Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated...

Lipopolysaccharide Is Radioprotective in the Mouse IntestineThrough a Prostaglandin-Mediated Mechanism

TERRENCE RIEHL,* STEVEN COHN,‡ TERESA TESSNER,* SUZANNE SCHLOEMANN,*and WILLIAM F. STENSON**Division of Gastroenterology, Washington University School of Medicine, St. Louis, Missouri; and ‡Division of Gastroenterology,University of Virginia, Charlottesville, Virginia

Background & Aims: The bone marrow and the intes-tine are the major sites of radiation-induced injury. Thecellular response to radiation injury in the intestine orbone marrow can be modulated by agents given beforeirradiation. Lipopolysaccharide is known to be radiopro-tective in the bone marrow, but its effect on theintestine is not known. We sought to determine iflipopolysaccharide is radioprotective in the intestineand, if so, to determine the mechanism of its radiopro-tective effects. Methods: Mice were treated with paren-teral lipopolysaccharide or vehicle and then irradiated(14 Gy total body irradiation in a cesium irradiator). Thenumber of surviving intestinal crypts was assessed 3.5days after irradiation using a clonogenic assay. Results:Parenteral administration of lipopolysaccharide 2–24hours before irradiation resulted in a 2-fold increase inthe number of surviving crypts 3.5 days after irradia-tion. The radioprotective effects of lipopolysaccharidecould be eliminated by coadministration of a selectiveinhibitor of cyclooxygenase 2. Lipopolysaccharide wasradioprotective in wild-type mice but not in mice with adisrupted cyclooxygenase 2. Parenteral administrationof lipopolysaccharide resulted in increased productionof prostaglandins in the intestine and in the inductionof cyclooxygenase 2 expression in subepithelial fibro-blasts and in villous, but not crypt, epithelial cells.Conclusions: Lipopolysaccharide is radioprotective inthe mouse intestine through a prostaglandin-depen-dent pathway.

The adult small intestinal epithelium is continuouslyand rapidly replaced by cell replication within the

crypts and the subsequent migration of their progenyonto the villous epithelium.1 Intestinal epithelial cells areultimately derived from multipotent stem cells locatednear the base of each intestinal crypt.2–5 In the adultmouse small intestine, these stem cells rarely divide toproduce a daughter stem cell and a more rapidlyproliferating transit cell (Figure 1). Transit cells in turnundergo a number of rapid cell divisions in the prolifera-tive zone in the lower half of each crypt. Their progenydifferentiate into mature epithelial cell types and migrate

onto the villus. Various noxious agents (chemical, physi-cal, infectious, and inflammatory) can kill epithelial cells,resulting in the destruction of the epithelial barrier. Afterinjury, stem cells proliferate to increase their number andgive rise to transit cells that proliferate to form regenera-tive crypts.6 Normal patterns of epithelial differentiationare reestablished by migration and differentiation of cellsproduced in these regenerative crypts.

Although many noxious agents cause epithelial injuryand initiate the process of epithelial regeneration, radia-tion is the most extensively characterized model ofintestinal injury.6–8 Radiation injury affects rapidly prolif-erating cell populations; after total body irradiation, themajor sites of injury are the bone marrow and theintestinal epithelium. Mice exposed to 7–15 Gy of totalbody irradiation exhibit death of most proliferating cellsin the bone marrow, but some stem cells will survive andrepopulate the marrow.9 With higher doses of irradiation,the number of surviving bone marrow stem cells isinsufficient to repopulate the marrow and the animal diesof sepsis. Similarly, after 8–14 Gy of total body irradia-tion, proliferating transit cells in the intestinal crypt arekilled, but some stem cells survive.6–8 These survivingcrypt epithelial stem cells play a central role in theregeneration of the mucosa after radiation injury. Theyproliferate and give rise to the transit cells that formregenerative crypts and eventually repopulate the mu-cosa. Higher doses of radiation kill more of the stem cellsand, consequently, reduce the number of regenerativecrypts. A functional assay for quantifying stem cellsurvival after radiation (or other forms of cytotoxic injury)has been developed; this assay is based on the ability of asingle surviving stem cell (clonogenic cell) to regenerate a

Abbreviations used in this paper: COX, cyclooxygenase; dmPGE2,dimethyl prostaglandin E2; IL, interleukin; LPS, lipopolysaccharide;PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; Tb,body temperature; TGF, transforming growth factor; TNF, tumornecrosis factor.

r 2000 by the American Gastroenterological Association0016-5085/00/$10.00

doi:10.1053/gast.2000.7959

GASTROENTEROLOGY 2000;118:1106–1116

crypt-like focus of cells termed a microcolony.8,10 Threeor 4 days after total body irradiation, the number ofcrypt-like foci of surviving epithelial cells is scored onhistological sections of intestine.

