Cytokine and endothelial-IL10

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Cytokine and endothelial cell adhesion molecule

expression in interleukin-10-deficient mice

SH IGEYUKI KAWACHI,1 STEPHEN J ENNI NGS,2 J ULIAN PANES,3 ADAM COCKRELL,1

F. STEPHEN LAROUX,1 LAURA GRAY,1 MICHAEL PE RRY,4 HENRY VAN DER HEYDE,2

EDWARD BALISH,5 D. NEIL GRANGER,1 ROBERT A. SPECIAN,1

AND MATTHEW B. GRISHAM1

Departm ents of 1 Molecular and Cellular Physiology and 2 Microbiology and Im m un ology, L ouisiana

S tate U niversity Medical Center, S hreveport, Louisiana 71130; 3 Departm ent of Gastroenterology,

University of Barcelona, Barcelona, S pain; 4S chool of Physiology an d Pha rm acology,

University of New South Wales, Sydney, Australia; and 5 Departm ent of S urgery,

University of Wisconsin, Madison, Wisconsin 53706

Ka w a c h i , S h i g e y u k i , S t e p h e n J e n n i n g s , J u l i a n P a n e s ,

Adam Coc kr e l l , F . Ste phe n Lar oux, Laur a G r ay, M i c hae l

P e r r y , H e n r y v a n d e r H e y d e , E d w a r d B a l i s h , D . N e i l

G r ange r , Robe r t A. Spe c i a n, and M atthe w B . Gr i s ham .

Cytokine and endothelial cell adhesion molecule expressionin interleukin-10-deficient mice. Am J Physiol Gastrointest

Liver Physiol 278: G734– G743, 2000.—The objectives of thisstudy were to quantify cytokine mRNAlevels and endothelialcell adhesion m olecule m essage an d protein expression inhealt hy wild-type and interleuk in-10-deficient (IL-10 / )micetha t develop sponta neous a nd chronic colitis. We found tha tcolonic messa ge levels of IL-1, IL-6, tu mor necrosis factor-,interferon-, lymphotoxin-, a nd t ra nsformi ng growt h fa c -tor- were elevated in colitic mice 10- to 35-fold comparedwith their healthy wild-type controls. In addition, colonicmessage levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and mucosaladdr essin cell a dhesion molecule-1 (MAdCAM-1) were foun dto be in creased 10-, 5-, and 23-fold, r espectively, in colitic

IL-10 / mice compar ed with t heir wild-type controls. Immu -noradiolabeling as well as immunohistochemistry revealedlarge and significant increases in vascular surface expressionof colonic ICAM-1, VCAM-1, and MAdCAM-1 in the mucosaas well as the submucosa of the colons of colitic mice. Thesedata are consistent with the hypothesis that deletion of IL-10results in the susta ined production of proinflammat ory cyto-kines, leading to the upregulation of adhesion molecules andinfiltration of mononuclear and polymorphonuclear leuko-cytes in to th e cecal an d colonic inters titium .

leukocytes; inflam mat ion; immu nology

INFLAMMATORY BOWEL DISEASE (IBD) is a recurrent in-flamm ation of the sma ll and/or lar ge bowel of unkn ownetiology. Increasingly, experimental and clinical data(17, 18, 21, 55) suggest tha t t he induction a nd pa thogen-esis of this disease is a multifactorial process involvingintera ctions a mong genetic, immun e, and environmen-tal factors. Indeed, there is substantial experimental

evidence to suggest tha t chronic gut inflam mat ion ma yarise from a dysregulated immune response to compo-n e n t s of t h e n or m a l g u t flor a (2 5, 3 6, 4 3 – 4 5). Th is

hypothesis is based on several different studies (16,4 9 – 5 2, 5 6) d e m on s t r a t i n g t h a t t a r g e t ed d e le t ion of ce r t a in g en e s k n o wn t o b e i m p or t a n t i n t h e i m m u n eresponse induces spontaneous colit is in mice that isdependent on the presence of normal enteric bacteria.One such model of IBD is the interleukin-10 deficient(IL-10 / ) model of colitis (4, 14, 31, 33, 4 6– 48). IL-10 isa T h elper cell 2 (Th2)-type cytokine with a sp ectru m of biological activities. It is produced by several differentcell types, including T cells, macrophages, and CD5 Bcells, an d functions to inhibit t he synth esis of T h elpercell 1 (Th1)-type and/or macrophage-derived cytokines[e.g., t um or necrosis factor- (TNF-), IL-12, inter-feron- (IF-)] by different populations of leukocytes

(23, 32). In addition, IL-10 at tenu ates th e forma tion of reactive oxygen and nitrogen metabolites as well asinhibiting surface expression of major histocompatibil-ity complex class II molecules and intercellular adhe-sion m olecule-1 (ICAM-1). Not sur prisin gly, I L-10 /

mice develop moderate to severe enterocolitis whenra ised un der conven tional hous ing conditions (4, 14, 31,3 3, 4 6 – 4 8). Th e col it i s d e ve lop s i n t h e ce cu m a n dproximal colon and is associated with the infiltration of l a r ge n u m b e r s of m on on u cl ea r l eu k ocy t es s u ch a slymphocytes, monocytes, and plasma cells (4, 14, 31,33, 46–48). Animals derived and raised under germ-free conditions fail to develop this distal bowel inflam-ma tion (4, 47, 48).

