www.elsevier.com/locate/issn/10434666

Cytokine 32 (2005) 85e93

Role of tumor necrosis factor receptors in an animalmodel of acute colitis

Carmencita Rojas-Cartagena a, Idhaliz Flores a, Caroline B. Appleyard b,*

a Department of Microbiology, Ponce School of Medicine, Ponce, PR 00732-7004, USAb Department of Physiology, PO Box 7004, Ponce School of Medicine, Ponce, PR 00732-7004, USA

Received 27 September 2004; received in revised form 29 June 2005; accepted 24 August 2005

Abstract

TNF-alpha is known to play an important role in inflammatory bowel disease (IBD); however, the pathophysiological role of its receptors isstill under study. Acute colitis was induced in rats by intracolonic administration of trinitrobenzene sulfonic acid (TNBS). Control rats receivedthe ethanol vehicle. Rats were sacrificed 72 h later and samples of tissue and fluids were collected. There was a significant increase in the proteinlevels of sTNF-alpha, sTNFRI, and sTNFRII in the peritoneal fluid (PF) of experimental rats. TNF-alpha, TNFRI, and TNFRII mRNA expres-sion was increased significantly in the colon of experimental animals compared to controls. TRAF3 and TRAF5 expression was also significantlyhigher, as was that of the adhesion molecules ICAM-1 and E-selectin. The increased expression of TNF-alpha, TNFRs, and the associated sig-naling factors in the colon of this rat model of IBD provides further evidence for their involvement in the promotion of inflammation and tissuedamage. In addition, increased levels of sTNFRs in the PF of experimental ratsdparticularly sTNFRIIdmay be involved in the development ofcolitis by serving as a reservoir of TNF-alpha, and thus provide a novel therapeutic target for IBD.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Colitis; TNF-alpha; TNFRs; TRAFs; Rat

1. Introduction

Tumor necrosis factor-alpha (TNF-alpha) is a multifunc-tional cytokine produced primarily by activated monocytes/macrophages; it plays a crucial role in the initiation and con-tinuation of inflammation and immunity [1]. This cytokine isinvolved in many cell processes including apoptotic cell death,metabolism, inflammation, thrombosis, and fibrinolysis [2].It is widely accepted that TNF-alpha plays a central role inmucosal inflammation and is likely to be a focal point of theinflammatory cascade in IBD [3,4].

TNF-alpha responses are mediated through binding toeither of its receptors, TNFRI or TNFRII. Both receptors arepresent on virtually all cells and can be down-regulated by

* Corresponding author. Tel.: C1 787 840 2575; fax: C1 787 841 1040.

E-mail address: cappleyard@psm.edu (C.B. Appleyard).

1043-4666/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cyto.2005.08.001

TNF-alpha in a time- and dose-dependent manner through in-ternalization and shedding [5]. Both TNF receptors can be pro-teolytically cleaved to yield soluble proteins that retain thecapacity to bind and release TNF-alpha [6]. Depending onthe concentration of soluble receptor and the biological systemstudied, the binding of TNF-alpha may either neutralize or in-crease its biological activity [1]; however, the exact physiolog-ical function of soluble TNFRs (sTNFRs) is not known. The55 kilodalton (kDa) receptor (also known as p55, TNFRI,CD120a) is widely expressed on many cell types. Its intracel-lular portion contains a ‘‘cell death domain’’, which is re-quired for the signaling of apoptosis and NF-kB activation[7]. Ligand binding to TNFRI can lead to either apoptotic orantiapoptotic cascades. Which pathway is activated dependson the recruitment of different cellular factors that bind tothe intracellular death domain [8,9]. The 75 kDa receptor(also known as p75, TNFRII, CD120b) is found predominantly

86 C. Rojas-Cartagena et al. / Cytokine 32 (2005) 85e93

on leukocytes and endothelial cells and lacks the cell death do-main [10]. Animal models of inflammatory diseases provideevidence of a physiological role for TNFRII since either up-or down-regulation of its expression can produce or exacerbateinflammation [11].

TNF-alpha has been hypothesized to be an important cyto-kine in the pathogenesis of inflammatory bowel disease (IBD).Several lines of evidence support this hypothesis: (i) anti-TNF-alpha monoclonal antibodies have produced beneficial re-sponses among some patients with Crohn’s disease [12]; (ii)deletion of elements from the 3# untranslated region of themouse TNF-alpha gene leads to transmural inflammation high-ly reminiscent of human Crohn’s disease [13]; and (iii) severalanimal models of IBD show significant amelioration of muco-sal inflammation when treated with anti-TNF-alpha antibodies[14]. Increased levels of sTNFRs have been reported in theurine of children with IBD [15], suggesting an importantrole for the receptors as indicators of a serious inflammatoryresponse. Recently it has been found that polymorphisms inthe TNFRII gene are associated with susceptibility to ulcera-tive colitis and Crohn’s disease [16]. Moreover, overexpres-sion of TNFRII has been reported in colonic epithelial cellsof IBD patients as well as in the lamina propia of animal mod-els of experimental colitis [17,18].

