5⬘GGGACTTTCC-3⬘). The active form of NF-␬B contained in nuclear extract specifically binds to this oligonucleotide. Primary antibodies that detect NF-␬B recognize an epitope on p65 that is accessible only when NF-␬B is activated and bound to its target DNA. A horseradish peroxidase conjugated secondary antibody was used for the spectrophotometric quantification. Cytokine immunoassays. For the IL-1␤, TNF-␣, and IL-6 analyses, the tissue samples were weighed and then homogenized in 10 mM PBS (pH 7.4) supplemented with protease inhibitors: 2 mM PMSF, 10 ␮g/ml pepstatin A, 1 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 0.5 mg/ml EDTA. Samples were then centrifuged at 10,000 g for 10 min, and the supernatants were stored at ⫺20°C for later measurement. The IL-1␤, TNF-␣, and IL-6 assays used ELISA kits (R&D Systems, Minneapolis, MN). The rabbit anti-rat IL-1␤ polyclonal antibody of the IL-1␤ kit recognizes both recombinant and natural rat IL-1␤. The manufacturer reports that this antibody does not cross-react significantly with recombinant (r) human (rHuman) IL-1RI, IL-1RII, or IL-1ra or rRat IL-1␣, IL-2, IL-4, IFN-␥, or TNF-␣, or rMouse IL-1␣ or IL-1ra. The rabbit anti-rat TNF-␣ polyclonal antibody of the TNF-␣ kit recognizes both recombinant and natural rat TNF-␣. No significant cross-reactivity was observed between this antibody and rHuman TNF-␣ or rRat IL-1␤, IL-2, IL-4, or IFN-␥. The anti-rat IL-6 antibody recognizes both recombinant and natural rat IL-6. No significant cross-reactivity of this antibody was observed with rat IL-1␤, IL-2, IL-4, IFN-␥, or TNF-␣. AJP-Gastrointest Liver Physiol • VOL

Result expression and statistical analysis. We used the comparative ⌬⌬CT-method (8) for the relative mRNA quantitation. The relative quantitation of target, normalized to an endogenous reference (HPRT) and a relevant unirradiated control, is given as relative quantitation ⫽ 2⫺⌬⌬CT, where ⌬⌬CT is defined as the difference between the mean ⌬CT (irradiated sample) and the mean ⌬CT (unirradiated sample), and ⌬CT is the difference between the mean CT (threshold cycle; cytokines, NF-␬B subunit, or AP-1) and the CT (HPRT) is the endogenous control. All data are expressed as means ⫾ SE for five animals. Comparisons among groups used one-way ANOVA, the Bonferroni’s t-test (applied to test the rationale gene expression), and Student’s t-test for nonpaired data. RESULTS

Effects of irradiation on proinflammatory cytokine levels. Figure 1 reports the effects of irradiation on tissue cytokine concentrations. The muscularis layer of the irradiated rats had significantly higher IL-1␤ lev-

Fig. 1. Effect of a 10-Gy abdominal ␥-irradiation on ileal procytokine levels. The immunoreactivities of IL-1␤, TNF-␣, and IL-6 were each measured by ELISA in ileal muscularis layers obtained 6 h, 24 h (D1), and 3 days (D3) postirradiation. Values are means ⫾ SE, n ⫽ 5, * P ⬍ 0.05, ** P ⬍ 0.01, *** P ⬍ 0.001 irradiated vs. control rats. 285 • SEPTEMBER 2003 •

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Fig. 2. Time course of abdominal irradiation-induced increases in mRNA levels of proinflammatory cytokines in vivo. IL-1␤ (A), TNF-␣ (B), IL-6 (C), and IL-8 [cytokine-induced neutrophil chemoattractant (CINC)] (D) mRNA levels were measured in the ileal muscularis layer by real-time quantitative RT-PCR 6 h, 24 h, and 3 days after a single abdominal irradiation (10-Gy). The results were expressed as a ratio to the reference gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA levels. Data are the means ⫾ SE (n ⫽ 5); * P ⬍ 0.05, ** P ⬍ 0.005, *** P ⬍ 0.001 significantly different from control value.