Recent studies suggest that this model of IBD ex-presses m ore of a Th1-type response, indicat ing un con-tr olled activat ion of T cells an d ma crophages (4, 47, 48).These proinflammatory cytokines are thought to ini-tiate and/or perpetuate chronic gut inflammation byvirt ue of th eir ability to upr egulate th e sur face expres-sion of different endothelial cell a dhesion molecules(ECAMs), thereby enha ncing the adh esion a nd infiltra -tion of different populations of leukocytes (13, 20, 24,27). Although the IL-10 / model of enterocolitis hasbeen used by several laboratories t o explore certainimmunologic aspects of chronic gut inflammation, no

The costs of publication of this a rticle were defrayed in pa rt by thep a ym e n t of p a g e ch a r g es . T h e a r t icle m u s t th e r e for e b e h e r e b ymar ked ‘‘advertisement ’’ in accordan ce with 18 U.S.C. Section 1734solely to indicate th is fact.

Am J Physiol Gastrointest Liver Physiol278: G734–G743, 2000.

0193-1857/00 $5.00 Copyright 2000 th e American Physiological SocietyG734 h t t p://www.a jpgi.or g



systematic or quantitat ive informat ion is availableregarding the relationship between cytokine and ECAMexpression in the IL-10 / model of colitis. Thus theobjectives of this study were to quantify message levelsfor a variety of different Th1 and Th2 cytokines andECAM message and protein expression in the colons of healthy wild-type and IL-10 / mice with distal boweldisease.

MATERIALS AND METHODS

Animals

IL-10 / mice were gener at ed by gene-targeting deletion inembryonic stem cells as described by Kuhn et al. (31). Themi c e use d i n t hi s s t udy we re bre d ont o a 129/ SvEv ba c k-ground. When raised in specific pathogen-free conditions,IL-10 / mice develop spontaneous colitis at 8–12 wk of age.Wild-type mice (129/SvEv) were used as controls at 8–12 wk of age and were obtained from Taconic Labs (Germant own,NY). De ve lopme nt of col it i s wa s a sse sse d a s pre vi ousl ydescribed by Berg et al. (4), using serum amyloid A proteinlevels as a n ind icator of ongoing inflamm at ion.

Colonic RN A Isolation and Qu antification of mR NA by RNase Protection Assay

Colons were r emoved, cleaned of intestinal content s, an dwe ighe d, a n d t ot a l l engt h wa s de t ermi ne d. The t i ssue wa sthen divided into pr oximal an d distal colon. Total RNA wasisolated from the t issues u sing TRIzol reagen t (GIBCO BRL),according to th e m anu factur er’s instructions. RNa se pr otec-tion assay was performed using [-32P]UTP-labeled MCK2ba nd MCK3b Ri boQua nt probe se t s (Pha rm i nge n) for t hedi ffe re nt Th1 a nd Th2 cyt oki nes. ICAM-1, va scul a r ce lladhesion molecule-1 (VCAM-1), and mucosal addressin celladhesion molecule-1 (MAdCAM-1) message levels were quan-tified using a custom probe set obtained from Pharmingen(San Diego, CA). Pr obe synt hesis, hybridization, RNase t reat -

me nt , gel pre pa ra t i on, a nd e le ct rophore sis were a l l pe r-form ed according to the man ufactur er ’s instr uctions. Aut ora-diographs of the resulting protected species were processedi nt o di gi t a l i ma ge s a na l yz e d a nd qua nt i fie d usi ng Ima ge -Quant software (Molecular Dynamics). The relative levels of mRNA for t he t wo di ffe re nt cyt oki nes a nd ECAMs weredetermined from the ratio of the density obtained for eachcytokine to th e density obtained for the mRNA of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase.Multiple exposur es of each reaction were genera ted to ensur eaccur acy of the compa rison of th e two signals un der differentsignal dens ity conditions.

