Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats

GASTROENTEROLOGY

1994;106:960-972

Tissue Interleukin 1 and Interleukin-1 Receptor Antagonist Expression in Enterocolitis in Resistant and Susceptible Rats ROBERT ROBERT

D. MCCALL,* STEPHEN HASKILL, t5 ELLEN M. ZIMMERMANN,* C. THOMPSON,” and R. BALFOUR SARTOR*!§

P. KAY LUND,”

Departments of *Internal Medicine, ?Obstetrics and Gynecology, 5Microbiology and Immunology, and ttPhysiology and Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina: and ‘Synergen, Inc., Boulder, Colorado

Background/Aims: Subserosal injection of purified group A streptococcal peptidoglycan-polysaccharide (PG-APS) induces chronic relapsing granulomatous enterocolitis and systemic inflammation in susceptible inbred Lewis rats but only transient intestinal injury in Buffalo and Fischer rats. Cecal interleukin 1 (11-l) and 11-l receptor antagonist (IL-lra) expression was measured in inbred rats displaying differential susceptibility to experimental enterocolitis. Methods: The ileum and cecum of Lewis, Buffalo, and Fischer rats were subserosally injected with purified PG-APS or albumin. 11-l and Il-lra messenger RNA (mRNA) and protein (11-l only) were measured 1 or 27 days later. PG-APS-injected Lewis rats were treated with recombinant human ILlra. Kinetics of 11-l and Il-lra mRNA expression were studied in peritoneal cells. Results: All rat strains developed acute inflammation with increased cecal concentrations of Il-lp and Il-lra mRNA. Lewis rats developed chronic enterocolitis and had higher 11-l and Il-lra mRNA tissue levels than Buffalo or Fischer rats, which displayed no chronic inflammation. Il-lp and Il-lra were produced by submucosal granulomas and correlated with inflammation. IL-la protein levels paralleled Il-lp mRNA expression. Il-lra treatment attenuated acute and chronic enterocolitis, adhesions, and arthritis. PG-APS induced IL-1 and Il-lra expression in peritoneal cells from Lewis and Fischer rats. Conclusions: Bacterial cell wall polymers stimulate 11-l and Il-lra expression in vivo and in vitro. These counterbalancing cytokines are increased in experimental enterocolitis and have important immunoregulatory roles in intestinal inflammation.

e

have developed a rat model of chronic relapsing granulomatous enterocolitis characterized by spontaneously relapsing inflammation, differential susceptibility in inbred rat strains, and extraintestinal manifestations, which make it a valuable model of inflammatory bowel disease.‘-i Sterile sonicated group A streptococcal peptidoglycan-polysaccharide (PG-APS), a bacterial cell wall polymer, is injected subserosally (intramu-

W

rally) into the distal inflammation

small

later in all rat strains diminishes

intestine

and cecum.

studied

Acute

24 hours

can be seen at the site of injection

to date; this inflammation

over several days. In susceptible

Lewis rats,

the quiescent phase is followed by a relapsing, chronic, and diffuse, granulomatous enterocolitis that develops in the distal small intestine and cecum approximately 2 weeks after PG-APS

injection.

terocolitis is accompanied tion, including peripheral anemia, model

The chronic

phase of en-

by extraintestinal arthritis, hepatic

inflammagranulomas,

and leukocytosis.2.3 is the differential

An important feature of this genetic susceptibility of inbred

rat strains. Lewis rats develop acute and chronic relapsing enterocolitis, develop

whereas

degrees

Lewis rats, but exhibit tous enterocolitis

Buffalo

of acute

and Fischer

inflammation

strains to

no evidence of chronic granuloma-

or extraintestinal

ence 2 and unpublished

F344

equivalent

inflammation

(refer-

data, R. B. Sartor).

Lewis rats are known to be susceptible to a number of chronic inflammatory conditions including experimental autoimmune APS-induced

encephalitis4; adjuvant, collagen, and PGarthritis>; and PG-APS-induced chronic relapsing enterocolitis.’ The mechanism for enhanced susceptibility of Lewis rats to these and other inflammatory processes has not been established, but two hypotheses have been advanced. First, Lewis rats have a defect in the acute hypothalamic/pituitary/adrenal response to PG-APS or interleukin 1 (IL-l) stimulation compared with Fischer F344 rats.’ Second, Lewis rats have increased production of IL-1 by PG-APS-stimulated macrophages compared with the Buffalo strain.’ Abbreviations used in this paper: DEPC, diethyl pyrocarbonate; ELAM-I, endothelial cell adhesion molecule I; HBBS, Hank’s balanced saline solution; HSA, human serum albumin; ICAM-I, intercellular cell adhesion molecule I; IL-l, interleukin I; IL-lra, interleukin-l receptor antagonist; PG-APS, group A streptococcal peptidoglycan-polysaccharide; rhll-lra, recombinant human IL-lra; RNase, ribonuclease; SDS, sodium dodecyl sulfate; TNFa, tumor necrosis factor a. 0 1994 by the American Gastroenterological Association 0016-5085/94/$3.00

April 1994

IL-1 AND IL-lra

IL-l@ and p are key proinflammatory

cytokines

syn-

thesized by immune, mesenchymal, and endothelial cells when activated by bacterial products, cytokines, or cell adhesi0n.a

IL-l has a number

ties relevant

of proinflammatory

to inflammatory

munoregulatory

cytokine

noids, cytokines,

and growth

induces

secretion

of eicosa-

factors by a variety

types as well as in the perfused lates proliferation

proper-

bowel disease.899 This im-

intestine.98’0

and differentiation

IL-l

of cell stimu-

of T and B lympho-

increasing

expression

of endothelial

sion molecule molecule

1 (ELAM-1)

1 (ICAM-l),

chemotaxis.“a’2 (mRNA)

and stimulation

colitis

RNA

and correlate in exper-

colitis.”