TNF receptor superfamily members interact with a familyof adaptor proteins referred to as tumor necrosis factor recep-tor-associated factors (TRAFs). To date, six distinct TRAFmolecules (TRAF1 to TRAF6) have been identified in mam-malian species [19]. The structural features of TRAF proteinssuggest that they function as cytoplasmic adaptors, promotingintracellular signal transduction and potentiating the recruit-ment of proteins, including each other, to a signaling complex.TRAF proteins can serve to modulate the ability of receptorsto trigger distinct signaling pathways that lead to activationof protein kinases and, subsequently, to the activation of vari-ous transcription factors including NF-kB, a key factor in in-testinal inflammation [20,21]. It has been postulated thatthree different variables could be involved in the differentialmodulation of the downstream events that either keep cellsalive or trigger apoptosis: differences in the expression levelsof TRAFs, the depletion of distinct TRAF molecules, or thenature of the multimerized receptor [19]. The expression ofthese factors might therefore change in association with in-flammation such as that seen in IBD.

TNF-alpha induces the expression of a wide variety of in-flammatory genes, including intracellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular endothelial growth factor(VEGF) [22,23]. During inflammation, ICAM-1 and E-selectinexpression is markedly upregulated, thereby contributing to theadhesion between lymphocytes and antigen presenting cells[23]. Enhanced expression of both has been reported in patientswith IBD [24]. In addition, it has been reported that patients withIBD experience an increase in VEGF and that TNF-alpha’s an-giogenic effects are mediated, in part, by modulating the VEGFsystem [25].

The central hypothesis of these studies is that TNF-alphaand TNF-alpha receptor expression and activities play a critical

role in IBD. To test this hypothesis, the expression of TNF-alpha, TNFRs, TRAFs, ICAM-1, E-selectin, VEGF, andVEGF receptors was determined in an acute model of colitis.

2. Materials and methods

The experiments reported herein were performed in accor-dance with the principles described in the ‘‘Guide for the Careand Use of Laboratory Animals’’, Publication No. DHMS(NIH) 86-23.

2.1. Animal model

Studies were performed with female SpragueeDawley ratsweighing 275e300 g (Southern Veterinary Service, PSM, PR).All animals were maintained in restricted-access rooms witha controlled temperature (23 �C) and a 12 h lightedark cycle.Standard laboratory chow and drinking water were providedad libitum. All experimental procedures involving animalswere approved by the Animal Care and Use Committee atPonce School of Medicine. Colitis was induced by the intraco-lonic administration of 2,4,6-trinitrobenzene sulfonic acid(TNBS, Fluka; 30 mg in 0.5 ml of 50% ethanol) as previouslydescribed [26]. Control animals (n Z 10) received 0.5 ml ofthe 50% ethanol vehicle under similar conditions. Rats wereeuthanized with 100 mg pentobarbital i.p., 72 h after TNBSadministration. Blood samples were taken by cardiac punctureand then centrifuged for 2 min (14,000 ! g); the plasma wasremoved and stored at �80 �C until analysis. Following lapa-rotomy, peritoneal fluid samples (200e500 ml) were obtainedfrom the abdominal cavity and stored at �80 �C until analysis.The number of white blood cells (WBC) in the peritoneal fluidwas determined microscopically (high power field !400).

2.2. Determination of macroscopic andmicroscopic damage

The whole colon was removed and examined for macro-scopic damage (ulceration, adhesions, colon thickness, and di-arrhea) using an established, well-defined scoring system [26].Segments of colon were fixed in 10% formalin. After routineprocessing, sections were stained with hematoxylin and eosinto determine the extent of inflammatory infiltration and the ap-pearance of the underlying muscle layers. Histological assess-ment of damage was performed using previously publishedcriteria [26]. Briefly, we evaluated the loss of mucosal archi-tecture, muscle thickness, crypt abscess formation, and gobletcell depletion.