The cellular response to radiation injury in either thebone marrow or intestine can be modulated by agentsgiven before irradiation.11 Agents that decrease theamount of radiation-induced injury are said to be radio-protective. Several factors with hematopoietic or immunestimulatory properties protect mice from radiation injury.Interleukin (IL)-1, IL-11, IL-12, tumor necrosis factor(TNF)-a, granulocyte-macrophage colony–stimulatingfactor, and stem cell factor are radioprotective in the bonemarrow.12–16 In the intestine, radioprotective agents suchas IL-1, IL-11, and transforming growth factor (TGF)-bgiven before radiation increase the number of survivingcrypts after radiation.17–19 Exogenous prostaglandins,including the stable prostaglandin (PG) E2 analoguesmisoprostol and dimethyl prostaglandin E2 (dmPGE2),are radioprotective in both the intestine and bonemarrow.20–22 Lipopolysaccharide (LPS) is radioprotectivein the bone marrow23; whether it is radioprotective in theintestine has not been investigated. LPS has a largenumber of biological effects including the enhancementof prostaglandin synthesis through the induction ofcyclooxygenase (COX)-2.24 In this study we used the

microcolony assay to determine if LPS is radioprotectivein the intestine and to determine if its radioprotectiveeffects are mediated through prostaglandins. We foundthat administration of parenteral LPS to mice increasesintestinal PGE2 levels and induces the expression ofCOX-2 in villous epithelial cells and subepithelialfibroblasts. Moreover, we found that LPS is radioprotec-tive in the intestine and that its radioprotective effects aremediated by prostaglandins produced through COX-2.

Materials and Methods

AnimalsFVB/N female mice (Taconic, Germantown, NY) and

C57BL/6Jx129 COX-12/2, COX-22/2 mice and their litter-mates were maintained on a 12-hour light/dark schedule andfed standard laboratory mouse chow. Mice were irradiated atage 6 weeks in a Gamacel 40-cesium irradiator at 0.96cGy/min. Animals were killed at various times after irradiationand rapidly dissected, as described previously.25 The proximaljejunum was fixed in Bouin’s solution and divided into eight5-mm segments before paraffin embedding and immunohisto-chemical analysis. The distal jejunum was snap-frozen in liquidnitrogen for analysis of PGE2 levels.

Genotyping COX-1 andCOX-2 Knockout Mice

Breeding pairs were obtained from R. Langenbach(National Institute of Environmental Sciences, Research Tri-angle Park, NC).26,27 Mice carrying the disrupted COX-1 orCOX-2 gene were screened by polymerase chain reaction (PCR)using DNA isolated from tail clippings. For identifyingCOX-1 knockout mice, PCR primer sequences amplified a600–base pair (bp) DNA fragment from the wild-type alleleand a 650-bp DNA fragment from the disrupted allele. Foridentifying COX-2 knockout mice, PCR primers 58-CTGGT-GCCTGGTCTGATGATGTAT-38 (forward primer COX-2 exon7) with 58-GCCTTTGCCACTGCTTGTA-38 (reverse primerCOX-2 exon 9) amplified a 1.0–kilobase pair DNA fragmentfrom the wild-type allele. The same forward primer with thereverse primer 58-ATCCCATCGAGCGAGCACGTACTC-GGA-38 (Neo 2A) amplified an 850-bp DNA fragment fromthe disrupted allele. Mice were also determined to be homozy-gous COX-22/2 by Southern blot analysis and by their elevatedblood urea nitrogen level (kit from Sigma Chemical Co., St.Louis, MO) caused by kidney disease expressed by theseanimals.26

Crypt Survival

Crypt survival was measured in animals killed 3.5 daysafter irradiation using a modification of the microcolonyassay.8,10 Each mouse received 120 mg/kg bromodeoxyuridine(BrdUrd) (Sigma) and 12 mg/kg fluorodeoxyuridine (Sigma) 2hours before death to label the S-phase cells. Five-micrometerparaffin sections were prepared from proximal jejunum ori-ented so that the sections were cut perpendicular to the long

Figure 1. Diagram of a small intestinal crypt showing location of stemcells and proliferative (transit) cells.

June 2000 LIPOPOLYSACCHARIDE AND RADIOPROTECTION 1107

axis of the small intestine. Deparaffinized sections wereincubated with a 1:2000 dilution of affinity-purified goatanti-BrdUrd28 at 4°C overnight after blocking of nonspecificprotein binding sites with phosphate-buffered saline (PBS)containing 2% bovine serum albumin, 0.2% nonfat dry milk,and 0.3% Triton X-100. Bound anti-BrdUrd was detectedwith gold-labeled rabbit anti-goat immunoglobulin (Ig) Gwith subsequent silver enhancement (Amersham, ArlingtonHeights, IL). For purposes of the microcolony assay, a regenera-tive crypt was determined to have survived irradiation based onits histological appearance. The viability of each survivingcrypt was confirmed by incorporation of BrdUrd into 5 or moreepithelial cells within each regenerative crypt. A minimum of 6complete cross sections were scored for each mouse.