IL-l receptor antagonist (IL-lra) is a 22-kilodalton glycoprotein that binds to type I and type II IL-l receptors but has no agonist

activity.

tions

downregulator

as an endogenous

by blocking

Therefore,

IL-lra

func-

of inflammation

the effects of IL-1.‘9-22 A variety of stimuli,

including lipopolysaccharide and adherent immunoglobulin G, stimulate IL-lra production.21X23X24 Treatment with recombinant human IL-lra (rh IL-lra) has been found to attenuate a number of inflammatory processes, including experimental colitis,” thus showing the importance response

of IL-l as a key mediator of the inflammatory and the potential role of endogenous IL-lra as

an inhibitor

of IL-l

bioactivity.

Immunohistochemical

studies

have shown

both

IL-1

from Mycobacterium tuberculosissarcoidosis, foreign bodies,25726 and

and IL- 1 ra in granulomas induced

infection,

rabbit lung granulomas.27 from patients with active

active in rat inflammation

enabled us to ask the following

questions. (1) Can the ubiquitous bacterial cell wall polymer PG-APS induce production of IL-lp and IL-lra? (2) What is the relative tissue abundance of IL-lp and ILlra mRNA

during

granulomatous

acute and chronic

enterocolitis?

phases of relapsing

(3) Does the relative

abun-

Which

messenger

they are also increased

The

cell adhe-

cell adhesion

in tissues of patients

disease and ulcerative

of Lewis rats to chronic inflammation.

recent availability of specific rat complementary DNA (cDNA) probes and the demonstration that rhIL-lra is

molecules

of IL-g-mediated

of IL-l

are increased

with disease activity13-17; imental

adhesion endothelial

and intercellular

Concentrations

and protein

with Crohn’s

including

961

dance of mRNA for these key proinflammatory and antiinflammatory cytokines correlate with genetic susceptibility to chronic enterocolitis in inbred rat strains? (4)

cytes by inducing IL-2, IL-6, and other cytokines. IL1 promotes the recruitment of inflammatory cells by and effector cell ligands,

predisposition

IN RAT ENTEROCOLITIS

In colonic biopsy specimens intestinal inflammation, our

group showed IL-l and IL-lra mRNA.‘7X28 IL-lra expression is deficient in unstimulated leukemic cells2” and is expressed preferentially in the intracellular form in endometrial cancer tissues. 3o The ratio of intracellular IL-lra to IL-la proteins is significantly increased in psoriatic skin.31 Because IL-l and IL-lra competitively bind to the same receptors, an imbalance in the relative amounts of these two peptides may be important in the perpetuation of inflammatory processes. The goals of the present study were to investigate the expression and biological significance of IL-l and IL-lra in acute and chronic phases of experimental enterocolitis and to determine whether differences in expression of these competing cytokines are responsible for the genetic

cells synthesize

inflammatory

model?

IL-1 and IL-lra

mRNA

And (5) can IL-lra

inhibit

in this acute

and chronic phases of granulomatous enterocolitis? To address these questions, we first studied the effect of in vitro PG-APS on rat peritoneal macrophages. We then measured cecal levels of IL-lfl and IL-lra mRNA in susceptible and nonsusceptible rat strains during acute and chronic phases of experimental enterocolitis and examined correlations between mRNA abundance and inflammatory scores. We measured IL-la protein levels in the same tissues. In situ hybridization studies localized sites of mRNA

expression.

Finally,

we treated PG-APS-

injected Lewis rats with subcutaneous injections of rhILlra and observed their effects on gross and histological inflammation.

Materials and Methods Rats Female, inbred specific pathogen-free

rats weighing

135- 150 g were obtained from Charles River (Raleigh, NC;

Lewis and Fischer strain) and Harland (Blacksburg, VA; Buffalo). Rats were fed Agway Prolab rat chow (Agway Inc., Syracuse, NY) ad libitum. All rat experiments were conducted in accord with the highest standards of humane animal care as outlined in the National Institutes of Health’s Guidefor the Care and Use of Laboratmy Animals and approved by the University of North Carolina Institutional Animal Care and Use Committee.

PG-APS Purified, sterile PG-APS fragments from the cell walls of group A, type 3, strain D58 streptococci (Streptococcus pyogenes) were prepared as described previouslyS and provided by Dr. John Schwab (Department of Immunology and Microbiology, University of North Carolina, Chapel Hill). Sonicated cell wall fragments prepared by this method have a heterogeneous spectrum of molecular weights ranging from 5 X 10” to 5 X 1O8.32 The final PG-APS concentration was calculated based on rhamnose

content.’

In Vitro Peritoneal Macrophage Incubations Six female Lewis and six Fischer rats were killed by an overdose of CO, inhalation. Each peritoneal cavity was washed

962

MCCALL ET AL.

with

a total

Mg*+-free

GASTROENTEROLOGY Vol. 106, No. 4

of 50 mL of heparinited

Hanks

balanced

(2 U/mL)

saline solution

(HBSS). Cells, 3 X

106/mL, were kept at 4°C until the addition bility

and purity

for 10 minutes

and resuspended

in Dulbecco’s

containing

autologous

mg/mL),

glutamine

(4 mmol/L),

Cells were incubated

(10 pg/mL).

guanidinium

modified

(0.1

At each time point and immediately

thiocyanate

flammation

(0, 1, 2, 4, and lysed with 4.0

(GIBCO,

MD; pH 7) and frozen for later mRNA

were fixed in formalin,

with

sections

mRNA studies. pathogen-free anesthetized

histological

20 VlOO

g by intramuscular

Moore Inc., Washington subserosally

Crossing, NJ), and intes-

using aseptic technique.

with PG-APS

Rats

(total dose, 37.5 pg/g

body wt) divided into seven subserosal injection sites in the ileal identical injections of 37.5 kg/g human serum albumin (HSA) (Baxter Health Care Corp., Glendale, CA). The rats in the acutephase group were killed 24 hours after injection. The rats in the group were killed 27 days after injection.

were killed by overdose inhalation rhlL-lra

treatment

All rats

of 100% CO*.

studies.

Female

Lewis rats were

injected subserosally with PG-APS using the protocol detailed above, then treated with either rhIL-lra (Synergen, Boulder, CO) in 0.3 mL phosphate-buffered PBS alone administered

subcutaneously.

rats per group were examined study,

and 2 mglkg intravenously) ately before PG-APS

were assigned

in the external

for both

described’

of O-4

(4 being

acute and chronic

and internal

were totaled.

inflammatory

study and 10 or 11

(8 mg/kg

experiment.

hours after PG-APS injection.

injection,

the chronic-phase

study,

subcutaneously

rhIL-lra

4 and 10

per dose), then every 12

(8 mg/kg

injection.