ECAM E xpression I n Vivo

Monoclonal antibodies used for experiments. T h e d u a l

ra diolabeled monoclonal an tibody (MAb) technique was u sedto quant ify ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1expression in different vascular beds (12, 19, 27). YN-1, a r atIgG2b directed against mouse ICAM-1 (Bayer, West Haven,CT); 3C4 (MIC2/4), a ra t IgG2a di rect e d a ga inst mouseICAM-2 (Pharmingen); MK1.9.1, a rat IgG1 targeted againstmouse VCAM-1 (Bayer), an d MECA-367, a rat IgG2a tar-geted against mouse MAdCAM-1 (Pharmingen) were used ast he MAbs. P-23, a muri ne IgG1 a ga i nst huma n P-se l e c t i n(Pha rm acia-Upjohn, Kalama zoo, MI), was u sed as th e contr olnonbindin g MAb.

The bindi ng MAbs dire ct e d a ga inst ICAM-1, ICAM-2,VCAM-1, an d MAdCAM-1 were r ad iolabeled with 125 I (Dupont

NEN , Boston, MA), whereas t he n onbinding MAb (P-23) waslabeled with 131 I . Ra dioiodi na t ion of t he MAbs we re pe r-formed by t he iodogen method as previously described (12,19, 27).

Anim al procedures. At 12 wk of age, both wild-type andIL-10 / mice were anesthetized with ketamine hydrochlo-ride (150 mg/kg im) an d xylazine (7.5 mg/kg im), an d th e right

jugular vein and carotid artery were cannulated with polyeth-ylene tubing.

For a ssessing th e ICAM-1 expression, a mixtu re of 10 µg of 125 I ant i-ICAM-1 MAb a nd 40 µ g of un labeled a nti-ICAM-1MAb was given with a n am ount of 131 I-labeled P-23 n ecessar yto ensure a total 131 I-injected activity of 400,000–600,000 cpmt hrough t h e j ugul a r ve in ca nnul a (t ot a l vol 200 µ l ). In t heI CAM -2 e xp er i m en t s , a m i xt u r e of 1 0 µ g of 125 I-labeledanti-ICAM-2 MAb and 60 µg of unlabeled anti-ICAM-2 MAbwas given with an appropriate amount of 131 I P -23 (400,000–600,000 cpm). In the VCAM-1 experiments, a mixture of 10µg of 125 I-labeled anti-VCAM-1 MAb and 20 µg of unlabeledant i-VCAM-1 MAb was administered with an appropriateamount of 131 I-labeled P-23 through the jugular vein. In theMAdCAM-1 experiments, only 10 µg of 125 I-labeled ant i-MAdCAM-1 MAb were administrated with an appropriateamount of 131 I-labeled P -23 t hrough the jugular vein. These

dosages were selected on th e basis of pilot stu dies demonstr at-ing optimum activity and receptor saturation in the tissuesexamined un der constitut ive and stimulated conditions.

A blood sa mpl e wa s t a ke n 5 mi n a ft er MAb i nje ct i on.Bicar bonate-buffered saline was isovolum ically excha nged byinfusion of buffer through the jugular vein and simultaneouswit hdra wa l of bl ood a nd buffe r from t he a rt e ry. Aft e r t hevascular system was completely flushed with bicarbonate-buffered saline, the small intestine, cecum, large intestine,and a var iety of other tissues were har vested and weighed.

Calculation of ECAM expression. A 14800 Wizard 3 gammacounter (Wallac, Turku, Finland), with automatic correctionfor background activity and spillover, was used to count 125 Ia n d 131 I a ct i vi t ie s i n e a ch orga n a nd i n a 100-µl pl a smasample. The injected activity in each experiment was calcu-

l a t ed by c ount i ng a 4-µl sa mpl e of mi xt ure cont a i ni ng t h era diolabeled MAbs. The am ount of ra dioactivity rema ining inthe t ube used to mix the MAbs and t he syringe used to injectthe mixture was subtracted from the total calculated injectedactivity. The accumulated activity of each MAb in an organwas expressed as the percentage of the injected dose (%ID)pe r gra m of t i ssue . ECAM e xpre ssi on wa s ca l cul a t e d bysubt ra cting th e nonspecific tissue a ccum ulat ion of MAb fromthe accumulation of binding MAb as follows: VCAM-1 expres-sion (%ID/g for 125 I) (%ID/g for 131 I) (%ID for 125 I inplasma)/(%ID for 131 I in plasma). This formula corrects thet i ssue a ccumul a t i on of nonbindi ng MAb for t he re la t i vepla sma l evel s of bot h bindi ng a n d nonbindi ng MAbs t oestimate the nonspecific tissue accumulation of MAb. Thisvalue, expressed a s %ID/g, was converted t o µg MAb/g tissu eby mu ltiplying the above value by th e t otal injected bindingMAb (in µ g), divided by 100.