2.3. Immunoassays

Total protein was extracted from the colon and smallintestine tissues after homogenization using Trizol Reagent,following the manufacturer’s specifications (Gibco BRL,

87C. Rojas-Cartagena et al. / Cytokine 32 (2005) 85e93

Gaithersburg, MD). To determine TNF-alpha levels in tissuehomogenates, and of TNF-alpha and sTNFR in peritoneal flu-id, aliquots of each were assayed by sandwich ELISA usingcommercial kits (R&D Systems, Minneapolis, MN), followingthe manufacturer’s protocol. The detection limit was less than5 pg/ml; intra- and inter-assay variations were less than 10%for TNF-alpha. For both sTNFRI and sTNFRII assays, thedetection limit was 5 pg/ml; intra- and inter-assay variationswere less than 8%.

2.4. Relative and quantitative gene expression

RNA was isolated using the Trizol protocol as describedabove. The quality of the extracted RNA was evaluated byformamide/agarose electrophoresis, and quantity was deter-mined by spectrophotometry at OD 260 in a GeneQuant�DNA/RNA calculator (Pharmacia�, Piscataway, NJ). Prior toRT-PCR, RNA from serum and peritoneal WBC was amplifiedas previously described using a commercial kit (Messa-geAmp�, Ambion�, Inc., Austin, TX) [27,28]. Quantitativereal time RT-PCR was conducted following the manufacturer’sspecifications using the QuantiTect� SYBR� Oregon Green�

RT-PCR kit (Qiagen�, Valencia, CA). PCR amplificationincluded a reverse transcription step (50 �C/20 min), an acti-vation step (95 �C/15 min), and 50 cycles of 94 �C/15 s,gene-specific annealing temperature/30 s, and 72 �C/1 min.Annealing temperatures were empirically determined foreach gene: 56 �C for TNF-alpha, 59 �C for TNFRI, and53 �C for TNFRII. Construction of standards for real timeRT-PCR was performed as described by Milligan and co-workers [29]. For gene-specific relative RT-PCR, first strandsynthesis was performed with the RETROscript� First StrandSynthesis Kit following the manufacturer’s specifications(Ambion�, Inc., Austin, TX). Briefly, 1 mg of total RNA wasincubated with random decamers for 3 min at 80 �C. After in-cubation, dNTPs, buffer, RNase inhibitor, and reverse tran-scriptase were added and the reaction incubated at 44 �C for1 h. This was followed by a 10 min incubation at 92 �C.The single-stranded cDNA obtained was amplified by PCRas described below. All reactions used 1.5 mM MgCl2, exceptfor E-selectin and VR2, for which 2.0 mM MgCl2 was usedand included 4 ml of 2:8 ratio primer/competimer for the18 s internal control. The primers and conditions used forthe amplification of TRAF1 [30], TRAF2 [31], TRAF3 [32],TRAF5 [21], TRAF6 [33], ICAM-1 and E-selectin [34],VEGF and its receptors VR1/VR2 [35] have been describedelsewhere. All primers were synthesized at the MolecularResource Facility, New Jersey Medical School, (Newark, NJ).

2.5. Statistical analysis

Data analysis was carried out using the SPSS Base 8.0.1statistical package (SPSS Inc., Chicago, IL, USA). Valuesare presented as the mean G SEM. Differences between themeans of each group were analyzed using the student’s

t-test. Correlations between variables were analyzed usingPearson’s correlation. Level of significance was fixed at 0.05for all statistical tests.

3. Results

3.1. Experimental rats showed significantly highermacroscopic and microscopic damage in thecolon and small intestine

Macroscopic damage was evaluated in the colon of theTNBS and vehicle (50% EtOH) treated controls. To determinethe extent of underlying tissue damage, histological prepara-tions were performed for colon and small intestine samples.TNBS rats exhibited significantly higher macroscopic colonicdamage (11.80G 0.67; p ! 0.0001) than the controls (3.62G0.38) (Fig. 1). The total microscopic damage (Table 1) wassignificantly increased in the colon (1.5-fold) ( p ! 0.05),and in the small intestine (1.6-fold) ( p ! 0.01) of the TNBSrats compared to the controls. The extent of disruption ofthe mucosal architecture was significantly worse in the colonand small intestine of the experimental rats than in the con-trols. Cellular infiltration as well as muscle thickeningdmarkers for inflammation and edemadwere also significantlyhigher in the colon of the TNBS rats ( p ! 0.05).

3.2. The number of white blood cells wassignificantly increased in the peritonealfluid of experimental rats

The number of white blood cells in the peritoneal fluid wasmeasured in order to determine the presence of peritonealinflammation in TNBS and controls; when the results werecompared, the TNBS rats showed a significantly higher num-ber of white blood cells (39.20 G 10.40 cells per high powerof view; p ! 0.05) than the controls (14.80 G 2.73) (Fig. 2).