els at 6 h (519 ⫾ 63 pg/mg of protein, P ⬍ 0.001; n ⫽ 5), at 1 day (1,064 ⫾ 300 pg/mg of protein, P ⬍ 0.01; n ⫽ 5) and at 3 days (593 ⫾ 72 pg/mg of protein, P ⬍ 0.001; n ⫽ 5) after irradiation than that of the controls (261 ⫾ 25 pg/mg of protein). The TNF-␣ concentration had increased 14-fold (3.93 ⫾ 0.1 pg/mg of protein, P ⬍ 0.01; n ⫽ 5) at only 6 h after irradiation, compared with control levels (0.28 ⫾ 0.1 pg/mg of protein). Although there was no change at 6 h, the IL-6 content increased significantly thereafter to 1.3-fold (3.55 ⫾ 0.30 pg/mg of protein, P ⬍ 0.05) at day 1, and twofold (5.62 ⫾ 0.30 pg/mg of protein, P ⬍ 0.001, n ⫽ 5) at day 3 after irradiation, compared with control levels (2.69 ⫾ 0.20 pg/mg of protein). No change was observed at 6 h after irradiation. Effects of irradiation on mRNA levels of proinflammatory cytokines. mRNA levels of the proinflammatory cytokines IL-1␤, TNF-␣, IL-6, and IL-8 (CINC) were quantified and expressed as a ratio to a reference gene, HPRT, in the ileal muscularis layer (Fig. 2). The HPRT mRNA level in the ileal tissue was unchanged after irradiation (data not shown). Abdominal irradiation (10-Gy) induced a significant increase of IL-1␤ (2.5fold, P ⬍ 0.05), TNF-␣ (4.1-fold, P ⬍ 0.005), and IL-6 (2.9-fold, P ⬍ 0.05) mRNA levels at 6 h. Levels of IL-1␤ and IL-6 mRNA remained significantly higher (3.5and 2.9-fold, respectively) in the irradiated than in the control tissue at 3 days after irradiation. IL-8 (CINC) mRNA appeared only at 3 days at a level 10.8 times greater than in the control tissue (P ⬍ 0.001). Effects of irradiation on mRNA levels of anti-inflammatory cytokines. Analysis of the temporal patterns of the IL-1ra mRNA levels showed increases by factors of 3.7 (P ⬍ 0.05) and 4.4 (P ⬍ 0.01) at 6 h and 3 days postirradiation, respectively (Fig. 3A). At day 1, on the other hand, the IL-1ra mRNA level did not differ from that in the control. The IL-1ra/IL-1␤ ratio, which is an indicator of the inflammatory balance, was significantly lower than the control at 1 day after exposure AJP-Gastrointest Liver Physiol • VOL

(⫺41%; P ⬍ 0.05), although there was no significant change at either 6 h or 3 days (Fig. 3B). Other cytokines with anti-inflammatory effects include IL-10 and TGF-␤. Analysis of the time course of IL-10 expression in the irradiated tissue showed that its mRNA levels were dramatically lower than in the control: 87% lower (P ⬍ 0.05) at day 1 and 93% lower (P ⬍ 0.01) at day 3 postirradiation (Fig. 4A). In con-

Fig. 3. Time course of abdominal irradiation-induced increases in the level of IL-1 receptor antagonist (IL-1ra) mRNA. The IL-1ra mRNA level (A) was measured in ileal muscularis layer by real-time quantitative RT-PCR at 6 h, 24 h, and 3 days after a single abdominal irradiation (10-Gy) and expressed as a ratio to the reference gene (HPRT) mRNA levels. The IL-1ra/IL-1␤ ratio (B) was calculated by dividing the quantity of IL-1ra mRNA by the quantity of IL-1␤ mRNA for each rat. Data are the means ⫾ SE (n ⫽ 5); * P ⬍ 0.05, ** P ⬍ 0.01 significantly different from control value. 285 • SEPTEMBER 2003 •

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Fig. 4. Time course of abdominal irradiation-induced modifications in mRNA levels of anti-inflammatory cytokines in vivo. IL-10 (A) and TGF-␤ (B) mRNA levels were measured in ileal muscularis layer by real-time quantitative RT-PCR 6 h, 24 h, and 3 days after a single abdominal irradiation (10-Gy). Results were expressed as a ratio to the reference gene (HPRT) mRNA levels. Data presented are the means ⫾ SE (n ⫽ 5); * P ⬍ 0.05, ** P ⬍ 0.01, *** P ⬍ 0.005 significantly different from control value.