Immunohistochemistry

For immunohistochemical localization of mouse COX-1or COX-2, deparaffinized sections of Bouin’s fixed tissue wereincubated with a 1:2000–1:8000 dilution of either rabbitanti-mouse COX-1 (a gift from J. Masferrer, Searle, St. Louis,MO)29 or rabbit anti-mouse COX-2 (Cayman Chemical, AnnArbor, MI).25 For COX-1 immunohistochemistry, endogenousperoxidase activity was quenched with 1% hydrogen peroxide.COX-2 immunohistochemistry was performed in the samemanner except that peroxidase activity was quenched with 3%hydrogen peroxide and nonspecific staining was blocked byincubating the sections sequentially in blocking buffer, avi-din-D solution (Vector Laboratories; Burlingame, CA), bio-tin-D solution (Vector Laboratories), and normal donkey serum(Sigma) before incubating the slides overnight at 4°C withrabbit anti-mouse COX-2 antibody (Cayman Chemical). Sec-tions incubated with normal rabbit serum or without primaryantibody serve as negative controls. Bound antibody wasdetected using 3,38-diaminobenzidine tetrahydrochloride (Vec-tor Laboratories) after incubation with anti-rabbit IgG conju-gated to horseradish peroxidase.

COX Inhibitors

Indomethacin (Sigma) was dissolved in ethanol anddiluted into sterile 5% sodium bicarbonate immediately beforeuse. The indicated doses of indomethacin were administered byintraperitoneal injection. NS-398 (Biomol, Plymouth Meet-ing, PA)29 was dissolved as for indomethacin and administeredintraperitoneally at the indicated doses and times. The choiceof dosages of NS-398 was based on the doses that inhibitedPGE2 production in vivo in COX-2–dependent animal mod-els29 without affecting synthesis of PGE2 through COX-1.

Measurement of PGE2 Levels

Lipids were extracted by homogenizing flash frozentissue in cold ethanol/0.1 mol/L sodium phosphate, pH 4.0(70%/30%, vol/vol) followed by shaking incubation at roomtemperature. An aliquot of the extract was dried down under astream of nitrogen, and the PGE2 concentration was deter-mined by a PGE2-specific enzyme-linked immunoassay (Cay-man Chemical) according to the manufacturer’s directions.25

Isolation of Mouse IntestinalEpithelial Cells

Epithelial cells were isolated from 5–6-week-old irradi-ated (14 Gy) and nonirradiated mice. Small intestines werewashed in ice-cold PBS, transferred to prewarmed (37°C)Hank’s basic salt solution containing 0.5 mmol/L EDTA andantibiotics (gentamicin, penicillin, and streptomycin), andincubated with gentle agitation for 15 minutes at 37°C.Samples were vortexed for 10 seconds, and tissue was removedfrom the suspensions of released epithelial cells. Fluorescence-activated cell sorter analysis indicated that these suspensionsconsisted of at least 95% epithelial cells with not more than5% intraepithelial lymphocytes.30 Cell suspensions were pel-leted at 1500 rpm for 5 minutes and resuspended in aproteinase inhibitor cocktail containing 25 µg/mL antipain, 25µg/mL aprotinin, 25 µg/mL leupeptin, 25 µg/mL chymostatin,50 µmol/L phenanthroline, 10 µg/mL pepstatin A, and 2nmol/L dithiothreitol in 20 mmol/L N-Tris (hydroxymethyl)-methyl-2-aminoethane sulfonic acid, pH 7.4.

Sodium Dodecyl Sulfate–PolyacrylamideGel Electrophoresis and Western BlotAnalysis of COX-1 and COX-2

Epithelial cells isolated from irradiated and nonirradi-ated mouse small intestine were assayed for COX-1 andCOX-2 by Western blotting. Samples for sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) weresolubilized in SDS-PAGE sample buffer (pH 6.8) containingTRIS (62.5 mmol/L), glycerol (10%), SDS (2%), bromophenolblue (1%), and b-mercaptoethanol (5%). Equal amounts ofproteins from the nonirradiated and irradiated mouse intestinalepithelial cell lysates and the positive controls (lysates of mouseperitoneal macrophages and of LPS-stimulated RAW cells)were separated by electrophoresis on 8% SDS-polyacrylamidegels. After electrophoresis, the separated proteins were trans-ferred to an Immobilon transfer membrane (Millipore Corp.,Bedford, MA). Rabbit antibodies against mouse COX-1 (a giftfrom J. Masferrer, Searle) and COX-2 (Cayman Chemical) wereused to detect bands corresponding to COX-1 and COX-2,respectively. Bound antibody was visualized using a donkeyanti-rabbit IgG linked to horseradish peroxidase and ECL(Amersham; Arlington Heights, IL) with fluorographic detec-tion on Kodak BioMax ML film (Kodak, Rochester, NY).