In

per dose) or PBS

every 12 hours beginning

8

days after intestinal PG-APS injection and continued until rats were killed 15 days after PG-APS injection. Assessment of inflammation

Cincinnati, (Beckman 20°C

surfaces

at cross

The maximum

possible

score was 32.

was performed

by a blinded

as outlined below. Joint diameters were measured with calipers as previously described.’

in 2 mL of 4 mol/L guanidinium a tissue

observer

in duplicate

Assessment of Inflammation Cardiac blood was obtained for cell counts. Gross inflammation was scored by a single blinded observer (R.D.M. for mRNA studies, R.B.S. for treatment experiments) using a modification of a previously described method.’ Values of O4 (4 being the most severe) were assigned to (1) the number

homogenizer

OH) and centrifuged Instruments

for 18 hours

treated

through

acetate,

in sterile

in a Beckman

cesium

DEPC-treated

SW50.1

CA) at 36,000 chloride

water

RNA

and quantified

rotor ‘pm at

cushions.

in 70% ethanol

pH 5.2. Precipitated

spectrophotometry

thiocyanate Tissuemizer,

in diethyl pyrocarbonate

water and precipitated

sodium

(Tekmar

Co., Fullerton,

RNA pellet was resuspended

The

(DEPC)-

and 0.3 mol/L

was resuspended by ultraviolet

(A2601280).

Northern Blots Samples

consisting

ceca of PG-APS-

during

(8 mg/

For the next 24 hours, injections

hours until rats were killed 3 days after PG-APS

with

identical

immedi-

then rhIL-lra

subcutaneously

were made every 8 hours (8 mg/kg

was administered

(GIBCO)

RNA

of 10 pg of RNA

or HSA-injected

in 1.2% formaldehyde

subcutaneously

or PBS were administered

intestinal

for 30 seconds

Five rats per treat-

in the chronic-phase

rhIL-lra

kg per dose) or PBS was injected

intestinal

score of in-

Total tissue RNA was prepared using a standard extechnique. 33,34Frozen cecal tissues were homogenized

traction

saline (PBS) (pH 7.4) or

ment group were used in the acute-phase In the acute-phase

A histological Values

samples

and sectioned

RNA Preparation

injec-

Peyer’s patches, terminal ileum, and colon.’ Control rats received

chronic-phase

in paraffin,

Groups of four to seven female, specific

tines were exposed by laparotomy were injected

(H&E).

and kept at

Other

of mid cecum and cecal tip. The acute and chronic

inbred Lewis, Buffalo, and Fischer F344 rats were (Innovar-Vet,

tion; Pitman

embedded

16. Cecal and

nitrogen

analysis.

modifications.

scores for each section

Animal and Treatment Protocols

and protein

staining

severe)

inflammation

Gaithersburg,

analysis.

in liquid

was made for each animal as previously

the following

the most

of cecal bowel wall

gross gut score being

for later mRNA

for histochemical

at 37°C in the presence

(3)

The “gross gut score” is the sum of these values, possible

and (4) extent

mesentery,

thickening.

-70°C

(IO pg/mL).

of contracted

of adhesions,

liver tissues were snap-frozen

Eagle

gentamicin

(2) the severity

the severity the maximum

at 1500 ‘pm

and polymyxin

nonadherently

8 hours), cells were pelleted mmol/L

sera (2.5%),

Via-

microscopically

blue. The cells were pelleted

medium

of PG-APS

of PG-APS.

of the cells were confirmed

using 4% trypan

of cecal nodules,

Ca*+- and

acute-phase

from

gels.35*36 On each blot, an aliquot

preparation

a Buffalo rat during

extracted

rats were electrophoresed

from

inflammation chronic-phase

the cecum

of

of a Lewis rat

and one from the cecum of were used as internal

stan-

dards. Ethidium bromide staining of gels confirmed equivalent amounts of 18s and 28s ribosomal RNA per lane and lack of RNA degradation.

The gel was photographed

using 665 nega-

tive film (Polaroid, Cambridge, MA). After electrophoresis, RNA was transferred to nylon membranes (Nytran, 0.45~pm; Schleicher

& Schuell, Keene, NH) by blotting

with 10X SSPE

(saline, sodium phosphate, and ethylenediaminetetraacetic acid) for 18 hours. RNA was fixed to the moist membrane with a UV Crosslinker

(Stratagene

cat. no. 400071;

La Jolla,

CA) set for 1200 J at 254 nm. Membranes were hybridized to [32P]deoxycytidine triphosphate-labeled cDNA probes (Random Primed DNA Labeling Kit; Boehringer Mannheim, Indianapolis, IN) encoding rat IL-lp (200 nucleotides, a generous gift of Dr. Alan Shaw, Glaxo Institute of Molecular Biology, Geneva; current address, Merck, Sharp and Dohme, West Point, PA) and rat IL-lra (237 nucleotides3’; a generous gift of Dr. Stephen Eisenberg, Synergen). Hybridizations were performed in 50% formamide, 5X SSPE, 5X Denhardt’s reagent,

IL-1 AND

April 1994

sperm

DNA

at 42°C for 18 hours.

washes were carried

to a stringency

SDS at 42°C. Filters (Kodak,

Rochester,

were exposed

screen. Autoradiographs era model

4810;

autoradiographs

were imaged

COHU,

was performed

1.49; National

version calibrated

by densitometry

Institutes

of Health,

was normalized ribosomal

of IL-lp-

MD)

Rochester, mRNAs

staining

for minor variations sample was included

each blot,

we were able to control

for variations

intensities

across blots that arose as a result of differences activities mRNA

or exposure sample

units relative to the signal intensity phase internal

times.

on

in signal in

Signal intensity

was expressed

figure 1. Nonelicited peritoneal cells (3 X 106/mL) were incubated nonadherently in the presence of the bacterial polymer PG-APS (10 pg/mL). Northern blots of RNA extracted from the cells were probed with cDNA probes for IL-la and IL-lra.

in

posi-

tive (acute Lewis cecal RNA) control

probe-specific

IL-1 ra

of the 18s

of RNA loaded per sample. Because an identical

each specific

IL-1 1:

of

Image

Bethesda,

Kodak,

bromide

band in each lane to control

amount

film cam-

(NIH

and IL-lra-specific

to the ethidium

01248hr

intensifier

CA). Quantitation

to a step tablet (no. 903ST258;

NY). The abundance

X-OMAT

a single

using a solid-state

San Diego,

Fist her

01248hr

SSPE and 0.1%

to Kodak with

963

Posthybridization

of 0.2X

NY) at -8O’C

IN RAT ENTEROCOLITIS

Lewis

0.1% sodium dodecyl sulfate (SDS), and 100 pg/mL denatured salmon

IL-lra

of

as densitometry

of the corresponding

acute-

standard.

containing

sodium

azide (0.05%).