trast, the TGF-␤ expression was weak but higher than in the control at 1 day (1.5-fold) and significantly higher at 3 days (1.8-fold, P ⬍ 0.005) (Fig. 4B). Influence of irradiation on levels of NF-␬B subunits. The predominant form of NF-␬B is a dimer of p50 and p65 subunit proteins that binds to I␬B inhibitory proteins in cytoplasm. Cytokines stimulate the release of I␬B and the consequent translocation of NF-␬B to the nucleus (39, 43). Nuclear levels of NF-␬B p65 and p50 proteins were evaluated by Western blot at 6 h, 24 h, and 3 days after radiation exposure (Fig. 5). Analysis of protein levels showed that irradiation induced NF-␬B translocation. The nuclear p65 protein concentration was five times greater than in the control at 6 h and 24 h postirradiation (P ⬍ 0.005) (Fig. 5A). Irradiation also induced p50 translocation, and the p50 protein level in the nucleus was greater than in the control tissue by a factor of 1.8 at 6 h (P ⬍ 0.05), of 1.3 at 24 h (P ⬍ 0.01), and of 1.4 at 3 days after irradiation (P ⬍ 0.005) (Fig. 5B). In parallel, analysis of cytoplamic protein levels showed that irradiation induced a fourfold decrease of p65 level at 6 h (P ⬍ 0.005) and a 2.5-fold increase 3 days after irradiation (P ⬍ 0.01) (Fig. 5C). No modification was observed on cytoplasmic p50 level induced by irradiation (Fig. 5D). After the NF-␬B p65 and p50 proteins translocate into the nucleus, I␬B is ubiquitinated and rapidly degraded by proteasomes; the cytoplasmic I␬B content is thus subAJP-Gastrointest Liver Physiol • VOL

stantially modified (2). New I␬B␣ synthesis is NF-␬B dependent (47). I␬B␣ is the only inhibitor protein regulated by NF-␬B, and its level increased progressively after the first day, reaching five times the control level (P ⬍ 0.001) at 3 days after irradiation (Fig. 5E). Effect of irradiation on NF-␬B activation. NF-␬B dimers, after translocation into the nucleus, activate appropriate target genes. Involvement of p65 and p50 subunits in NF-␬B activation was determined by electrophoretic mobility shift assays of nuclear extracts. Activation of NF-␬B peaked at 6 h and declined at day 1 and day 3 (Fig. 6A). The composition of the NF-␬B complex was determined with p65 and p50 antibodies. The anti-p65 produced a supershift band, indicating that the activated complex contained predominately the p65 subunit. The p65 DNA-binding activity was confirmed by the Trans-AM NF-␬B analysis (Fig. 6B) showing a 3.5-fold increase of activity (P ⬍ 0.01, n ⫽ 5) 6 h after irradiation. This activity disappeared in the presence of an excess amount of soluble oligonucleotide containing a wild-type NF-␬B consensus-binding site (data not shown). Effect of irradiation on gene expression of NF-␬B and AP-1 complex components. The relative p105 mRNA level was half that of the control at 6 h after irradiation (P ⬍ 0.001). A small but significant decrease in p65 mRNA expression was observed at 1 day (P ⬍ 0.05) and 3 days (P ⬍ 0.01) after irradiation (Fig. 7A). AP-1 is a homo- or heterodimeric transcription factor composed of members of the Jun and Fos families of DNA-binding proteins (7, 21). The level of c-fos mRNA in the ileal muscularis layer was double that for control tissue (P ⬍ 0.005) at 3 days after irradiation, the only time point with a significant difference (Fig. 8A). The c-jun mRNA level of the tissue from irradiated and control rats did not differ (Fig. 8B). DISCUSSION