TNBS0

5

10

15

***

Mac

rosc

opic

dam

age

scor

e

50 EtOH

Fig. 1. Macroscopic colonic damage score. The macroscopic colonic damage

was significantly increased in animals treated with TNBS. Values are means G

SEM of 10 animals per group. ***p ! 0.0001 between TNBS and 50%

EtOH.

88 C. Rojas-Cartagena et al. / Cytokine 32 (2005) 85e93

Table 1

Microscopic damage in the colon and small intestine

Criteria Mean score

Colon Intestine

50% EtOH TNBS 50% EtOH TNBS

Loss of mucosal architecture 1.30G 0.34 2.60G 0.16** 1.90G 0.23 2.60G 0.22*

Cellular infiltration 1.40G 0.30 2.30G 0.21* 1.40G 0.34 2.00G 0.26

Muscle thickening 1.10G 0.28 2.20G 0.29* 0.30G 0.21 0.90G 0.31

Goblet cell depletion 0.70G 0.15 0.40G 0.16 0.10G 0.00 0.30G 0.15

Crypt abscess formation 0.40G 0.16 0.00G 0.00 0.00G 0.00 0.30G 0.15

Total damage score 4.90G 0.86 7.50G 0.48* 3.70G 0.56 6.10G 0.46**

Values are meansG SEM, n Z 10 rats per group. Significance is the difference from the corresponding EtOH-treated group *p! 0.05; **p! 0.01.

3.3. Peritoneal fluid levels of TNF-alpha and bothsoluble receptors were higher in experimental animalsthan in the controls; plasma levels of sTNFRII weresignificantly correlated with colonic macroscopicdamage score

Peritoneal fluid and plasma samples were collected to de-termine the protein levels of TNF-alpha and its soluble recep-tors (Table 2). The levels of TNF-alpha in both plasma( p Z 0.055) and peritoneal fluid ( p ! 0.01) were higher inexperimental animals than in the controls (Table 2). Levelsof sTNFRI and sTNFRII in both plasma and peritoneal fluidof the experimental rats were found to be increased comparedwith those of the controls; however, only the difference inperitoneal fluid levels reached statistical significance( p ! 0.01). A significant correlation was found between theperitoneal fluid levels of TNF-alpha and sTNFRIIdbut notsTNFRIdof the experimental animals (r2 Z 0.87;p ! 0.001). In contrast, plasma levels of TNF-alpha andsTNFRIdbut not sTNFRIIdwere significantly correlated(r2 Z 0.54; p ! 0.05). Plasma levels of sTNFRII were signif-icantly correlated with both colonic macroscopic damagescore (r2 Z 0.85; p ! 0.001; Fig. 3A) and peritoneal levelsof sTNFRII (r2 Z 0.57; p ! 0.05; Fig. 3B), yet this was notthe case for sTNFRI.

TNBS0

10

20

30

40

50 *

No.

of

wbc

/hig

h po

wer

fie

ld o

f vi

ew

50 EtOH

Fig. 2. Number of white blood cells in the peritoneal fluid. The number of

white blood cells in the peritoneal fluid of experimental rats treated with

TNBS was increased. Values are means G SEM of 10 animals per group.

*p! 0.05 between TNBS and 50% EtOH.

3.4. Protein levels of TNF-alpha in the colon weresignificantly correlated with colonic macroscopicdamage in experimental rats

The colon and small intestine were examined in order todetermine the levels of TNF-alpha protein. It was foundthat the TNF-alpha protein level was significantly higher inboth the colon ( p ! 0.01; Fig. 4A) and small intestine( p ! 0.001; Fig. 4B) of the experimental animals than ofthe controls. A correlation was found between the TNF-alphaprotein levels in the colon and the colonic macroscopic dam-age in rats that had been treated with TNBS (r2 Z 0.55,p ! 0.05).

3.5. TNF-alpha and TNFRs gene expression weresignificantly increased in the colon of rats withexperimental colitis

The expression of TNF-alpha, TNFRI, and TNFRII was de-termined using quantitative real-time RT-PCR. The copy num-ber of mRNA for each gene was assessed in the colon, smallintestine, plasma, and peritoneal white blood cells (Fig. 5). Ex-pression of TNF-alpha was significantly increased only in thecolon ( p ! 0.05) of the TNBS rats and not in that of the con-trols; this correlated with its protein levels (r2 Z 0.46;p ! 0.05). The expression of TNFRI was significantly in-creased in the colon ( p ! 0.01) and small intestine( p ! 0.05) of the TNBS rats when compared to controls. Incontrast, the expression of TNFRII was significantly increasedonly in the colon ( p ! 0.05) of the experimental rats. No sig-nificant differences were found in the expression of TNF-alpha, TNFRI, and TNFRII in the plasma and peritoneal whiteblood cells of the TNBS animals compared to controls.