LPS Treatment and Body Temperature

LPS from Escherichia coli (K-235; Sigma) was dissolvedin pyrogen-free PBS (Cellgro, Herndon, VA) and injected at adose of 0.15, 0.5, or 1.5 mg/kg. Control mice were injectedwith pyrogen-free PBS. Body temperature (Tb) was measured incontrol and LPS-treated mice with a temperature probe (series400; YSI Inc., Yellow Springs, OH) connected to a digitalthermometer (VWR Scientific Products, St. Louis, MO) andinserted into the rectum. Tb was measured before administra-tion of LPS or pyrogen-free PBS (controls) and at 30 minutesand every hour during the postinjection period for 9 hours. To

1108 RIEHL ET AL. GASTROENTEROLOGY Vol. 118, No. 6

minimize stress, mice were adapted to temperature measure-ment on a daily basis for 7 days before the experiment.

Results

We first sought to determine if LPS is radioprotec-tive in the intestine. Crypt survival was assessed in mice3.5 days after total body irradiation using the micro-colony assay. Mice receiving 14 Gy total body irradiationhad an average of 9.3 surviving crypts per intestinal crosssection (Figure 2). Administration of LPS (0.5 mg/kg) 14hours before radiation increased the number of survivingcrypts 2-fold to 18.4 per cross section. We also assessedthe effects of LPS (0.5 mg/kg) given either 2 or 24 hoursbefore radiation and found that in each of these condi-tions the increase in crypt survival was only slightly lessthan that seen with LPS given 14 hours before radiation(Figure 3). This suggests that the intermediate biochemi-cal events induced by LPS have developed by 2 hours andpersist until at least 24 hours after LPS administration.

COX inhibitors were used to determine if the radiopro-tective effects of LPS were mediated through the induc-tion of prostaglandin synthesis. Administration of indo-methacin, which inhibits both COX-1 and COX-2, orNS-398, which inhibits only COX-2, had no effect oncrypt survival (Figure 2). However, when either indo-methacin or NS-398 was administered with LPS, theeffects of LPS on increasing crypt survival were lost.These findings suggest that the effects of LPS as aradioprotective agent are mediated through prostaglan-dins produced through COX-2.

Having shown that inhibitors of prostaglandin synthe-sis block the radioprotective effects of LPS, the ability ofLPS treatment to induce an increase in intestinal prosta-

glandin synthesis was measured. To implicate PGE2 as amediator of LPS-induced radioprotection, it is importantto know PGE2 levels in the intestine at the time ofradiation. In this experiment the mice were not radiatedbut were killed 14 hours after LPS administration.Administration of LPS increased PGE2 levels 4-fold from18 to 72 pg/mg tissue (Figure 4). Coadministration ofeither indomethacin or NS-398 with LPS reduced PGE2

levels back to baseline values. The ability of NS-398, aselective COX-2 inhibitor, to block the increase in PGE2

levels induced by LPS suggests that the increased PGE2

synthesis induced by LPS is produced through the

Figure 2. LPS treatment before radiation increases crypt survival in aPGE2-dependent manner. FVB/N mice received vehicle (control),indomethacin (1.5 mg/kg every 8 hours), NS-398 (1 mg/kg every 8hours), LPS alone (0.5 mg/kg), LPS plus indomethacin, or LPS plusNS-398 beginning 14 hours before irradiation (14 Gy). Mice were killed3.5 days after radiation, and the number of surviving crypts per crosssection was determined. Data are the mean 6 SEM for 9 animals.*P , 0.001 compared with control; **P , 0.001 compared with LPSalone.

Figure 3. LPS pretreatment as little as 2 hours before radiationincreases crypt survival. FVB/N mice received LPS (0.5 mg/kg) orvehicle (control) at the indicated time before irradiation (14 Gy). Thenumber of surviving crypts per cross section was determined 3.5 daysafter radiation. Data are the mean 6 SEM for 3 animals. *P , 0.01compared with control.

Figure 4. LPS treatment increases PGE2 production in the mousesmall intestine. FVB/N mice were treated with vehicle (control),indomethacin (1.5 mg/kg every 8 hours), NS-398 (1 mg/kg every 8hours), LPS (0.5 mg/kg), LPS plus indomethacin, or LPS plus NS-398.After 14 hours, mice were killed and the PGE2 content of intestine wasdetermined by enzyme immunoassay. Data are the mean 6 SEM of3–15 animals. *P , 0.001 compared with control; **P , 0.001compared with LPS alone.