Imm unohistochemical Assessment of ECAM Expression

Se pa ra t e groups of a ni ma l s (n 3 for each group) wereanesthetized with ketamine hydrochloride (150 mg/kg im)and xylazine (7.5 mg/kg im), and t he r ight jugular vein a ndcarotid artery were cannulated with polyethylene tubing. Forassessing th e immu nolocalization of th e four ECAMs, th esame t otal amount of MAb against the different ECAMs wasused as in the dual radiolabeled MAb technique describedabove. However, the MAbs were not radiolabeled; 50 µg of ICAM-1 MAb, 70 µ g of ICAM-2 MAb, 30 µ g of VCAM-1 MAb,

G735CYTOKINE AND ADHESION MOLECULES IN COLITIS



and 10 µ g of MAdCAM-1 MAb were infused in each experi-ment. F ive minu tes a fter MAb injection, th e vasculature wascompletely flushed with bicarbonate-buffered saline, an dtissu es were excised exactly as described for t he r adiolabeledMAb experiments.

The colons were divided into proximal and distal portions,a n d t h e con t e n t s w er e r e m ov ed b y g en t l e fl u s h i n g w i t hZamboni’s fixative usin g a syrin ge and a 30-gauge n eedle. Thesa mpl es we re t he n pl a ce d i n Za mboni ’s fixa t i ve a t 4°Covern ight. The following da y, th e sam ples were placed in 80%ETOH to remove picric acid from the solution, cleared inDMSO, a nd wa she d t hre e t i me s i n PBS. The sa mpl es we reth en stored in P BS cont aining 30% sucrose and 0.01% sodiumazide at 4°C overnight. Samples were placed in OCT embed-di ng me di um, a nd t he blocks we re froz en wit h i sope nt a nechilled in liquid nit rogen. Frozen sa mples were sectioned in acryostat at 10-µm thickness and collected on poly-L-lysine-coa t e d sli des. The sli des we re t h e n dri ed over P 2O5 in avacuum desiccator for 30 m in.

Col on se ct i ons we re blocke d wit h 10% norma l donke yserum and then incubated with a donkey anti-rat secondaryan tibody against E CAMs conjugated t o Cy3 fluorochrome in ahu mid box for 1 h. The slides were wash ed in PBS th ree timesfor 10 min each time, and t he sections ar ound an d on the back

we re dri e d before mount i ng. Sl ide s we re t he n mount e d i nMowiol-glycerol conta inin g 2.5% 1,4-diazabicyclo[2.2.2]octan e(pH 8.5). Sections were also stained with hematoxylin andeosin for sta nda rd observation of histology.

Histopathology

Colons from three representative wild-type and IL-10 /

mice were recovered from animals at necropsy and fixed inZamboni’s fixative. The tissues were rinsed in 0.1 M ph os-phate buffer and dehydrated with 95% ethanol. The tissueswe re t he n e mbedde d i n glycol me t ha cryla t e (JB-4; Poly-sciences, Warrington, PA). Sections (1–2 µm) were cut onglass knives, stained with hemat oxylin and eosin, dried on awa rmi ng pl a t e , a nd mount e d i n Pe rmount . Sa mpl e s we re

evaluated, and photograph s were taken with a SenSys digitalcamera an d processed with Metamorph.

Statistical Analysis

Al l v a lu e s a r e p r e se n t ed a s m e a n s S E . D a t a w er eanalyzed using standard statistical analyses, i .e., one-wayANOVA and a paired an d u npaired St udent’s t -test. Statisti-cal significance was set a t P 0.05.

R ES U LTS

Histopathology

Of the IL-10 / mice, 100% developed moderate tos e ve r e i n fla m m a t i on wit h m a r k e d t h i ck e n in g of t h e

intestinal wall, cecum, and proximal colon at 3 mo of age. Corresponding to this spontaneous colit is was adramatic increase in serum amyloid Aprotein levels (73vs. 2,381 µ g/ml for wild-type vs. IL-10 / mice; P 0.01). The colon in IL-10 / mice was significantlylonger tha n that of wild-type m ice (9.23 0.18 vs.7.02 0.22 cm), and the weight of colon in IL-10 /

mice was significant ly heavier than that of wild-typemice (0.57 0.03 vs. 0.20 0.04 g). Histopathologyrevealed moderate to severe inflam mat ion of the colon.The colonic mu cosa exhibited epithelial hyperplasiawith infiltration of chronic inflammatory cells such as

lymphocytes, plasma cells, and macrophages but alsocontained small-to-moderate numbers of neutrophils.This infiltrate was pr esent in both the m ucosa a nd th esubmucosa. Tran smura l inflamma tion, multiple smallsup erficial erosions of th e mucosa covered by inflam ma -t or y e xu d a t e , m u cu s , a n d ce ll u la r d e br i s, a n d cr y ptabscesses were pr esent a s well.