3.6. Gene expression of TRAF3 and TRAF5 wassignificantly increased in the colonic tissue ofanimals with experimental acute colitis

Gene-specific relative RT-PCR was performed in order todetermine the expression of TRAFs in both the colon and thesmall intestine. Though no significant differences were foundbetween experimental and control animals in the expression

89C. Rojas-Cartagena et al. / Cytokine 32 (2005) 85e93

Table 2

TNF-alpha and sTNFRs levels in plasma and peritoneal fluid

Plasma Peritoneal fluid

50% EtOH TNBS 50% EtOH TNBS

TNF-alpha 2.77G 0.99 8.34G 2.53 1.22G 0.44 3.00G 0.66*

sTNFRI 254.01G 224.95 264.01G 175.57 700.75G 284.63 1193.23G 357.47**

sTNFRII 621.32G 187.33 1402.00G 232.11 544.84G 65.31 2354.83G 535.05**

The levels of TNF-alpha and of its receptors are reported in pg/ml. Values are meansG SEM, nZ 10 rats per group. Significance is the difference from the

corresponding 50% EtOH-treated group. *p! 0.05; **p! 0.01.

of either TRAF1 or TRAF2 (Table 3), TRAF3 expressionwas found to have significantly increased in the colon ofthe TNBS treated rats ( p! 0.01) compared to 50% EtOH.TRAF5 expression was also found to be significantly in-creased in the colon ( p! 0.001) as well as in the small in-testine ( p! 0.05) of the experimental rats. TRAF6expression was not detected in either the colon or the smallintestine.

A

0 500 1000 1500 20000

10

20

Plasma sTNFRII (pg/ml)

Mac

rosc

opic

col

onic

dam

age

B

0 10000 20000 30000 400000

1000

2000

Peritoneal Fluid sTNFRII (pg/ml)

Pla

sma

sTN

FR

II (

pg/m

l)

Fig. 3. Correlation between plasma sTNFRII and (A) macroscopic colonic

damage score or (B) peritoneal fluid levels of sTNFRII. Significant correla-

tions were found between plasma levels of sTNFRII and macroscopic colonic

damage (r2 Z 0.85; p ! 0.001) or peritoneal fluid levels of sTNFRII

(r2 Z 0.57; p ! 0.05) in the TNBS animals.

3.7. The expression of the TNF-alpha inducible genesICAM-1, E-selectin, and VR1 was increased in thecolonic tissue of animals with experimental acute colitis

Gene specific relative RT-PCR was performed in order todetermine the expression of ICAM-1, E-selectin, VEGF, andthe VEGF receptors (VR1 and VR2) in the colon and small in-testine. The expression of ICAM was significantly increased inthe colon ( p ! 0.05) but not in the small intestine of experi-mental rats compared with the controls (Table 4). Likewise, E-selectin expression was found to be significantly increased inthe colon ( p ! 0.05) but not in the small intestine of the

A

TNBS

TNBS

0

10

20

30

40

50

*

TN

F-a

lpha

(pg

/ml)

TN

F-a

lpha

(pg

/ml)

B

0

10

20

30

40

50

**

50 EtOH

50 EtOH

Fig. 4. TNF-alpha protein levels in colon (A) and small intestine tissue (B).

The protein levels of TNF-alpha in the colon and small intestine of experimen-

tal rats treated with TNBS was increased. Values are meansG SEM of 10

animals per group. *p! 0.01 **p! 0.001 between TNBS and 50% EtOH.

90 C. Rojas-Cartagena et al. / Cytokine 32 (2005) 85e93

TNBS rats. In terms of the expression of VEGF, there was nodifference between TNBS rats and controls in either the colonor small intestine. However, in the colon of the experimentalrats the expression of VR1 was significantly increased( p! 0.01), whereas that of VR2 was significantly decreased( p! 0.01; Table 4). No differences in the expression of eitherVR1 or VR2 were observed in the small intestine.