COX-2 pathway. We found that LPS was radioprotectivewhen given 2, 14, or 24 hours before irradiation. Tocorrelate LPS-induced radioprotection with LPS-inducedPGE2 production, we also measured intestinal PGE2

levels at 2 and 24 hours after LPS treatment as well as at14 hours (Figure 5). At 2 hours, PGE2 levels were up7-fold over baseline, gradually decreasing to 3-fold at 24hours. Thus, all the time points associated with LPS-induced radioprotection were also associated with at leasta 3-fold increase in PGE2 levels.

A dose-response curve for PGE2 synthesis in responseto LPS administration showed that the dose required toincrease PGE2 levels in the intestine was between 0.15and 0.5 mg/kg (Figure 6A). Increasing the dose above 0.5mg/kg did not further increase PGE2 levels.

LPS has a wide variety of biological effects, all of whichare dose related. If LPS is to be considered as aradioprotective agent, the doses used would have to befree of intolerable side effects. We assessed the effects ofthe LPS doses used in this study on Tb, a biologicalparameter known to be affected by LPS; at low doses LPSinduces hyperthermia and at higher doses it induceshypothermia.31 At all 3 doses used in this study (0.15,0.5, and 1.5 mg/kg), LPS induced hypothermia (Figure6B). These effects were dose related, with the higherdoses of LPS inducing a greater and more sustaineddecrease in body temperature. All the mice in all the LPS

dosing groups survived, and by 24 hours after injectionTb was back to baseline (data not shown).

Western blotting was used to determine if LPStreatment increased COX-1 or COX-2 protein expressionin intestinal epithelial cells. COX-1 was expressed inepithelial cells from both control and LPS-treated mice,and LPS treatment had no effect on the level of COX-1expression (Figure 7). Treatment with LPS results inincreased prostaglandin production through the COX-2pathway. A Western blot of a homogenate of controlintestinal epithelial cells revealed no band correspondingto COX-2; however, a homogenate of intestinal epithelialcells from an LPS-treated mouse revealed a band of 72kilodaltons corresponding to COX-2.

Immunohistochemical localization of COX-1 expres-

Figure 5. Time course for LPS-induced PGE2 synthesis. FVB/N micereceived LPS (0.5 mg/kg) at the time indicated before death. Controlmice received no LPS. The mice were killed, and the PGE2 content ofsmall intestine determined by enzyme immunoassay. Data are themean 6 SEM for 3 animals. *P , 0.001 compared with control.

Figure 6. (A) LPS increases PGE2 synthesis in a dose-dependentmanner. FVB/N mice were given the indicated doses of LPS intraperito-neally 14 hours before death. After death, the small intestine wasassayed for PGE2 by enzyme immunoassay as described in Materialsand Methods. Data are the mean 6 SEM for 3 animals. *P , 0.01compared with control. (B) Body temperature response of FVB/N miceinjected with LPS (r, 0.15 mg/kg; j, 0.5 mg/kg; m, 1.5 mg/kg) orpyrogen-free PBS (d). Data are the mean 6 SEM for 3 animals.


sion is identical in intestines from control and LPS-treated mice (Figure 7A and B). COX-1 is expressed incrypt epithelial cells; as the epithelial cells migrate up onto the villus they stop expressing COX-1. The epithelialexpression of COX-1 roughly corresponds to the prolifera-tive zone in the crypt. As epithelial cells migrate onto thevillus they differentiate, lose their ability to proliferate,and stop expressing COX-1. Immunohistochemistry forCOX-2 in the proximal jejunum of control mice revealedstaining of a few scattered villous epithelial nuclei (Figure8C). One hour after intraperitoneal administration of LPSthere was strong staining for COX-2 in the villousepithelial cells (Figure 8D). There was a sharp demarca-tion of COX-2 expression at the crypt-villus junction.Higher-power examination revealed that COX-2 expres-sion in the villous epithelial cells of LPS-treated mice wasnuclear and perinuclear with additional expression in aband just below the microvilli (Figure 8E). COX-2 wasnot expressed in the epithelial cells of the tip of the villus.Intraepithelial lymphocytes do not express COX-2. Cryptepithelial cells did not express COX-2 in LPS-treatedmice; however, immunoreactive COX-2 was found inpericryptal fibroblasts (Figure 8F). Thus, in LPS-treatedmice, crypt epithelial cells do not express COX-2 butCOX-2 expression seems to be activated in epithelial cellsas they migrate up into the villus. The villous epithelialcells stop expressing COX-2 as they near the tip of thevillus and are about to be shed into the lumen. InLPS-treated mice, the epithelial cell populations express-ing COX-1 and COX-2 seem to be mutually exclusive.