L phenylmetholsulfonyl antiproteases

antipain,

Homogenates

fluoride, aprotinin,

tative rat IL-lb

protein

80 (l%),

1 pg/mL

leupeptin,

were kept at -8O’C

the radioimmunoassay

Tween and

2 mmol/

each of the

and pepstatin

A.

until assay. Because quanti-

assays are not yet available,

kit for rat IL-la

Boston, MA) to measure duplicate

(Cytokine

2: 1 dilutions

we used Systems,

of the homoge-

nate.

In Situ Hybridization Statistics In situ hybridization ing a previously APS-

histochemistry

described

technique.‘”

and HSA-injected

pound,

frozen

Tissue

sections,

subbed

slides and kept

acetic

at -80”.

Sections

K (1 pg/jtL)

anhydride

were dehydrated

through

were synthesized (Promega labeled

rats was imbedded

hybridization containing

pGEM

75% formamide

vectors

riboprobes

were prepared

by in

or SP6 polymerase.

was a standard

hybridization

The

buffer’s

and 1 X lo6 cpm of ?S-labeled

cRNA probe per slide in 50 /.tL. Slides were incubated

at 55°C

for 18 hours followed

(RNase)

by treatment

A (200 /.tglmL; Sigma Chemical bridization

washes

were then

with ribonuclease

Co., St. Louis, MO). Posthyperformed

to ‘a stringency

of

0.2X SSC for 1 hour at 55°C. Slides were coated with emulsion and kept at -20°C D19

developer.

for 20 days and then developed

Slides were stained

to allow histological

correlation

with Kodak

with Mayer’s hematoxylin

and photographed

negative

sense strand hybridization

probes,

all of which

Cecal tissues

weighing

approximately

in 1 mL of ice-cold

Student’s

t test. Statistical

at the 95% confidence

level. Paired

relative expression of IL-1 or IL-lra vs. gross inflammation were evaluated by linear regres-

sion analysis.

In Vitro incubations of PG-APS-Treated Peritoneal Macrophages Neither IL-l or IL- lra mRNA were detected in unstimulated peritoneal macrophages from Lewis or Fischer rats. After the addition of PG-APS, IL-l mRNA was detectable at 1 hour and peaked by 2 hours in both strains (Figure 1). IL-lra mRNA expression was also induced by PG-APS, but its appearance was slightly delayed relative to IL-l. Of interest, high expression of IL1 was maintained for 8 hours in Lewis peritoneal cells but was declining by 8 hours in cells from Fischer rats.

Gross, Histological, and Hematologic Observations

gave uniformly

signals.

IL-1 Protein Assay were homogenized

was established

two-tailed

levels

Groups were com-

under light-

and dark-field illumination. Negative controls included RNase treatment of tissue sections before hybridization and hybridization with

for each group as indicated.

scores,

and protein

Probes

3 riboprobe

triphosphate

with T7 polymerase

mixture

An ace-

significance

data comparing and histological

mRNA,

the sections

WI). Sense and antisense

[?j]uridine

in

error for inflammatory

IL-1 and IL-lra

pared using the unpaired

(0.1 mol/L) and 0.15% then

The mean and standard joint diameters, were calculated

in PBS, and

graded alcohols and air-dried.

from linearized

vitro transcription

were postfixed

washed

was performed;

Corp., Madison, with

in OCT com-

for 10 minutes.

step with triethylammonium

(vol/vol)

Cecal tissue from PG-

for 30 minutes,

with proteinase

tylation

us-

in isopentane at -3O”, and stored at -80’. lo-/.tm thick, were placed on polylysine-

4% paraformaldehyde treated

was performed

100 mg each

TE buffer

(pH

7.6)

Twenty-four hours after injection, all PG-APSinjected rats had gross inflammation at the small intestinal and cecal injection sites characterized by bowel wall and adhesions, whereas the thickening, hemorrhage, HSA controls had only minimal changes (Table 1). All

964

GASTROENTEROLOGY Vol. 106, No. 4

MCCALL ET AL.

Table1. Hematologic

Profile and Intestinal n

Inflammation

in inbred Rats 1 Day or 27 Days After PG-APS or HSA Injection

Acute phase Lewis PG-APS Lewis HSA Buffalo PG-APS Buffalo HSA Fischer PG-APS Fischer HSA Chronic phase Lewis PG-APS Lewis HSA Buffalo PG-APS Buffalo HSA Fischer PG-APS

Gross score

Hemoglobin (g/dL)

WBC (xI@/mL)

4.7 12.2 5.4 6.8 4.6 7.7

(0.3)” (2.0) (0.3)” (0.2) (0.2)a (0.3)

13.8 14.3 12.6 12.9 14.8 14.7

(0.6) (0.2) (0.1) (0.1) (0.2) (0.4)

8.3 (0.4)” 1.8 (0.5) 8.2 (0.5)a 2.1(0.1) 7.4 (0.3)a 1.3 (0.2)

28.9 10.8 26.9 5.1 19.6 2.3

(1.7)a (3.0) (1.6)” (0.4) (2.7) (0.3)

33.3 8.6 12.4 6.8 10.2

(5.9)“b (1.3) (l.l)a (0.2) (0.9)

11.0 14.2 12.5 13.1 15.1

(0.3)a.b (0.3) (0.3) (0.2) (0.2)

9.7 (1.7)“b 2.1(0.4) 2.0 (0.4)” 0.4 (0.2) 2.0 (0.3)