Cytokine m RN A E xpression

The pattern of cytokine mRNA expression in wild-type an d colitic IL-10 / mice revealed enh an ced expres-sion of IL-1, IL-1, interleukin-1 receptor an tagonist,and IL-18 as well as IL-6, IFN-, TNF-, and lympho-toxin (LT)- compar ed with their health y cont rols (Fig.1). LT- was present at low levels in colitic colons yetsignificantly above the levels detected in the healthywild-type mice (Fig. 1). IL-10 mRNA was t otally absentin colitic IL-10 / mice, as expected; however, TGF-1mRNA levels were increased approximately threefold(Fig. 1). Colitic IL-10 / mice also expressed low butdetectable levels of mRNA for the IL-12 p35 and p40

subunits (Fig. 1).

ECAM Expression

Colonic message levels of ICAM-1, VCAM-1, an dMAdCAM-1 in colitic IL-10 / mice were found to besignifican tly increased by 10-, 5-, a nd 23-fold, r espec-tively, compared with their wild-type controls (Fig. 2).Corresponding to these large and significant increasesin ECAM mRNA was enhanced surface expression of colonic ICAM-1, VCAM-1, a nd MAdCAM-1 (Fig. 3).Colonic VCAM-1 and MAdCAM-1 expression were en-h a n ce d e ig h t - a n d fou r f ol d , r e s p e ct i v el y, wh e r e a sICAM-1 surface expression was increased by 50% (Fig.

3). Imm un olocalization of each ECAM using t he samemethod described for the radiolabeled MAb techniquer e ve a l ed e n h a n c ed v a s cu l a r s t a i n in g of I C AM -1 ,VCAM-1, and MAdCAM-1 in colitic mice comparedwith their wild-type controls (Fig. 4). Constitutive expres-sion of VCAM-1 in healthy wild-type mice was belowthe sensitivity of the immunolocalization method; how-ever, we were able to observe enh an ced vascular expr es-sion of VCAM-1 in the IL-10 / mice (Fig. 4). Weobserved low levels of cons tit ut ive expr ession of ICAM-1that increased in submucosal and mucosal vessels inthe IL-10 / mice (Fig. 4). Enhanced vascular expres-sion of MAdCAM-1 was also observed in colitic mice;however, the staining pattern for MAdCAM-1 was quite

different from those of the other ECAMs. We observedstaining of large mucosal and submucosal vessels thatappeared approximately two to three times larger thanthose vessels from wild-type anima ls with constitu tiveexpression of MAdCAM-1 (Fig. 4). Furthermore, thestaining appeared t o be junctional in na ture. BecauseICAM-2 is constitutively expressed and not upregu-l a t ed i n r e s pon s e t o a v a r ie t y o f p r o in fla m m a t or ymediators and/or cytokines (40), we quantified thisECAM to assess vascular su rface area in wild-type vs.colitic mice. We found, using either the dual radiolabelor immunohistochemical techniques, that ICAM-2 ex-

G736 CYTOKINE AND ADHESION MOLECULES IN COLITIS



Fig. 1. Relat ive messa ge levels of different colonic cyto-kines in wild-type and interleukin-10-deficient (IL-10 / ) mice normalized to glyceraldehyde-3-phosphatedehydrogenase (GAPDH). Three representative wild-type mice (filled bars) and three colitic IL-10 / mice(open bars) were used for these studies. Relative in-creases in cytokine mRNA were determined from ratioof density obtained for each cytokine to density deter-mined for housekeeping gene GAPDH. Specific proto-cols a re described in MATERIALS AND METHODS. D a ta a r eexpressed as m ean values. LT- or -, lymphotoxin- or

-; T N F -, tumor necrosis factor-; IL-1Ra, interleu-kin-1 receptor anta gonist; IFN-, interferon-; TGF,transforming growth factor; MIF, macrophage migra-tory inhibitory factor.

Fig. 2. Colonic message levels of inter-cellular adh esion molecule-1 (ICAM-1),v a s cu l a r ce l l a d h e s ion m o le cu l e -1(VCAM-1), and mucosal a ddressin celladhesion molecule-1 (MAdCAM-1) inwild-type a nd IL-10 / mice. Specificmethods for RNA isolation and RNaseprotection a ssay ar e described in MATE -R I AL S A N D M E T H OD S. L 3 2, r ib os om a lstructural protein. B : filled bars, wild-type mice; open bars, IL-10 / mice.




pression was similar in wild-type and IL-10 / mice(Figs. 3 and 4). In addition to the colon, we observedsignificant increas es in expr ession of ICAM-1, VCAM-1,and MAdCAM-1 in the cecum of colitic IL-10 / mice,wh e r ea s on l y VCAM -1 wa s e n h a n ce d i n t h e s m a llbowel of the colitic anima ls (Figs. 5 an d 6).