Having shown that a specific COX-2 inhibitor, NS-398, blocks the radioprotective effects of LPS, we nextsought to determine if LPS is radioprotective in mice

with a disrupted COX-2 gene (COX-22/2). In theabsence of LPS, the number of surviving crypts afterradiation injury in the COX-22/2 mice was the same asthat in their wild-type littermates (Figure 9). Treatmentof COX-22/2 mice with LPS 14 hours before irradiationhad no effect on crypt survival, but the same treatmentdoubled the number of surviving crypts in their wild-type littermates. Thus, disruption of COX-2 blocks theincrease in crypt survival induced by LPS but has noeffect on crypt survival in the absence of LPS, just asNS-398 blocks the increase in crypt survival induced byLPS but has no effect on crypt survival in the absence ofLPS. As we previously reported,32 the number of surviv-ing crypts in mice with a disrupted COX-1 gene wasdiminished compared with controls. However, treatmentwith LPS increased crypt survival in mice with disruptedCOX-1 just as it increased crypt survival in theirwild-type littermates.

Discussion

In this study we show that LPS is radioprotectivein the intestine and that its radioprotective effects aremediated through the induction of prostaglandin synthe-sis through COX-2. Exogenous prostaglandins, includ-ing misoprostil and dmPGE2, have been shown to beradioprotective,20–22 but this is the first demonstrationthat endogenous prostaglandin synthesis can be radiopro-tective and that the radioprotective effects of anotheragent are mediated through prostaglandin synthesis.Treatment with LPS increased total intestinal PGE2

levels 4-fold, suggesting that fairly modest increases inintestinal prostaglandin levels can increase crypt sur-

A B

Figure 7. Detection of (A) COX-1and (B) COX-2 in epithelial cellsisolated from small intestines ofcontrol and LPS-treated mice byWestern blotting. LPS (0.5 mg/kg) was given to FVB/N mice 1hour before death, intestinal tis-sue was harvested, epithelialcells were isolated, and the ex-pression of COX-1 and COX-2proteins in the epithelial lysateswas determined by Western blot-ting using anti-mouse antibod-ies. A mouse macrophage cellline (RAW cells) was used as anegative control. RAW cellsstimulated with LPS (LPS-in-duced mouse macrophage) wereused as a positive control.


vival.25 We had previously shown that administration ofindomethacin, an inhibitor of COX-1 and COX-2, 1hour before radiation had no effect on crypt survival.Together, these data show that increasing intestinalprostaglandin levels to superphysiological levels immedi-ately before radiation increases crypt survival, but decreas-ing prostaglandins levels to subphysiological levels hasno effect.

Prostaglandins are synthesized through 2 COX iso-forms, COX-1 and COX-2.33 In the normal intestine,

COX-1 is expressed constitutively in epithelial cells inthe crypt but not in the villus.25 COX-2 is not expressedin appreciable amounts in epithelial cells in the normalintestine. LPS has been shown to induce COX-2 inmacrophages, astrocytes, and gastric epithelial cells.24,34,35

In this study, LPS induced the expression of COX-2 invilolus epithelial cells and in subepithelial fibroblasts buthad no effect on the expression of COX-1. This is the firstdemonstration of COX-2 induction by LPS in intestinalepithelial cells. Furthermore, LPS induced COX-2 expres-

Figure 8. Immunohistochemical localization of COX-1 and COX-2 in the proximal jejunum of control and LPS-treated mice. The cellular localizationof COX-1 in epithelial cells of crypts and lower regions of the villi was the same in (A) control and (B) LPS-treated mice. (C) Control mice showedCOX-2 staining of a few villous epithelial nuclei. (D) One hour after LPS (0.5 mg/kg intraperitoneally) treatment, there was COX-2 staining of villousepithelial cells. Arrows in A, B, and D indicate the crypt-villus junction. Higher power views of the intestines from LPS-treated mice showed stainingof villous epithelial cells with sparing of the villus tip. There is no staining of intraepithelial lymphocytes (arrow in E ). There is staining of sub-epithelial fibroblasts (arrows in F ) but no staining of crypt epithelial cells. (Original magnification: A, B, and D, 4003; C, 2003; E and F, 10003).


sion in a distinct subset of epithelial cells becauseincreased expression was seen in villous but not cryptepithelial cells. Thus in the normal mouse crypt, epithe-lial cells express COX-1 but stop expressing it as theydifferentiate and migrate onto the villus.25 In contrast,the ability of LPS to induce COX-2 expression isassociated with villous epithelial cells but not crypt cells.Whether crypt epithelial cells have some factor thatprevents COX-2 expression or lack some factor requiredfor COX-2 expression is not clear. The induction ofCOX-2 in villous epithelial cells after parenteral adminis-tration of LPS is interesting in that villous epithelial cellsare constantly exposed to LPS from luminal bacteria.Thus, parenteral LPS can induce changes in villousepithelial cells that are not produced by LPS derived fromluminal bacteria. The response of villous epithelial cellsto parenteral but not luminal LPS may be a result of thehigher dose, or the differential responsiveness of theapical and basolateral surfaces of these cells to LPS.