22.6 0.3 8.2 2.0 3.4

(3.4)=+ (0.3) (l.l)a (0.5) (1.2)

NOTE. Results are expressed as mean (SEM). Inbred rats were injected subserosally with group A streptococcal day (acute) or 27 days (chronic) before necropsy. WBC, white blood cells. “P < 0.01 vs. HSA-treated controls. ? < 0.01 vs. chronic PG-APS-injected Buffalo and Fischer rats.

rat strains

had similar

degrees

Histology score

of acute

intestinal

in-

mRNA

abundance

flammation

APS

cal

significantly

with no difference in their gross or histologirats had inflammatory scores. PG-APS-injected

injection

PG-APS or HSA (37.5 kg/g) 1

in cecal tissues

was also increased between

inbred

24 hours after PGand

did

not

rat strains (Figure

differ

5). HSA-

slight decreases in total white cell counts but no change in hemoglobin values. Twenty-seven days after PG-APS

injected rat cecal tissues had little or no detectable IL1 p and IL-lra mRNA at 24 hours. Twenty-seven days

injection,

after

six of seven Lewis rats had evidence

granulomas,

extensive

adhesions,

of cecal

bowel wall thickening,

jetted

injection

inflammation

hepatic granulomas, and arthritis; one Lewis rat had no evidence of spontaneously reactivating chronic intestinal

els, compared

or systemic

single

mation

inflammation.

The chronic

in Lewis rats was characterized

markedly

increased

histologically

phase of inflamby anemia

total white cell counts (Table

by chronic

transmural

granulomatous

and

1) and in-

flammation in the cecum with areas of focal necrosis, neutrophil infiltration, extensive fibrosis, and bowel wall thickening (Figure 2). Twenty-seven days after PG-APS injection, Buffalo and Fischer rats showed no active cecal inflammation, only minimal fibrosis or leukocytosis, and no hepatic granulomas, anemia, or arthritis. Gross and histological inflammatory scores were significantly higher in Lewis rats compared with Buffalo or Fischer rats 27 days after PG-APS injection (P < 0.01; Table 1). Control Lewis, Buffalo, and Fischer rats had little or no gross or histological evidence of active inflammation 27 days after HSA injection.

Cecal

IL-Q

and IL-lra

Expression

A representative Northern blot for IL-1 and ILlra cecal mRNA in the Lewis, Buffalo, and Fischer rats is shown in Figure 3. Twenty-four hours after PG-APS injection, the abundance of IL-lp mRNA by densitometry was increased in all rat strains (Figure 4). IL-lra

(chronic

phase),

only

Lewis rats that developed

granulomatous

IL-1 p and IL-lra mRNA levwith almost undetectable mRNAs in the

had elevated

nonreactivated

HSA-injected

the PG-APS-in-

chronic

PG-APS-injected

Lewis rat, the

Lewis rats, and the PG-APS-injected

and Fischer rats (P < 0.005).

IL-lb

and IL-lra

Buffalo mRNA

abundance correlated well with the degree of gross inflammation in the 27-day Lewis rats (Figure 6; Y = 0.88 and 0.82,

respectively)

inflammation

and with

the presence

of histological

(r = 0.88 and 0.83, respectively;

data not

shown). In all cecal tissue samples, there was a strong correlation between the abundance of IL- 1 p and the abundance of IL-lra

mRNA

IL-1-IL-lra

(Y = 0.95; data not shown).

mRNA Ratio

The ratio of IL-1 p to IL-lra mRNA densitometry units was calculated for each rat in which detectable levels were present. Although each value is simply a relative number and does not reflect the true ratio of mRNA for these counterbalancing cytokines, this ratio does reflect the relative increases of IL-l and IL-lra mRNA in the presence of acute and chronic inflammation. The mean IL-l/IL-lra ratio in the acute phase of inflammation was similar for the Lewis (1.07 ? 0.1 l), Buffalo (1.12 + 0.15), and Fischer rats (0.82 + 0.15). In the chronic phase of inflammation, the IL-l/IL-

IL-1 AND IL-lra

April 1994

IN RAT ENTEROCOLITIS

965

Figure 2. (A) Cross section of a normal-appearing Buffalo rat cecum 27 days after PG-APS injection. (B) Cross section of Lewis rat cecum 27 days after a single PGAPS injection showinggranulomatous mononuclear cell infiltrates (large arrow) and multinucleated giant cells (small arrow) (H&E; original magnification x25).

lra

mRNA

reactivated

ratio

(1.54

?

Lewis

rats

than

APS-injected however, pared IL-lra

Lewis

and

0.15)

was higher

in the acute-phase Fischer

rats

it did not reach statistical

with

the acute

for Buffalo

Buffalo

and

because

not above

rats.

Fischer

phase (27 days after PG-APS culated,

in the

hybridization

PG-

(P <

0.05);

significance

com-

Ratios

rats

injection) signals

of IL-l

in the

to

chronic

were not calwere generally

In all rat strains, cecal levels of IL-1CC protein were highest 24 hours after PG-APS injection as shown in Figure tissue IL-l concentrations in the PG-APS-injected

rats. PG-APS-injected rhIL-lra to determine

with HSA-injected

inhibit

enterocolitis

IL-1 Protein Levels

7 (P < 0.05 compared

(Figure 8). No IL-l p or IL-lra mRNA was detected in the intestinal epithelial cells. Sense strand controls and RNase pretreatment controls gave negative hybridization signals. rhlll-lra treatment of PG-APS-injected Lewis

would

background.

Cecal

ing IL-l p and IL-lra to focal collections of chronic inflammatory cells within granulomas in the submucosa of the gut

tissues). Cecal

remained elevated at 27 days only Lewis rats (P < 0.05 vs. pooled

PG-APS-injected Buffalo and Fischer rats). IL-la protein levels correlated with gross and histological scores (Y = 0.67 and 0.72, respectively) and IL-lp mRNA levels (Y = 0.59) 27 days after PG-APS injection. In Situ Hybridization In situ hybridization of sections of ceca from Lewis rats 27 days after PG-APS injections localized mRNA encod-

Lewis rats were treated with if increasing tissue levels of IL- lra

acute and chronic and extraintestinal

phases of experimental inflammation.