D IS C U S S ION

The distal bowel is continuously exposed to numer-ou s e n vi r on m e n t a l a n t i ge n s a n d b a ct e r ia l p r od u ct s ,resulting in a constan t threat of a potentially danger-ous and debilitating inflammatory response. For thisreason, immune responses of the intestine and colonare under very strict regulatory control (17, 43, 54). A

variety of recently developed mouse models have pro-v id e d i m p or t a n t i n si gh t s i n t o t h e m e ch a n i s m s t h a tcontr ol th e immu ne response in th e int estinal tra ct andt h e a b er r a t ion s t h a t le a d t o a d is ea s e s t a t e . D a t aob t a in e d fr om a v a r ie t y of d iffe r en t s t u d ie s u s in ggenetically engineered or imm un e-ma nipulat ed modelsof colitis suggest a regulatory n etwork in which IL-10and TGF- regulate expression of potentially damagingcytokines, such a s IFN - a n d TNF- (17, 43, 48). One of the best-studied m odels of spontaneous and chroniccolitis is the IL-10 / m o d e l b r e d t o t h e 1 2 9 s t r a i nbackgroun d (4, 47, 48). All of thes e m ice d evelop colitiswhen housed under conventional conditions, due princi-pally t o th e absence of th e major regulatory cytokine

IL-10 (4, 47, 48). St udies with this model, as well a sot h e r s , s u g ge s t t h a t ch r o n ic col it i s r e s u lt s fr om adysregulated immune response to components of thenorma l gut flora (43, 48). One of the ear liest m an ifesta-tions of th is un cont rolled activation of T cells within t hececal and colonic interstitium is the sustained produc-tion of proinflamma tory mediators such as the Th1 a ndmacrophage-derived cytokines. In the present study,we fou n d l a r ge a n d s ig n ifica n t i n cr e a s es i n I L-1,IL-1, I L- 6 , I FN-, a n d T N F - mRNA in colons of IL-10 / mice compared with their healthy controls(Fig. 1). Each of these cytokines was strongly associ-

ated with ongoing colitis. These findings are consistentwith previous reports (2, 7, 14, 38, 39, 44) implicatin gIFN- and TNF- as ma jor mediators in th e path ophysi-ology of chronic of colitis.

However, the pat tern s of oth er cytokine species wereunexpected. In the IL-10 / colitis model, it was ex-pected th at no IL-10 mRNA would be detected, an d th iswas found to be true. This finding is also consistentwith t he concept t hat IL-10 production is essential forthe development an d expression of fun ction of regula-tory CD4 T cells, designated Tr-1 (25). Interestingly,it has been reported that IL-10 is an important growthand differentiation factor for Tr-1 CD4 T cells, whichexert their regulatory function principally through the

production of the inhibitory cytokine TGF- (25). How-ever, the level of mRNA for TGF-1 was significantlyelevated, yet pr ofound disease wa s st ill present . Theser e s u l t s s u g g e s t t h a t TGF- alone, in the absence of IL-10, or in the presence of elevated levels of othercytokines is not sufficient to limit the development of IBD. The cellular origin of TGF- in this model is notk n own . I t i s l ik e ly t h a t i t i s d e r i ve d fr om a n on -Tlymph ocyte source within t he site of inflamm at ion.

Another un expected observation mad e in the pr esentstudy was the divergence in expression of LT- a n dLT- mRNA species. In health y wild-type m ice, low butdiscernible levels of both mRNA species are present.

However, in colitic m ice, LT- mRNA was very high,whereas LT- mRNA was very low to undetectable.This was an un expected observat ion, since th e mat ur e,secreted form of th e lymph otoxin m olecule is composedof a heter otrimeric complex consisting of one chain of LT- an d t wo cha ins of LT- (1). The a bsen ce of th e LT-mRNA species may indicate th at, dur ing chronic inflam-ma tion, fun ctiona l LT is not secret ed by activated CD4

T cells within the site of inflammation. However, LTplays a critical role in the initiation of disease, sinceIBD does not develop in mice in wh ich t he signa ling viathe LT- receptor (LT-R) i s b lock e d b y a s ol u bl e

Fig. 3. Endothelial cell adh esion molecule (ECAM) ex-pression in colons from wild-type (wt) and IL-10 /

mice. Vascular sur face expression oft he different ECAMswas quantified using the dual radiolabeled monoclonalantibody (MAb) technique described in MATERIALS AND

METHODS. ECAMs were quantified in mice at 3 mo of age. A : ICAM-1. B : VCAM-1. C : MAdCAM-1. D: ICAM-2.* P 0.05 vs. wild type.




