Interleukin 1 (IL 1) gene expression, synthesis, and effect of specific IL 1 receptor blockade in rabbit immune complex colitis

Interleukin 1 (IL-1) gene expression, synthesis,

and effect of specific IL-1 receptor blockade in

rabbit immune complex colitis.

F Cominelli, … , R C Thompson, C A Dinarello

J Clin Invest.

1990;

86(3)

:972-980.

https://doi.org/10.1172/JCI114799

.

Interleukin 1 (IL-1) may be a key mediator of inflammation and tissue damage in

inflammatory bowel disease (IBD). In rabbits with immune complex-induced colitis, IL-1

alpha and beta mRNA levels were detectable at 4 h, peaked at 12 but were absent at 96 h

after the induction of colitis. Colonic IL-1 tissue levels were measured by specific

radioimmunoassays. IL-1 alpha was significantly elevated at 4 h (9.4 +/- 1.5 ng/g colon),

progressively increased at 48 h (31 +/- 5.8 ng/g) and then decreased by 96 h (11.5 +/- 3.4

ng/g). IL-1 beta levels were 2.0 +/- 0.5 ng/g colon at 4 h, 5.0 +/- 1.6 ng/g at 48 h and

undetectable by 96 h. By comparison, colonic levels of PGE2 and LTB4 were unchanged

during the first 12 h and did not become elevated until 24 h. IL-1 alpha levels were highly

correlated with inflammation (r = 0.885, P less than 0.0001), edema (r = 0.789, P less than

0.0001) and necrosis (r = 0.752, P less than 0.0005). Treatment with a specific IL-1 receptor

antagonist (IL-1 ra) before and during the first 33 h after the administration of immune

complexes markedly reduced inflammatory cell infiltration index (from 3.2 0.4 to 1.4

+/-0.3, P less than 0.02), edema (from 2.2 +/- 0.4 to 0.6 +/- +/-0.3, P less […]

Research Article

Interleukin

1

(IL-1) Gene Expression, Synthesis,

and Effect of

Specific

IL-1

Receptor Blockade

in Rabbit Immune

Complex

Colitis

Fabio Cominelli, Cynthia C. Nast,* Burton D.

Clark,*

Ralf

Schindler,'

RafaelLlerena,Viktor E.Eysselein,

Robert C.

Thompson,*

and Charles A. Dinarello

DepartmentsofMedicine and *Pathology and Inflammatory BowelDiseaseCenter,Harbor-Universityof CaliforniaatLosAngeles

Medical Center, Torrance, California 90502; *Synergen Inc., Boulder, Colorado80301;§DepartmentofMedicine and New England Medical Center, Tufts University, Boston, Massachusetts 02111

Abstract

Interleukin

I

(IL-1)

may be a key

mediator

of

inflammation

and

tissue

damage in

inflammatory

bowel disease

(IBD).

In rabbits with

immune complex-induced

colitis, IL-la and (3 mRNA levels were

detectable

at 4 h, peaked at 12 but were absentat

96

hafter the induction of colitis. Colonic

IL-1

tissue levels were measured by

specific radioimmunoassays.

IL-la was

significantly elevated

at 4 h (9.4±1.5 ng/g

colon),

progres-sively increased

at

48

h

(31±5.8 ng/g)

and then

decreased

by

96

h

(11.5±3.4 ng/g).

IL-lft

levels were 2.0±0.5 ng/g colon at 4h,

5.0±1.6 ng/g

at 48 h and

undetectable

by 96 h. By

compar-ison, colonic

levels

of PGE2

and LTB4 were

unchanged

during the

first

12 h and

did

not become

elevated until

24 h. IL-la levels were highly

correlated

with

inflammation

(r=0.885, P

<

0.0001),

edema

(r

=

0.789,

P <

0.0001)

and

necrosis

(r = 0.752, P<

0.0005).

Treatment with a

specific

IL-1

receptor

antagonist

(IL-Ira)

before and

during

the

first 33

h

after

the

administration of

immune complexes markedly reduced

in-flammatory

cell

infiltration

index

(from

3.2±0.4 to

1.4±0.3,

P

<

0.02),

edema

(from

2.2±0.4 to

0.6±0.3,

P <0.01) and

ne-crosis

(from 43±10%

to

6.6±3.2%,

P<

0.03)

compared to

vehicle-matched

colitis

animals.