The mechanism for the induction of epithelial cellCOX-2 by LPS is not clear. It may be a direct effect ofLPS on epithelial cells or an indirect effect mediatedthrough a second cell type, perhaps lamina propriamacrophages. Although most intestinal macrophages donot express CD14, the plasma membrane receptor forLPS-binding protein, LPS-induced activation of macro-phages is well described.36

COX-22/2 mice have a histologically normal intesti-nal epithelium.26,27 LPS was not radioprotective in theintestine in these mice, providing additional support forthe suggestion that the radioprotective effects of LPS inthe intestines of normal mice are mediated throughCOX-2. Whether COX-22/2 mice would be resistant toother biological effects of LPS has not been investigated.Our present data show that LPS-induced radioprotection

is mediated by prostaglandins produced through COX-2but do not directly address the question of which specificprostaglandin is the mediator. Three pieces of indirectevidence suggest that PGE2 is the mediator. The first isthat PGE2 and stable analogues of PGE2 (dmPGE2 andmisoprostil) are radioprotective in the intestine; thesecond is that PGE2 is the most abundant prostaglandinin the intestinal epithelium37; and the third is that PGE2

is known to modulate stem cell survival after radiation.25

Although COX-2 is not expressed in appreciableamounts in normal intestinal or colonic epithelial cells, itis expressed in colonic adenomas and adenocarcinomasand in human inflammatory bowel disease.38 In Crohn’sdisease of the ileum, COX-2 expression is induced invillous epithelial cells but not crypt epithelial cells, thesame pattern of distribution as seen in the intestines ofmice given LPS.39 This similarity suggests that thevillous epithelial cells are capable of expressing COX-2 inresponse to various agents, whereas crypt epithelial cellscannot. The expression of COX-2 in villus but not cryptepithelium in Crohn’s ileitis suggests that the distribu-tion of COX-2 in epithelial cells in LPS-treated mice is aproduct of the ability of villous but not crypt cells toexpress COX-2 rather than the product of their relativesensitivity to LPS.

Crypt survival after radiation requires the survival andproliferation of stem cells in the lower portion of thecrypt.8 In this study, we found that LPS induces enhancedCOX-2 expression in villous epithelial cells and insubepithelial fibroblasts but not in crypt epithelial cells.The mediation of LPS-induced radioprotection of stemcells in the crypt by prostaglandins produced throughCOX-2 can be reconciled with the geographic distribu-tion of COX-2 expression induced by LPS in 2 ways. Thesimpler and more likely explanation is that PGE2

produced by villous epithelial cells or by subepithelialfibroblast acts on stem cells to induce radioprotection.The alternative explanation is that PGE2 production invillous epithelial cells or subepithelial fibroblasts causesthose cells to produce a second substance, which in turninduces radioprotection in stem cells. If, as is likely,LPS-induced radioprotection is mediated by the effects ofPGE2 on stem cells, it is difficult to say whether PGE2 ismore likely to come from subepithelial fibroblasts orvillous epithelial cells. Immunohistochemistry suggeststhat COX-2 is expressed in greater abundance in villousepithelial cells than in subepithelial fibroblasts. However,prostaglandins are degraded very rapidly and usually acton cells near the cells in which they are produced.Subepithelial fibroblasts are immediately adjacent tocrypt epithelial cells, whereas villous epithelial cells aresomewhat removed from the crypts. This suggests that

Figure 9. COX-2 expression is required for LPS-induced enhancementof crypt survival. Bl6/129 F2 COX-1 knockout mice (COX-1 K.O.),COX-2 knockout mice (COX-2 K.O.), or their wild-type (W.T.) littermatesreceived vehicle or LPS (0.5 mg/kg) 14 hours before 14-Gy irradiation.The number of surviving crypts per cross section was determined 3.5days after irradiation. Data are the mean 6 SEM of 3 animals. **P ,

0.002 compared with wild-type irradiated control; *P , 0.005 com-pared with COX-1 knockout irradiated control.


LPS-induced radioprotection may be mediated by PGE2

produced by subepithelial fibroblasts.LPS has a wide variety of biological effects including

the induction of a battery of cytokines such as IL-1,TNF-a, IL-6, IL-8, interferon gamma, and TGF-b.40

Some of these, including IL-1, TNF-a, and TGF-b, areradioprotective and have been implicated in mediatingthe radioprotective effects of LPS in the bone marrow.The roles of IL-1 and TNF-a in LPS-induced radioresis-tance in the bone marrow were only observed when LPSwas given 20 hours before radiation.23 In contrast, theradioprotective effects of LPS in the intestine are medi-ated through prostaglandins and occur when LPS is givenas little as 2 hours before radiation.