Intestinal inflammation such as in IBD is accompanied by hyperplasia, hypertrophy, and muscle hyperresponsiveness, and IL-1␤ mRNA induction and changes in motility are related (12, 26). We previously observed (16) that abdominal irradiation altered motor function and contractile activity associated with modification of substance P, an inflammatory mediator. Interactions between intestinal smooth muscle cells and immune cells may be particularly important in the external neuromuscular layer. The increased cytokine expression observed as early as 12 h after bacterial infection in the rat neuromuscular layer suggests that smooth muscle cells play an active part in the inflammatory process (12, 26). We hypothesized that the muscularis layer may initiate and sustain an inflammatory response to radiation that may contribute to altering muscle function during the acute phase. Although early cytokine response after radiation exposure has been reported previously in alveolar macrophages (34) and in organs such as the lungs, spleen, and brain (11, 20, 37, 52), this study is the first to demonstrate in vivo that abdominal irradiation in285 • SEPTEMBER 2003 •

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Fig. 5. Effect of ␥-irradiation on NF-␬B p65 and p50 subunit nuclear translocation and cytoplasmic p65, p50 and I␬B␣ levels in vivo. The nuclear protein levels of the p65 (A) and p50 (B) and cytoplasmic protein level of p65 (C), p50 (D), and I␬B␣ (E) subunits were measured by Western blotting at 6 h, 1 day, and 3 days after a single abdominal irradiation (10-Gy). Twenty micrograms of protein extract were separated on SDS-polyacrylamide gel; p65 was detected with anti-p65 antibodies (F-6), and P50 with anti-p50 antibodies (E-10), which reacted to the nuclear localization signal domain in the p65 and p50 subunits, respectively. I␬B␣ was detected with anti-I␬B␣ antibodies (C-21). The density of bands was quantitated by transmittance densitometry and analyzed with the Biocom analyzer. Results are expressed in relative units. Data are the means ⫾ SE (n ⫽ 5); * P ⬍ 0.05; ** P ⬍ 0.01; *** P ⬍ 0.005 significantly different from control value.

duces alterations in the expression of genes involved in acute intestinal inflammatory response and thereby modifies the balance between pro- and anti-inflammatory cytokines. Increase in IL-1␤, TNF-␣, and IL-6 expression occurred early at 6 h after irradiation and the high levels of IL-1␤ and IL-6 mRNA persisted for 3 days. Increased cytokine expression and protein production were observed simultaneously. A cascade of inflammatory events ensued, with the proinflammatory cytokines (IL-1, TNF-␣, and IL-6) increasing the ability of endothelial cells, macrophages, smooth muscle cells, and fibroblasts to secrete IL-8 (15). The accumulation of neutrophils at the inflammatory site is known to be caused mainly by the chemotactic cytokine IL-8 (36). Recently, by using immunohistological analysis, we reported (31) a marked neutrophil infiltration characterized by an increase of myeloperoxidase-positive cells and myeloperoxidase activity in the ileum 3 days after 10-Gy abdominal irradiation. The IL-8 (CINC) was highly expressed 3 days after irradiation (Fig. 2). Although the exact role of IL-8 in the inflammatory pathogenesis is not totally clear (25), its level (mRNA and protein) has been positively correlated with the intestinal inflammation score. AJP-Gastrointest Liver Physiol • VOL

The intestinal immune response is carefully regulated in normal tissue so that an inflammatory event is quickly and appropriately counterbalanced by local anti-inflammatory mechanisms. An imbalance between pro- and anti-inflammatory cytokines leads to intense inflammation and tissue destruction. The biological effects of IL-1␤ are regulated by naturally produced inhibitors, including IL-1ra (9). In our study, the IL-1ra/IL-1␤ ratios calculated at 6 h and 3 days after irradiation were nearly identical to those for the nonirradiated rats, suggesting that at these times, ileum produces appropriate amounts of IL-1ra to counterbalance the IL-1␤ excess. However, 24 h after irradiation, the IL-1ra/IL-1␤ ratio was half that of the control. In human IBD, both tissue IL-1ra and IL-1␤ levels increase with inflammation, but the IL-1ra/IL-1␤ ratio decreases (9). The deficit of endogenous IL-1ra production is an important factor in IBD pathogenesis and may explain why the acute inflammatory response in some individuals develops into chronic persistent inflammation rather than resolving. Our results suggest that the imbalance observed here is not specific to IBD but extends to irradiation-induced inflammatory effects. TGF-␤1 is a cytokine with pleiotropic properties and local proinflammatory effects. Among other things, it 285 • SEPTEMBER 2003 •