LT-R-lg fusion molecule (34). A potent ial consequ enceof the uncontrolled production of Th1 and macrophage-derived cytokines is the tra nscriptional activation of ECAM expression within the colonic microvasculature.A nu mber of different experimental a nd clinical stu dies(24) suggest tha t leukocyte-endothelial cell interac-tions play a critical role in initiating an d/or perpetu at-

ing the chr onicgu t inflamm at ion observed in experimen-t a l a s w el l a s h u m a n I BD . T h e m icr ov a scu la t u r eoccupies a crit ical position in the inflammatory re-sponse by virtu e of its ability to different ially r egulatethe infiltration of specific populations of leukocytes. Inthis respect, the postcapillary venules ma y define thetype of inflammatory cell that may gain access to th e

Fig. 4. Imm un olocalizat ion of different E CAMs in colons obta ined from wild-type (wt) and IL-10 / mice. At 3 mo of

age, unlabeled rat anti-mouse MAb specific for the different ECAMs was injected (iv), and tissues were processed asdescribed in MATERIALS AND METHODS. Fr ozen sections were stained with donkey ant i-rat secondary antibodyconjugated to Cy3 fluorochrome. Note vascular immun olocalization of each MAb, indicatin g little or no st aining of extravas cular cells and t issue (magnificat ion, 100).




colonic interstitium a nd dictat e whether an acute an d/orchronic inflamm ation will ensue.

I n t h e p r e se n t s t u d y, we d e m on s t r a t e t h a t ch r on i center ocolitis in IL-10 / mice is a ssociated with en-hanced expression of ICAM-1, VCAM-1, and MAdCAM-1message and protein in the colon (Figs. 2 and 3). Thevascular localization of these E CAMs was confirm edusing immunohistochemistry (Fig. 4).

ICAM-1 and ICAM-2 are ECAMs, which are mem-bers of the immunoglobulin supergene family (11).ICAM-1 contains five immunoglobulin-like extracellu-lar domains of which the first N H 2-terminal immuno-globulin-like domain recognizes CD11a/CD18 an d th eth ird immun oglobulin-like domain recognizes CD11b/

CD18 (15). ICAM-1 is basally expressed on endothelialcells, and its expression is increased in response toactivation of endothelial cells with certa in Th 1 a nd/orma crophage-derived cytokines or ba cterial pr oducts.

Maximal expression of ICAM-1 is achieved within 4–8h after activating the endothelial cells and is associatedwith m axima l levels of leukocyte a dher ence. Indeed, weobserved a significant increase in both message levelsas well a s su rface expression of ICAM-1 in t he IL-10 /

colitic mice (Figs. 2 an d 3). The dispa rity between themagnitu de of th e increase in m RNA vs. sur face expres-sion of ICAM-1 (10-fold vs. 50%, respectively) mostprobably reflects t he large increase in infiltr at ing ICAM-1-cont ainin g cells (e.g., lymphocytes, monocytes),whereas the more modest increase in ICAM-1 surfaceexpression reflects increases in ICAM-1 only on vascu-lar endothelium (Fig. 6). Several different reports (29,35, 37, 41) have demonstrated enhanced staining for

ICAM-1 in biopsies obtained from patients with activeulcerative colit is and Crohn’s disease. Fur thermore,recent experimental and clinical studies (3, 58) havedemonstrated that infusion of an antisense oligonucleo-

Fig. 5. ECAM expression in cecum s from wild-type (wt)

and IL-10 / mice. Vascular surface expression of thedifferent ECAMs was quant ified using the dua l radiola-beled MAb technique described in MATERIALS AND METH -ODS . ECAMs were quantified in mice at 3 mo of age. A:ICAM-1. B : VCAM-1. C : MAdCAM-1. D: ICAM-2. * P

0.05 vs. wild type.

Fig. 6. ECAM expression in small intest ines from wild-

type (wt) and IL-10 /

mice. Vascular surface expres-sion of the different ECAMs was qua ntified using th edual radiolabeled MAb technique described in MATERI-ALS AND METHODS. ECAMs were quantified in mice at 3mo of age. A: ICAM-1. B : VCAM-1. C : MAdCAM-1. D:ICAM-2. * P 0.05 vs. wild type.




tide directed a gains t ICAM-1 messa ge produces clinicalimpr ovemen t in a mouse m odel of colitis an d in ster oid-resistan t Crohn ’s disease patients.

ICAM-2 is a truncated form of ICAM-1 containingonly two immunoglobulin-like extracellular domains.Like ICAM-1, ICAM-2 also is basally expressed onendothelial cells, but its expression is not increased inresponse t o cytokine exposur e (40). We found t ha t theexpression of ICAM-2 wa s similar between IL-10 /

and wild-type mice, suggesting that the density of thecolonic vascular bed (i.e., vascular surface area) wassimilar between colitic an d cont rol mice (Figs. 3 and 4).Tog et h e r, t h e s e d a t a s u g ge s t t h a t t h e ob s er v ed i n -creases in ICAM-1, VCAM-1, and MAdCAM-1 expres-sion a re du e to increases in expression on t he vascularendothelium rather than increases in colonic vascular-ity.