As illus-

trated in Figure 9, subcutaneous administration of rhILlra significantly attenuated both acute (3 days after PGAPS) and chronic (15 days) phases of PG-APS-induced enterocolitis. Although all components of the gross inAammatory

score were affected, rhIL-lra

particularly

in-

hibited adhesions (1.4 + 0.3 rhIL-lra-treated vs. 2.2 ? 0.3 for PBS controls at 15 days; P < 0.02). IL-lra therapy only minimally influenced weight gain, hepatic granulomas, anemia, and leukocytosis (P > 0.05) but significantly attenuated PG-APS-induced arthritis (Figure 10).

Discussion Our results indicate that both IL-lb and IL-lra mRNA levels are increased in inflamed cecal tissues dur-

966

GASTROENTEROLOGY Vol. 106, No. 4

MCCALL ET AL.

2.5 2.0-

**

*

1T

T

IL-1 1; IL-1 ra Lewis

28s 18s Figure 3. Representative from Lewis, Buffalo, and APS or HSA. Acute-phase injection. All results were

induced

by transmural

injection

kines appear to be produced localized

foci of granulomatous

be induced

in peritoneal

enterocolitis

of PG-APS.

by inflammatory

Both cytocells within

inflammation

cells by PG-APS.

Fmh

Lewis

Acute (ld)

Northern blot of IL-1B and IL-lra cecal mRNA Fischer rats injected subserosally with PGtissues are from rats killed 24 hours after confirmed in triplicate.

ing acute and chronic phases of experimental

Buff

Buff

Chronic (27d)

Figure 5. Abundance of IL-lra mRNA in cecal tissues from Lewis, Buffalo, and Fischer rats killed 1 day (Acute) or 27 days (Chronic) after PGAPS (D) or HSA (0) injection. IL-lra mRNA abundance was assessed by densitometry of hybridizing mRNA bands. Each sample was normalized as in Figure 3. Results are shown as mean ? SEM. *P < 0.05, significant difference between PG-APS- vs. HSA-injected Lewis rats at 24 hours; **P < 0.005, between acute PGAPS-injetted vs. HSA-injected Fischer rats at 24 hours; ***P c 0.05, between reactivated PG-APS-injected Lewis rats at 27 days vs. HSAinjected Lewis rats and PG-APS-injected Buffalo and Fischer rats at 27 days.

and can Blockade

of endogenous

IL-1 by systemic

injection

of rhIL-lra

pressed acute and chronic phases of enterocolitis 2.5 Y =

*

ciated

1T

arthritis.

These

key immunoregulatory

2.0-

inflammation

$5 1.5-

@ T;t Z!m 5 0

l.O-

results

tory cytokines

suggest

that

role in intestinal

and support

of these important

%E

Fisch

the concept

proinflammatory

can influence

sup-

and assoIL-1 has a

and systemic

that tissue ratios and antiinflamma-

the degree and chronicity

of

inflammation. The increased in inflamed OS-

IL-1 mRNA

and protein

PG-APS-injected

concentrations

rat cecal tissues are con-

sistant with findings in other models of experimental intestinal inflammation and with observations of in-

Od-' -.Lewis

Buff

Fisch

Acute (ld)

Lewis

Buff

Fisch

Chronic (27d)

Figure 4. Abundance of IL-lj3 mRNA in cecal tissues from Lewis, Buffalo (Buff), and Fischer (Fisch) rats killed 1 day (Acute) or 27 days (Chronic) after subserosal PG-APS (W) or HSA (Cl) injection. IL-1 mRNA abundance was assessed by densitometry of hybridizing mRNA bands. Each sample was normalized for minor variations in total mRNA based on the intensity of the 1% band by ethidium bromide staining of the same samples. Abundance of IL-1 in each group is expressed as a ratio relative to the abundance in the internal acute-phase control. Results are shown as mean 2 SEM. *P < 0.005, significant difference between PG-APS vs. HSA-injected Lewis rats at 24 hours; **P < 0.05, between acute PG-APS-injected vs. HSA-injected Fischer rats at 24 hours; ***P 5 0.005, between reactivated PG-APS-injected Lewis rats at 27 days vs. HSA-injected Lewis rats and PGAPS-injetted Buffalo and Fischer rats at 27 days.

creased IL-1 in intestinal tissues from patients inflammatory bowel disease and self-limited tory colitis.’ Tissue IL-1 mRNA

and protein

with active inflammalevels corre-

lated with the presence of gross and histological inflammation in our study. Similar increases in tissue IL-l expression have been reported with trinitrobenzene sulfonic acids9 and immune complex-induced” colitis. IL1 mRNA and protein concentrations correlate with the degree of inflammation in tissues from patients with inflammatory bowel disease.“X’5X’7 Furthermore, the localization of IL-l to foci of inflammatory cells in our model is consistent with observations that lamina propria macrophages from inflammatory bowel disease patients produce high levels of IL-l .14340We found no evidence of IL-1 production by colonic epithelial cells, in

April 1994

IL-1 AND IL-lra IN RAT ENTEROCOLITIS 967

GROSS

INFLAMMATION

SCORE

GROSS

INFLAMMATION

SCORE

flgure 6. Linear regression of gross inflammation score vs. cecal IL-@ mRNA abundance (A) and IL-lra mRNA abundance (6) in Lewis rats killed 27 days after subserosal injections with PG-APS (0) or HSA (W). One PG-APS-injected rat had no evidence of chronic inflammation. (A) r = 0.88; (B) r = 0.82.

agreement

with

results

in patients

bowel disease,40 but in contrast

with

inflammatory

to observations

in rabbit

complex colitis4* and in the acetic acid rat model

immune

of acute experimental

colitis.42

One explanation

of this

peared to be some discrepancy between mRNA tein levels in acute-phase PG-APS-injected tissues.