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Fig. 6. Time course of 10-Gy abdominal irradiation-induced NF-␬B p65 DNA-binding activity. NF-␬B activity was determined by EMSA (A). Migration positions of NF-␬B/DNA complex from control, 6 h, 1 day, and 3 days after irradiation are indicated. The composition of NF-␬B complex was determined by using anti-p65 and anti-p50. The FP lane represented the NF-␬B probe without nuclear extract and the C10X lanes were incubated in the presence of 10-fold molar excess of an unlabeled NF-␬B probe. The p65 binding activity was quantitated by the Trans-AM NF-␬B kits (B). Data are the means ⫾ SE (n ⫽ 5); * P ⬍ 0.01 significantly different from control value. C, control; OD, optical density.

stimulates chemotaxis of granulocytes and macrophages as well as the release of proinflammatory cytokines (IL-1, TNF-␣, IL-6). Turner et al. (49) show that it induces IL-6 as well as IL-1ra production. However, analysis of the temporal pattern of cytokine expression

in our study shows that IL-6 and IL-1ra expression begins some hours before TGF-␤1 expression. In our study, TGF-␤1 expression increased on and after day 1 postirradiation. TGF-␤1 is a critical profibrogenic factor

Fig. 7. Effect of abdominal irradiation-induced modification in mRNA levels of NF-␬B subunits in vivo. Levels of p105 (A) and p65 (B) mRNA were measured in the ileal muscularis layer by real-time quantitative RT-PCR at 6 h, 24 h (D1), and 3 days (D3) after a single abdominal irradiation (10-Gy). The results were expressed as a ratio to the reference gene (HPRT) mRNA levels. Data are the means ⫾ SE (n ⫽ 5); * P ⬍ 0.05, ** P ⬍ 0.01, *** P ⬍ 0.005, **** P ⬍ 0.001 significantly different from control value.

Fig. 8. Effect of abdominal irradiation-induced modification in activator protein-1 (AP-1) transcription factor mRNA levels in vivo. Levels of c-Fos (A), and c-Jun (B) mRNA were measured in the muscularis ileal layer by real-time quantitative RT-PCR at 6 h, 24 h, and 3 days after 10-Gy abdominal irradiation. The results were expressed as a ratio to the reference gene HPRT mRNA levels. Data presented are the means ⫾ SE (n ⫽ 5); * P ⬍ 0.005 significantly different from control value.

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that induces the synthesis and deposit of collagen and other matrix components. It appears to play a particularly prominent role in the chronic phase of injury and is consistently overexpressed in areas of the intestinal wall that have histopathological lesions. Our data corroborate the increase in TGF-␤1 protein observed by immunohistochemistry in the small intestines of the rat 1 day after fractionated exposure to X-radiation (38). IL-10 is a potent anti-inflammatory cytokine that downregulates the synthesis of TNF-␣ and IL-1␤ and upregulates IL-1ra synthesis (10). It probably counteracts the production of proinflammatory cytokines. In contrast to the substantial expression and concentration of IL-10 in IBD patients during the acute phase (42), IL-10 mRNA levels fell drastically 1 day after irradiation. This response seems to be specific for radiation exposure. This IL-10 gene repression is important, because the cytokine has a direct anti-proliferative effect through its modulation of T-cell functions and antifibrotic properties. Increased TGF-␤ expression associated with decreased IL-10 levels characterizes a fibrotic state. The importance of these anti-inflammatory cytokines on the pathophysiology of acute radiation-induced inflammatory processes is underlined by findings that IL-10 gene knockout mice develop gastrointestinal inflammation (27) and that exogenous IL-1ra improves colitis in animal models (13). NF-␬B and AP-1 activity play critical roles in the activation of several cytokines (IL-1, TNF-␣, IL-6, IL-8, and IL-10). In particular, we have reported that in vivo the treatment by the caffeic acid phenethyl ester, an inhibitor of the NF-␬B-DNA binding properties (33), inhibited totally the increase of the IL-6 and its specific receptor expressions induced by ␥-irradiation, demonstrating the implication of NF-␬B (30). In the present study, NF-␬B activation induced by irradiation appeared to be fully supershifted by the p65 antibody, suggesting that the NF-␬B complex implicated in the irradiation effect concerned more particularly the p65 subunit. However, Zhou et al. (52) used p50⫺/⫺ mice to demonstrate the involvement of the p50 subunit in the irradiation-induced expression of cytokines in vivo. There was a minimal NF-␬B activation in these knockout mice and low mRNA levels for IL-1␤, TNF-␣, and IL-6 after irradiation (8.5-Gy), but NF-␬B activation in intestine was not detected. In the intestinal ischemia model, however, Yeh et al. (50) showed that the activation of p50/p50 homodimers is unique to the intestine, whereas p50/p65 dimers are activated in other tissues. The time course of NF-␬B nuclear translocation and activation after irradiation (Figs. 5 and 6) is in agreement with previous observations of cells in which p65 binding activity was maximal 2 to 3 h after irradiation and decreased after 5 h (28). Western blot studies (Fig. 5) showed that cytoplasmic p65 levels decreased at 6 h, whereas nuclear levels increased. However, although p65 nuclear levels return to control values at 1 and 3 days after irradiation, a marked increase in cytoplasmic p65 levels was observed, but it did not correlate with p65 mRNA levels (Fig. 7). In fact, p65 and p105 (p50 precursor) expression decreased AJP-Gastrointest Liver Physiol • VOL