4. Discussion

TNF-alpha plays a central role in mucosal inflammation;quite likely it is a focal point of the inflammatory cascade inIBD [3,4]. Systemic inhibition of soluble TNF-alpha by

Colon Intestine Serum PF0.0

0.1

0.2

510152025

*

TN

F(#

cop

ies

x 10

10/µ

g to

tal R

NA

) T

NF

RI

(# c

opie

s x

1010

/µg

tota

l RN

A)

TN

FR

II(#

cop

ies

x 10

10/µ

g to

tal R

NA

)

Colon Intestine Serum PF

Colon Intestine Serum PF

0.00

0.02

0.04

0.06

100

300

500

700

**

*

0

5

10

100

300

500

700*

Fig. 5. TNF-alpha and TNFR gene expression in colon, intestine, serum and

peritoneal fluid. The expression of TNF-alpha, TNFRI and TNFRII was signif-

icantly increased in the colon of experimental rats treated with TNBS. Values

are means G SEM of 10 animals per group. *p ! 0.05 **p ! 0.01 between

TNBS (solid bars) and 50% EtOH (hatched bars).

chimeric anti-TNF-alpha mAbs (Infliximab) has been shownto induce remission in up to 50% of patients with Crohn’s dis-ease and to significantly improve clinical symptoms in mostpatients [36]. TNF-alpha responsesdincluding apoptosis,cell survival, and inflammatory responsesdare mediatedthrough binding to either of its receptors, TNFRI or TNFRII,which have been shown to have functional differences depend-ing on the target cell and their relative concentrations at thesite of action [9]. While it is well established that TNF-alphais a key factor in the pathogenesis of Crohn’s disease, the roleplayed by its two receptors in this condition is still unknown.Although TNF and its receptors have been studied in vitrothere is little information available from in vivo experimentswhich would lead to an increased understanding of the diseasepathogenesis [37]. The aim of this study was to better definethis role by looking at the differential expression of TNF-alphaand its receptors in an animal model of acute IBD.

Rats with experimental acute colitis had high colonic dam-age scores as well as increased numbers of peritoneal fluidwhite blood cells. This indicates the presence of an active in-flammatory process, not only in the colon, but also in the peri-toneal environment of the experimental animals. As expected,the experimental animals had higher peritoneal fluid andplasma TNF-alpha levels than control animals (3-fold and2.5-fold, respectively), although this difference was only sig-nificant in peritoneal fluid. This observation is in agreementwith other studies in which no significant increase in circulat-ing TNF-alpha concentration was found in animals with exper-imental colitis [38,39], and a very recent study examiningintraperitoneal cytokine production in patients with IBD[40]. Gene expression levels of TNF-alpha in peritoneal fluidand plasma white blood cells did not differ significantly be-tween experimental animals and controls. Thus, it appearsthat TNF-alpha is released locally in the colon, where it exertsa paracrine effect. It can be concluded, therefore, that circulat-ing levels of TNF-alpha are a poor indicator of its biologicaleffects in IBD. Indeed, our study shows that animals with ex-perimental acute colitis had increased protein levels of TNF-alpha in the colon and small intestine compared to controls,providing additional support to the notion that the gastrointes-tinal tract is the most important source of this cytokine. Wefurther observed that TNF-alpha mRNA expression in thecolon of the experimental rats was 100-fold higher than incontrols and that TNF-alpha protein and mRNA levels weresignificantly correlated. Increased expression of TNF-alphamRNA has been shown in biopsy specimens obtained fromIBD patients [41]. Overexpression of TNF-alpha is known toaugment the expression of its gene in gastrointestinal cellsand leads to a positive regulatory loop that amplifies its effectson the colon. As a result, the inflammatory process is perpet-uated and extensive tissue damage may occur. We observeda positive correlation, in fact, between protein levels ofTNF-alpha in the colon and macroscopic damage scores thatstrongly indicates that TNF-alpha overproduction can explain,in part, the augmented damage seen in the tissues of animalswith acute colitis. This damage may result from excessiveproduction of matrix degrading enzymes, loss of mucosal

91C. Rojas-Cartagena et al. / Cytokine 32 (2005) 85e93

Table 3

TRAFs expression in the colon and small intestine

50% EtOH TNBS

Colon Intestine Colon Intestine

TRAF1 0.32G 0.02 0.40G 0.02 0.39G 0.04 0.48G 0.04

TRAF2 0.59G 0.05 0.83G 0.10 0.56G 0.10 0.78G 0.08

TRAF3 0.78G 0.12 0.27G 0.04 2.16G 0.32** 0.35G 0.07

TRAF5 1.10G 0.10 1.52G 0.17 1.89G 0.15*** 3.2G 0.69*

TRAF6 N/D N/D N/D N/D

The levels of the TNF receptor adaptor factors (TRAFs) are reported as OD per mg total RNA. Samples were normalized against 18 s. Values are meansG SEM of

10 animals per group. *p! 0.05; **p ! 0.01; ***p! 0.001 between 50% EtOH and TNBS rats. N/D, not detected.

integrity and ulceration, and activation of a variety of immuneand non-immune cells due to overproduction of inflammatorycytokines (including TNF-alpha) [42]. Overexpression ofTNF-alpha can also contribute to the worsening of ulcerationby downregulating trefoil factor, a key protein in woundhealing [43].