The signal transduction pathways by which prostaglan-dins induce radioprotection is not known. All thebiological effects of prostaglandins are thought to bemediated through plasma membrane receptors.41 Thereare 4 known receptors for PGE2 (EP1, EP2, EP3, andEP4). Two of these (EP2 and EP4) are tied to intracellularsignaling mechanisms that increase adenosine 38,58-cyclic monophosphate (cAMP) levels. Whether cryptepithelial cells express EP2 or EP4 receptors is notknown. Mediation of the radioprotective effect of PGE2through EP2 or EP4 receptors is suggested becausephosphodiesterase inhibitors, which also increase cAMPlevels, are also radioprotective.42

Among the potential mechanisms for radioprotectionis an increase in the number of stem cells or synchroniza-tion of the stem cells so that they are all at a point in thecell cycle where they are resistant to radiation. TNF-aand IL-1 spare hematopoietic stem cells by regulatingtheir cell cycling status. TNF-a inhibits stem cells fromcycling by arresting them in the radiation-resistant Go

phase, whereas IL-1 induces hematopoietic progenitorsinto late S2 phase, which is also radioresistant.23,43 Theseeffects are only seen if TNF-a and IL-1 are given at least20 hours before radiation to allow time for synchroniza-tion. However, the synthetic prostaglandins, dmPGE2

and misoprostil, are radioprotective when given as littleas 1 hour before irradiation, suggesting that they do notinduce radioprotection by inducing stem cell prolifera-tion or by synchronizing stem cells. Another possiblemechanism for radioprotection is the induction of bio-chemical and enzymatic pathways that prevent radiation-induced damage or promote DNA repair. These mecha-nisms include production of scavengers of reactive oxygenintermediates and repair of radiation-induced DNAstrand breaks. Induction of the antioxidant enzymemanganese superoxide dismutase is radioprotective inbone marrow44; however, there is no evidence for prosta-glandin-induced radioresistance being mediated through

scavenging reactive oxygen intermediates. There are,however, suggestions for a link between prostaglandin-mediated radioprotection and the repair of DNA strandbreaks. Misoprostil is radioprotective in spermatogonialstem cells in mice with intact DNA break repair enzymesbut is not radioprotective in SCID mice that lack theability to repair DNA breaks.45 These data are compat-ible with either a prostaglandin-sensitive DNA repairmechanism or a prostaglandin-sensitive step, whichrequires the ability to repair DNA breaks.

In an earlier study we showed that endogenous PGE2

produced through COX-1 was involved in the process ofcrypt survival and proliferation after irradiation.25 Treat-ment of mice with indomethacin in the period afterirradiation decreased the number of surviving crypts. Wenow show that prostaglandins produced through COX-2mediate the radioprotective effects of LPS. These findingsdo not imply that prostaglandins produced throughCOX-1 and COX-2 have different biological roles. Inboth the unirradiated and the irradiated mouse intestine,COX-1 is expressed in epithelial cells, whereas COX-2 isnot. In contrast, in LPS-treated mice both COX-1 andCOX-2 are expressed in intestinal epithelial cells (al-though in different epithelial cell populations) and mostintestinal prostaglandins are produced through COX-2.Thus, the apparent differences in the biological roles ofprostaglandins produced through COX-1 and COX-2may reflect differences in COX-1 and COX-2 expressionin control and LPS-treated animals.

In conclusion, we showed that LPS is radioprotectivein the intestine and that its radioprotective effects aremediated by the induction of prostaglandin synthesisthrough COX-2. This raises the possibility that prosta-glandins may mediate the effects of other radioprotectiveagents in either the intestine or bone marrow. Otheragents that induce increased intestinal PGE2 levels mayalso be radioprotective. There may be therapeutic poten-tial for radioprotective agents in protecting the intestinalepithelium in patients receiving radiation therapy. LPShas been given parenterally to human volunteers but hasnot been used as a therapeutic agent. Whether LPS orother agents that induce prostaglandin production in theintestinal mucosa could be used as practical radioprotec-tive agents in humans remains to be seen.

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Received August 13, 1999. Accepted January 10, 2000.Address requests for reprints to: William F. Stenson, M.D., Division

of Gastroenterology, Campus Box 8124, Washington UniversitySchool of Medicine, 660 South Euclid Avenue, St. Louis, Missouri63110. Fax: (314) 362-8959.

Supported by National Institutes of Health grants DK33165 andDK55753 (to W.F.S.).


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