However,

is localized

24 hours after injection,

to the sites of injection

greater

complexes

noted in the chronic phase of cecal inflammation

more limited IL-la

acid produce

acute

inflammation

to the mucosa.

protein

tion, paralleling

levels were increased IL-l p mRNA

with

inflamma-

levels, although

there ap-

best correlation

1

*

between

between mRNA

rats, where the inflammation uted throughout the cecum.

sites. The

and protein

levels was in Lewis

is more uniformly distribIL-la levels in acute PGin rabbit

complex colitis in which both IL-la and p are elevated.‘* Moreover, in the immune com-

plex colitis model tissue, IL-la and p concentrations closely correlated,‘” even though these two cytokines

*

be produced dently.”

30-

to

injection

APS colitis in rats were close to those reported immune proteins

40

variation

inflammation

and is susceptible

difference is the chronic, transmural, and granulomatous nature of PG-APS-induced colitis, whereas immune and acetic

sampling

and proFischer

by different

cells and are regulated

are can

indepen-

The ability of rhIL-lra to significantly inhibit acute and chronic phases of experimental enterocolitis provides direct evidence of a key role for IL-l in the pathogenesis of intestinal inflammation, Moreover, these results imply that endogenous IL-lra has the potential to modify the inflammatory response. Previous studies have shown that rhIL-lra administration attenuates acute experimental colitis 24 or 48 hours after the inciting event.41343 Our data demonstrate that sustained administration of rhIL-

Lewis Buff Acute (1 d)

Fisch Lewis

Buff

Fisch

Chronic (27d)

Figure 7. IL-lo protein levels in cecal tissues from Lewis, Buffalo, and Fischer rats killed 1 day (Acute) or 27 days (Chronic) after PGAPS (W) or HSA (0) injection expressed as mean -e SEM. *PG-APSand HSA-treated rats of the same species were different (P < 0.05); ‘reactivated Lewis rats (6 of 7 injected) were different vs. HSA-injected Lewis rats (P < 0.05); and PGAPS-injected Lewis rats were different from pooled PG-APS-injected Buffalo and Fischer rats (P < 0.05).

lra can inhibit both acute (3 days after PG-APS injection) and chronic (15 days) phases of experimental enterocolitis. The observation that adhesions were significantly diminished is consistent with the ability of rhIL-lra to prevent collagen deposition in murine models of pulmowe showed that rhIL-lra nary fibrosis.44 Furthermore, can treat extraintestinal complications of experimental enterocolitis, as evidenced by a 50% inhibition of arthritis. This response is very similar to that described by

968

GASTROENTEROLOGY Vol. 106, No. 4

MCCALL ET AL.

Figure 8. Localization of IL-18 and IL-lra mRNA to submucosal inflammatory infiltrates (arrowheads) by in situ hybridization in cecal tissue sections from Lewis rats killed 28 days after subserosal PG-APS injection. (A) Bright-field, hematoxylin-stained tissue section hybridized with antisense IL-18 probe. (B) Sense strand IL-lra shows negative control. Same negative results were obtained with sense IL-18 probe or with sections pretreated with RNase (data not shown). (C) Dark-field view of same section as A hybridized with antisense IL-1 probe. (Photography exposure time, 0.85 seconds). (D) Darkfield section hybridized with antisense IL-lra probe (exposure time, 0.64 seconds; original magnification of A-D x25).

Schwab

et al. 45 in a reactivation

induced arthritis ministration flammation chronic,

in Lewis rats. We elected

of rhIL-lra

until the quiescent

(8 days after PG-APS

whether PG-APS relapsing

model

injection)

therapy could significantly phase of granulomatous

with associated extraintestinal

inflammation

of PG-APS-

mize the production

of anti-human

IL-lra

to delay ad-

the rats over a 15-day

period.

phase of in-

rationale for therapeutic

use of rhIL-lra

to determine prevent the enterocolitis and to mini-

of chronic

granulomatous

These

enterocolitis

antibodies

by

results provide in the treatment with

attendant

fibrosis and arthritis. To our knowledge, mRNA

expression

our report

in experimental

is the first on IL-lra colitis. In the chronic,

a

IL-1 AND IL-lra IN RAT ENTEROCOLITIS 969

April 1994

there was a trend

toward

an increased

IL-l/IL-

during

the chronic phase of granulomatous

These

results

are consistent

tions in humans concentrations

with

lra ratio

enterocolitis.

preliminary

observa-

in whom the ratio of IL-l/IL-lra is significantly

increased

protein

in active ulcera-

tive colitis and Crohn’s disease tissues relative controls data).

28, 46; R. B. Sartor,

(references We

have

previously

shown

that

to normal

unpublished patients

Crohn’s disease have a relative under-expression mRNA

relative

specimens

to IL-l

compared

suggest the lo-

Acute

Chronic

Flgure 9. Treatment of PG-APS-injected Lewis rats with recombinant human IL-lra (0) resulted in attenuation of acute and chronic intestinal inflammation 0, PBS. rhll-lra was administered subcutaneously beginning at the time of intestinal PG-APS injection in the acute phase (3 days, see Materials and Methods for dosing schedule) or 8 days after PGAPS injection in the chronic phase (8 mg/kg every 12 hours until rats were killed 15 days after PG-APS injection). Control rats received PBS subcutaneously on the same dosing schedule. *P < 0.05 vs. PBS-treated controls.

phase of our model,

is localized

matory

cells, and levels closely correlate

histological suring

inflammation.

rat cecal IL-lra

munosorbent anti-human reactivity

IL-lra

mRNA,

of submucosal

protein

antibodies

with gross and in mea-

by enzyme-linked blot

because

with the rat. Rat and human

like

inflam-

We were unsuccessful

assay or by Western

75% homologous possible

to aggregates

IL-lra

IL-lp,

using

im-

existing

block IL-l

can inhibit

monocyte

activation

including

posttranscriptional

compartmentalization

Our do not

bioactivity.8 molar

by 50%.47 Other factors, regulation

may be quite

cecal IL-lra

However,

excess of IL-lra and

protein

important

concentrations.

in de-

However,

it is

evident that tissue IL-l mRNA and protein concentrations are persistently elevated only in Lewis rats. Moreover, the kinetics lra mRNA differential sponding

granulomatous

controls.”

whole cecal mRNA

data show that a twofold

termining

biopsy

1 OO-fold IL-l ra excess over IL- 1 required

in vitro to completely recent

with mucosal

from normal and inflammatory

results based on homogenized

with

of IL- lra

of in vitro stimulation

of IL-l and IL-

by PG-APS in peritoneal cells also suggests regulation of IL-l in high- and low-rerat strains.