To our knowledge, we are the first to measure the levels ofsoluble TNF-alpha receptors in the peritoneal fluid of an ani-mal model of IBD. The peritoneal fluid levels of sTNFRI andsTNFRII were increased 1.7-fold and 4.3-fold, respectively, inrats with acute colitis as compared to controls. Soluble TNF-alpha receptors are derived from cleavage of cell surfaceproteins and are thought to play a key physiological role bycontrolling the actions of TNF-alpha in the host during periodsof excessive cytokine production [8,10,13]. For instance, solu-ble receptors have been shown to either inactivate or prolongthe action of TNF-alpha by blocking its bioactivity throughbinding, or by providing a reservoir from which small amountsof TNF-alpha are released [44]. Which activity is exerted isdetermined in part by the local concentration of TNF-alphaand the soluble receptors. Based on our observations, it canbe concluded that the increased levels of sTNFRs in the peri-toneal fluid of the experimental rats help to establish a reser-voir of TNF-alpha, which may lead to the exacerbation ofthe damage seen in the colon. By promoting a slow releaseof TNF-alpha, sTNFRs may prolong their detrimental effectsand disrupt the normal homeostasis of the colon. Thus, the in-creased levels of sTNFRs in the peritoneal fluid of animalswith acute colitis suggest that an up-regulation of sTNFRs inthe peritoneal environment may be taking place in the earlystages of the development of IBD and play an important rolefor the TNF-alpha system in the initiation phase of the disease.

Peritoneal fluid levels of sTNFRII were positively correlatedto both TNF-alpha peritoneal fluid levels and to sTNFRIIplasma levels, suggesting that the increased shedding ofTNFRII is caused by the overexpression of TNF-alpha. More-over, plasma levels of sTNFRII and macroscopic colonic dam-age were significantly correlated, indicating that circulatingsTNFRII may be associated with colonic alterations in this an-imal model. This observation offers the intriguing possibilityof using sTNFRII plasma levels as a marker for colonic dam-age. The levels of sTNFRI in the peritoneal fluid, on the otherhand, did not correlate with those of TNF-alpha, probably dueto the fact that TNFRI can be subject to internalization as wellas shedding [5].

Gene expression of both TNFRI and TNFRII was signifi-cantly increased in the colon of the experimental animals ascompared with controls (3.4-fold and 68-fold, respectively).The substantial difference in TNFRII expression observed inthis model of acute IBD suggests that this receptor is playingan important role in the initiation of the disease. It is interest-ing to note that TNF-alpha is a strong inducer of TNFRII ex-pression in various cell types [45] and that the overexpressionof TNFRII potentiates TNF-alpha induced apoptosis [46]. Ourobservations are supported by reports of TNFRII overexpres-sion in the colonic epithelial cells of patients with Crohn’s dis-ease and in a murine model of chronic intestinal inflammation,with TNFRII levels being proportional to the severity of colitis[18]. Moreover, polymorphisms in the TNFRII gene have beenfound in patients with Crohn’s disease, and these polymor-phisms appear to contribute to an increased risk of the disease[16]. Recent studies carried out on the rodent model of TNBScolitis showed that TNFR2�/� mice had improved survival,which indicates that signaling through TNFR2 promotes the

Table 4

ICAM-1, E-selectin, VEGF, VR1, and VR2 expression in the colon and small intestine

50% EtOH TNBS

Colon Intestine Colon Intestine

ICAM-1 0.89G 0.06 0.83G 0.16 2.06G 0.33* 1.50G 0.40

E-selectin 0.47G 0.05 0.84G 0.12 1.87G 0.28* 1.11G 0.18

VEGF 0.47G 0.04 0.37G 0.04 0.63G 0.10y 0.54G 0.09

VR1 0.66G 0.14 0.91G 0.21 1.93G 0.32** 1.15G 0.37

VR2 0.41G 0.03 0.50G 0.04 0.24G 0.02* 0.44G 0.03

The levels of ICAM-1, E-selectin, VEGF, VR1, and VR2 are reported as OD per mg total RNA. Samples were normalized against 18 s. Values are meansG SEM

of 10 animals per group. *p! 0.05; **p! 0.01; ypZ 0.09 between 50% EtOH and TNBS rats.