VCAM-1 is an inducible ECAM, which is known tomediate t he firm adhesion of monocytes an d lympho-cytes to endoth elial cells (22). Thu s th is ECAM ma y actto promote adh esion and immigrat ion of chronicinfla m-

ma tory cells into colonic inter stitiu m (22). Pr oinfla mm a-tory cytokines, such as TN F- an d IL-1, both of whichar e upr egulated in th is model, induce VCAM-1 expres-sion in various organs via activation of nu clear fac-tor-B (24). It is known that lymphocytes, monocytes,and eosinophils possess a 1 integrin, called very lateactivation antigen-4 (41), which binds to VCAM-1(and MAdCAM-1) (22). The kinetics of VCAM-1 expres-sion on vascular endothelial cells closely resemble thatseen with ICAM-1.Although a great deal of interest hasbeen generat ed regard ing the possibility tha t VCAM-1may be important in lymphocyte infiltration in IBD,several investigators (29, 30, 37) have failed to demon-strat e enhan ced expression of VCAM-1 in biopsies

obtained from patients with active colitis. These obser-v a t ion s a r e p a r t i cu l a r ly s u r p r is in g i n v ie w of d a t aob t a in e d i n t h e p r es en t s t u dy a n d in t h r e e r e ce n treports (5, 6, 26), which demonstrated that primarycultures of microvascular endothelial cells isolatedfrom human intestine and colon respond to differentproinflammatory cytokines with enhanced surface ex-pression of VCAM-1. The reasons for these differencesbetween experimental and huma n IBD are n ot kn ownbut m ay represent t he inheren t variability known to beassociated with immun ohistochemistr y compar ed withth e m ore objective, radiolabeled MAb meth od to qua n-tify ECAM expression in vivo. Alter na tively, the qu alityof the antibodies used for the immunolocalization stud-

ies ma y represent an importan t determinan t for a ccu-ra te det ermina tion of VCAM-1. In fact, our data woulds u g ge s t t h a t t h e d u a l r a d iol a be le d M Ab t e ch n i qu eprovides the necessary sensitivity to accurately deter-min e low-level const itu tive expr ession of VCAM-1 inth e absence of any immun ohistochemical evidence forVCAM-1-positive vessels in healthy controls (Fig. 4).

One of the more interesting findings in the presentstudy was the localization of MAdCAM-1 to dilatedmucosal and submucosal vessels in the colon, whichapp ear ed to be jun ctiona l in n at ur e (Fig. 4). MAdCAM-1was first described as an endothelial cell surface mol-

ecule that is selectively expressed in mucosal organsand required for lymphocyte homing to mucosal lym-phoid tissue (9, 10, 53). Murine endothelial cells can beinduced to express high levels of MAdCAM-1 in re-sponse t o proinflam ma tory cytokines (8, 9). Recently, ithas been shown (28, 57) that the adhesive interactionsmediated by MAdCAM-1 and its lymph ocyte receptor,47, are involved in leukocyte recruitment to chronic

inflamm at ory diseases of the bowel. MAbs specific for7 and MAdCAM-1 not only blocked recruitment of lymphocytes to the colitic colon but also reduced theseverity of colonic inflamma tion in severe combinedimmun odeficient m ice reconst ituted with CD4 T cellsenriched for the CD45RBhigh subpopulation (42). Ourstudies indicate that MAdCAM-1 expression in colon issignifican tly increased in colitic IL-10 / mice com-pared with wild-type mice. Together, these resultssuggest t hat MAdCAM-1 ma y also play an importan trole in initiating an d/or ma inta ining chr onic inflam ma-tion of colon in IL-10 / m i ce a n d m a y b e a r e le va n tther apeut ic tar get for patients with IBD.

In summary, this study represents the first demon-stration that ICAM-1, VCAM-1, and MAdCAM-1 areu p r e gu l a t ed i n t h e ch r on i ca l ly i n fla m e d ce cu m a n dcolon of IL-10 / mice. Furthermore, this study indi-cates that the transcriptional activation of certain Th1and macrophage-derived cytokines is associated withthe enhanced surface expression of ECAMs and maymediate the infiltration of immune modulating and/orpotentially injurious leukocytes. The specific cyto-kine(s) or E CAM(s) responsible for t he in itiat ion a nd/orperpetuation of the chronic gut inflammation in thismodel of colitis is presen tly un der in vestigat ion.

This study was supported by grants from the Arthritis Center of Excellence, Feist Foundation of Louisiana State University MedicalCenter in Shreveport, the Cr ohn’s an d Colitis Foundat ion ofAmerica,and the National Institute of Diabetes and Digestive and KidneyDisease s (DK-47663 an d DK-43785).

Address for reprint requests a nd other correspondence: M. B.Grisham, Dept. of Molecular and Cellular Physiology, LouisianaStat e Univ. Medical Cen ter, P.O. Box 33932, Sh reveport, LA, 71130(E-mail: m grish@lsum c.edu).

Received 1 November 1999; accepted in final form 11 January 2000.

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