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slightly 1 and 3 days after irradiation. Taken together, our results showed that 1) despite the low NF-␬B subunit expression, there are enough subunit proteins already present in the cytoplasm to produce the early irradiation response as suggested earlier (28); and 2) the expression of the NF-␬B subunits increased later after irradiation, but their activation is limited by the I␬B system. Interestingly, Beg et al. (5) reported that NF-␬B is constitutively inhibited by I␬B␣, activated by I␬B␣ degradation, and then inhibited once again by the resynthesis of I␬B␣. NF-␬B rapidly induces I␬B␣ synthesis in an effective negative feedback loop that controls the inflammatory process. Indeed, we observed an increase of I␬B␣ levels in the cytoplasm 24 h after irradiation, suggesting that NF-␬B activation induces the synthesis of its inhibitor I␬B␣ to regulate cellular activation, providing a negative feedback loop that regulates the inflammatory process induced by irradiation. The precise mechanism of NF-␬B activation by ␥-radiation remains unknown. The release of reactive oxygen species (ROS) that characterizes the response to ionizing radiation occurs very early, and in vitro studies (29) showed that ROS could activate NF-␬B in some cell types. However, direct involvement of ROS in radio-induced NF-␬B activation has been challenged (28). AP-1 transcription factors consist of the Jun and Fos protein families. Studies of several tissues show that c-jun and c-fos can be induced by ionizing radiation (19). In this study, only the level of c-fos mRNA was elevated on day 3 postirradiation, whereas no effect on c-jun was observed from 6 h through 3 days. However, the time course of expression may differ according to the tissue. For example, expressions of c-fos and c-jun increase in vivo in skin 2 h after an 8-Gy ␥-irradiation, with a maximal effect at 6 h (32), but do not increase in the gut (1). Sherman et al. (44) showed that the increase in c-fos expression peaked at 3 h and was associated with downregulation of c-fos RNA levels 24 h after irradiation. Irradiation induced a very early (15 min after) expression of c-fos in the brain, whereas the c-jun level was not changed up to 24 h (19). Accordingly, in our study, the absence of increased expression between 6 h and 3 days may be explained by a repression of expression. Indeed, expression of c-fos does not appear to parallel that of c-jun (19). The molecular cascades initiated by ionizing irradiation are complex and involve more molecules than those studied here. In particular, cytokine function is mediated through cytokine receptors that can be also secreted in a soluble form and that contribute to limiting cytokine action. The effect of radiation on these pathways must also be studied, because they are potential targets for therapeutic action. The existence of chronic cytokine-driven cascades raises the question of what perpetuates the response. The critical question that needs to be answered now is whether the acute responses we observed are responsible for acute and late intestinal damage, and if so, how efficient would the modification of proinflammatory cytokine expression be on the development of late complications after radiotherapy? 285 • SEPTEMBER 2003 •

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