92 C. Rojas-Cartagena et al. / Cytokine 32 (2005) 85e93

disease process, although the specific mechanisms involved re-main undefined [47]. Overexpression of TNF-alpha and of itsreceptorsdespecially TNFRIIdmay make the colonic tissuemore sensitive to the cytotoxic effects of this cytokine, eitherby exacerbating the immune response or by augmenting pro-grammed cell death in the colon. In addition, the sustainedproduction of TNF-alpha can result in chronic activation of in-flammation and tissue damage. Consequently, the increasedproduction of TNF-alpha and of its receptors in the colonmay play an important role in cell death and tissue damage,which makes them possible therapeutic targets for IBD.

TRAFs were originally identified as signal transducingmolecules for the TNF receptor superfamily members [19].Recently, it has been recognized that these cytoplasmic adap-tors differentially modulate the downstream events that cankeep cells alive or trigger apoptosis [48]. We determined thegene expression levels of all TRAFs (except TRAF4) in thecolon and small intestine of experimental rats and controlsand found that TRAF5 was overexpressed in the colon andthe small intestine of the experimental rats, while TRAF3was overexpressed only in the colon. No significant differen-ces were found in the expression of TRAF1 or TRAF2, andTRAF6 expression was not detected at all. TRAF5 has beenreported to be involved in the activation of NF-kB throughTNFRs [21] and can partially substitute for TRAF2 in the re-cruitment of I-kB kinases [49]. However, mice deficient inTRAF5 did not present defects in TNF-alpha induced NF-kB activation [50]. This suggests that TRAF5 is either notessential or plays a redundant role in NF-kB activation by TNF-alpha. At minimum, it would seem that the overexpression ofTRAF5 is enough to induce the expression of adhesion mole-cules and other inflammatory factors that contribute to thepathogenesis of IBD. This hypothesis is supported in part bythe overexpression of ICAM-1 and E-selectin observed inthe colon of the experimental rats. Interestingly, these twomolecules are known to be induced in response to TNF-alphathrough NF-kB activation [22,23]. TRAF3, on the other hand,is found broadly expressed in all tissues. The domain organi-zation of TRAF3 is similar to TRAF2 and TRAF5, but itsoverexpression does not activate NF-kB; rather, TRAF3 canblock TRAF2-mediated activation of NF-kB [20]. It hasbeen shown that T cells isolated from TRAF3-deficient miceare impaired in their ability to respond to antigens; thus,TRAF3 appears to be important in cellular activation andsurvival.

Based on these findings it is tempting to speculate that theoverexpression of TRAF3 in this model may induce cell deathmechanisms and promote an exacerbated activation and sur-vival of T cells, which may explain the inflammation and tis-sue damage observed. Taken together, these data suggest thatthe overall outcome of the TNF/TNFRs signaling pathway inthe colon of animals with acute colitis favors cell death andtissue damage; damage that comes as a result of the over-expression of TNF-alpha, its receptors, and TRAF3. Theseresults may however be specific to this model of colitis duringthe early stages of the inflammatory process. In other experi-mental models the receptors may play more divergent roles,

as has been demonstrated by the ability of the TNF/TNFR2signaling cascade to elicit anti-apoptotic effects in a knockoutmurine model of chronic colitis [18,37]. The specific mecha-nisms underlying TNFRs overexpression and TRAFs regula-tion therefore still require further investigation.

To conclude, the increased expression of TNF-alpha,TNFRs, and the associated signaling factors in the colon ofthis rat model of IBD provides further evidence of theirinvolvement in the promotion of inflammation and tissuedamage. Also, increased levels of sTNFRsdparticularlysTNFRIIdin the peritoneal fluid of experimental rats maybe involved in the development of colitis by serving as a reser-voir of TNF-alpha. An intriguing possibility suggested by ourdata is that sTNFRII levels in plasma may serve as a markerfor colonic damage as well as a novel therapeutic target forthe treatment of IBD.

Acknowledgements

The authors wish to acknowledge the technical assistanceof Olga I. Santiago, Cariluz Santiago, and Jainarine Lalla.Thanks also go to the RCMI Publications office and theMolecular Biology Core, supported by RCMI Grant # 2 G12RR03050-18. These studies were supported by NIGMS F31-GM68392 (C.R.C.) and S06-GM08239 (C.B.A. and I.F.)from the National Institutes of Health.

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