express high amounts

Cells of Lewis rats continued of IL-1 mRNA

to

at 8 hours, whereas

cells from Fischer rats had declining levels. The concept of differential regulation of IL-l in high- and low-responding

rat strains

al.,’ who recently production

is further

supported

demonstrated

by Bristol

enhanced

by PG-APS-stimulated

IL-l

macrophages

et

protein from

of lack of crossIL-lra

. f

are only

at the amino acid leve1.37 It is certainly

that tissue concentrations

not correlate with mRNA

of IL-lra

protein

may

levels because of posttranscrip-

tional regulation. However, our mRNA data are in agreement with observations of increased IL-lra mRNA concentrations in mucosal biopsy specimens of patients with self-limited colitis”; enhanced IL- 1 ra protein levels in tissues from patients

with active ulcerative

Crohn’s disease (R. B. Sartor, immunohistochemical staining

colitis and

unpublished data); and of IL-lra in pulmonary

granulomas.26 This study was designed to provide insights into the mechanisms of differential susceptibility to experimental chronic intestinal and systemic inflammation in inbred rat strains. Our results support, but do not provide definitive proof of, the hypothesis that disordered immunoregulation predisposes the Lewis rat to chronic inflammation.’ Although IL-lra mRNA was clearly upregulated in acute and chronic phases of enterocolitis in Lewis rats,

-1.04

I

3

I

I

0

Days

After

I

tt PQ-APS

I

12

14

‘

15

In)actlon

Figure 10. Effect of rhll-lra treatment on PGAPS-induced arthritis. rhll-lra (0; 8 mg/kg every 12 hours) or PBS (0; controls) was administered subcutaneously for 7 days beginning 8 days after subserosal (intramural) PG-APS injection. Change in joint diameter is calculated as serial joint diameter minus value immediately before PG-APS injection. *P < 0.05 compared with PBS controls; **P < 0.001 vs. controls.

970

GASTROENTEROLOGY Vol. 106, No. 4

MCCALL ET AL.

Lewis rats compared in steady-state

with Buffalo rats, but no difference

IL-l

mRNA

increased concentrations

levels. Finally,

of IL-lra

the ability of

to decrease inflamma-

tion shows that the balance of IL-lra

relative to IL-l

modulate

in Lewis rats. The

activity

alternative

hypothesis

ity to inbred cortisol

of inflammation

to explain differential

rats to inflammation,

in Lewis rats stimulated

relevant to our observations, IL-l

production

can

susceptibil-

a relative

with PG-APS6

same

cell,

and IL-lra

the

activated

produced.

monocyte/macrophage,

these

regulated.

Both transcrip-

regulation

affect the levels

Picogram

amounts

and production

G stimulates

In addition, 1.50-52 Wahl factor

expression

but inhibit

et a1.53 suggest

nants of differential hibits

production

in agreement

regulatory

by selective

play important

factor

C! (TNF-a),

synergistically

roles,

with IL-l

the inflammatory

by only 28% pathways

IL-2,

production

suggests

are involved. cytokines

including

and IL-8.

and an IL-8-like

inthat

Other

almost

tumor

These

gene is higher

of mRNA

necrosis

proteins

act evi-

for TNF-a

IL-S,

the expression

8. 9.

in Lewis than Buffalo

or Fischer cecal tissues.54 We are in the process of further characterizing

7.

cer-

and with each other to amplify expression

6.

rat strains

response. 9 We have preliminary

dence that constitutive

5.

with our results

of inbred

and effector

tainly

4.

of IL-

The fact that rhIL-lra

enterocolitis

3.

growth

are the sole determi-

susceptibility

proinflammatory

2.

cells.

and IL-lra

enterocolitis.

chronic

important

p and IL-4

is mediated

peritoneal

It is unlikely that IL-l

parallel

factor

that transforming

of IL-lra

in PG-APS-stimulated

to experimental

and regulation

gro, and other proinflammatory

10.

of TNF-a,

cytokines

in this

enterocolitis,

a valu-

11.

model. In conclusion, able animal

PG-APS-induced

model for Crohn’s

by increased tissue expression mRNA

concentrations

tion of rhIL-lra

IL-l

activity

attenuated

showing

is characterized

with tissue

inflamma-

Lewis rats selectively

4 weeks after PG-APS

ade of endogenous arthritis,

disease,

12.

for both IL-l /!iand IL-lra.

correlate

tion, such that susceptible these cytokines

and laying by altering

proinflammatory

chronic

inflammation

testable hypotheses

of inflammatory the foundation

bowel

in

gethis

applicable disease

for treating

the ratio of counteracting

of

and anti-

in

inflam-

mediators.

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production.24

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is delayed relative to IL-l,

and IL-lra

adherent

but not IL-l growth

p induction

stimulation

IL-lra

of LPS induce

of both IL-l

transforming

induce IL-lra

mation

the activity

characterization

will enable us to understand to

model, thereby generating

in vivo effect on IL-

by human monocytes.8X23324 However, globulin

peptides

susceptibility

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13.

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and

of these

15.

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to chronic granulomatous enterocolitis troenterology 1993; 104374OA.

in inbred rats (abstr). Gas-

Received February 1,1993. Accepted October 19,1993. Address requests for reprints to: R. Balfour Sartor, M.D., Division of Digestive Diseases, University of North Carolina School of Medicine, 326 Burnett-Womack Building, CB 7080, Chapel Hill, North Carolina 275997080. Fax: (919) 9666842. Supported by a Crohn’s and Colltis Foundation of America research fellowship award (R.D.M.); U.S. Public Health Service grants DK 40249 (R&S.), DK 34987, and Al26774 (S.H.); Synergen Inc.; and the molecular biology and radioimmunoassay cores of the University of North Carolina Center for Gastrointestinal Biology and Disease. The authors thank Drs. Alan Shaw and Stephen Stimpson (IL-@) and Stephen Eisenberg (IL-lra) for providing cDNA probes; Lisa Holt, Diane Bender, Roger Brown, Matt Pardo, and Todd Boyette for technical help; and Shirley Willard for secretarlal assistance.