These studies

demonstrate

that

(a)

IIL-

gene expression

and

synthesis occur

early in

the

course

of

immune

complex-induced colitis; (b)

are

significantly

elevated

for 12 h

before

the

appearance of PGE2

and

LTB4;

(c)

tissue

levels of

IL-1

correlate with the

degree

of tissue

inflam-mationand;

(d) specific

blockade

of IL-1

receptors reduces the

inflammatory

responses

associated

with

experimental

colitis.

(J. Clin.

Invest.

1990. 86:972-980.)

Key words: interleukin

1.

interleukin

1 receptor

antagonist

* colitis

Introduction

Interleukin-

1 (IL- 1)

is

a

polypeptide

cytokine produced during

inflammation and injury, that possesses a wide spectrum

of

Presentedin partatthe Second InternationalWorkshoponCytokines,

Hilton Head Island, SC, December 1989,andattheWesternSection of the American Federation of ClinicalResearch, Carmel, CA,February 1990.AddressreprintrequeststoDr.Cominelli,Division of Gastroen-terology, C-ITrailer, Harbor-UCLA MedicalCenter, 1124W.Carson Street, Torrance, CA 90502.

Receivedfor publication26 October 1989 andinrevisedform 17

May 1990.

immunologic

and

nonimmunologic

activities (1).

Two

distinct

gene

products of IL-I

have

been identified:

IL-lI3,

which is

secreted

by cells,

and

IL-la,

which is

primarily

a

cell-asso-ciated

protein

(2-4).

Both

forms share

the samespectrum

of

biological activities

and

recognize

the same

cell

surface

recep-tors

(reviewed in

1). Interest has

focused

on the role

of

IL- 1 as an

inflammatory mediator

in

chronic

and acute

diseases (5, 6).

Elevated IL-I levels

have been

reported

in

synovial

fluid and

plasma from patients with various arthritides (7-9).

In

addi-tion, patients with

acute

meningitis, septic

shock,

orburn

inju-ries

often

show

increased circulating levels of IL-I

(10, 11).

Increased

IL-I production

by

blood mononuclear

cells has been

described

in

several

pathologic

conditions

including

rheumatoid arthritis and Crohn's

disease

(12-14).

Several of

the

biological properties

of

IL-I are relevant to

inflammatory bowel disease.

When

injected

in

rabbits,

IL-I

induces fever, neutrophilia,

decreased plasma

iron,

zinc and

albumin levels, and synthesis of acute-phase proteins (1). IL-I

attracts leukocytes

into inflamed tissues

(15)

and

stimulates

prostaglandin synthesis

by

its

ability

to

induce cyclooxygenase

gene

expression (16).

Thus,

systemic

and

local

IL-I

production

may

initiate

or

contribute

to the

inflammatory

response

in

colitis and could play

a role

in

the

pathogenesis

of

this disease.

Our

recent

studies

are

consistent with this

hypothesis,

asIL-I

perfused

in or

incubated with rabbit colons

induced

prosta-glandin

and

thromboxane production

(17).

We have

previously used

the

rabbit model of

formalin-im-mune

complex colitis

to

investigate the

role

of arachidonic

acid-derived inflammatory mediators

and to

evaluate

thera-peutic strategies

in

colitis (

18-20).

The

availability

of

specific

cDNA

probes

and

radioimmunoassays for rabbit

IL-i

a and IL-

1(3

(21, 22),

as well as a

specific

IL-I receptor

antagonist

(IL-Ira)

(23, 24) offers

an

opportunity

to

study

the

role

of

IL-I

in

this model.

Therefore,

the

aims

of

the present

study

wereto

examine

the time course

of

IL- I gene

expression

and

synthesis

in

the

colon of rabbits after induction of formalin-immune

complex colitis

andto

determine whether selective blockade of

IL- Ireceptors would reduce the

subsequent colonic

inflamma-tory response. Here we report that IL- I

is

produced just

before

the sequence

of

histological

events and

eicosanoid

synthesis

characteristic of this colitis

model and that

specific

inhibition

of

IL-I

activity

significantly

reduces

inflammation

and

ne-crosis,

suggesting

that IL- I

plays

a

major

role in the

pathogene-sis of colonic inflammation.

Methods

Rabbit colitis model. Inflammationwasinducedin thedistal colon of

male NewZealand rabbits(2.2-2.5 kg) using amodification ofthe

immunecomplex method of colitis(25, 26).4mlof 0.45%

(vol/vol)

unbufferedformaldehyde(Electron Microscopy

Sciences,

Fort

Wash-J.Clin.Invest.

©TheAmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/90/09/0972/09 $2.00

ington, PA) was administered by a catheter inserted 10 cm into the

distalcolonof anesthetized rabbits(xylazine and ketamine). 2 h later,

animals received 0.85 ml of immune complexes in antigen excess

through an earvein. Thecomplexes were prepared by incubating human serum albumin (500

gg/ml)

with rabbit antihuman antisera (ICN Immunobiologicals, Costa Mesa, CA), decanting the superna-tant, and redissolving the precipitated immune complexes with an

albumin solution (6mg/ml) asdescribedpreviously(18).

Time coursestudy. Animalswerekilled with 40 mg/kg of nembutal (AbbotLaboratories,NorthChicago,IL) 0, 4, 12, 24, 48, and 96 h after

induction of colitis andthedistalcolon removed. Longitudinal

sec-tions from thelast 10cmofthe distal colon were obtained and

pro-cessedfor histologicassessmentof inflammation,IL- ImRNAanalysis

andIL-1proteinmeasurements. Noninvolved small intestine was also removed for study. Rectal dialysis was performed for 2 h immediately

before each timepoint formeasurement ofPGE2 and leukotriene B4

(LTB4)'aspreviously described(18).

Treatment withrecombinant human IL-Ira. Recombinant

inter-leukin-1 receptor antagonist, rIL-1ra, was prepared from lysates of

Escherichia coli transformed with a plasmid containing a modified

IL-1racDNA undercontrolofthe tacpromoter (23). Expression of

IL-lrawasinduced by the addition ofisopropylthio-fl-D-galactoside for several hours, and the cellswerecollectedbycentrifugationand

lysed ina pressure cell. The recombinant protein waspurified by

successive cationandanion exchange chromatographyand was>95% pure bySDS-PAGEand by reverse-phase HPLC. Itcontained <0.2

endotoxin U/mg ofprotein. The homogenous protein (18 kD) was

suspendedin0.5mlPBS (0.1Mphosphate, 0.15MNaCl,pH7.4). The rIL-lra inhibitsIL- 1 activityinvitrobybindingtocellsurface recep-torsandcompetitively inhibiting IL-l binding. This effect has been

demonstrated in the murine thymoma cell line EL4-6.1 (24). The

ability ofrIL- Ira toblock thebiological actions ofIL-Ihas been

dem-onstrated in human foreskin fibroblasts (23), inhuman rheumatoid synovial fibroblastsandchondrocytes(27),and in humanendothelial cells(28). Testswiththenatural IL-1rahaveshown thatit doesnot

block the action ofTNFondermalfibroblasts(29).When adminis-teredintravenouslyat 100timesthe doseofIL-1it blocksthedropin blood pressure andthe drop in circulating whiteblood cells in the

rabbit (30). Animalswere treatedintravenously with IL-lra (5 mg/kg;

n=8)orthevehicle(n= 1O)atsix timepoints:2 hbeforeand1,9, 17, 25,and 33haftertheadministration oftheimmunocomplexes. The

rabbitswerekilled 48haftertheinductionof colitisand colontissue analyzed for inflammation.

Histologic quantitationof

inflammation.

Histologic evaluationwas

performed on a minimum oftwo longitudinal sections from each colonaspreviouslydescribed(18).Allcolonsampleswereexaminedin ablind fashionbyasinglepathologist(C. C.Nast).Themucosaand

submucosawereseparatelyevaluated forinfiltration ofacute

inflam-matorycells(neutrophilsandeosinophils).Asemiquantitativescoreof leukocytes (L)per highpowerfield(HPF) wasdetermined for each

areaexaminedusingthefollowingquantitations:0=0or1;0.5=2-9;

1 = _10-20; 1.5 = 21-30; 2 = 31-40; 2.5 = 41-50; 3 = 51-65; 3.5

=66-80;4=.81L/HPF.Ataminimum, eightHPFs ofmucosaand submucosa from each specimen were separately evaluatedin each section. Theinflammatory indexwascalculated by addingthe aver-agedscoreforthemucosalandsubmucosal evaluations. Edemawas

semiquantitativelyassessedon ascaleof0to4.Necrosiswasexpressed

asthepercentageofmucosainvolved.

IL-Ia andIL-I 3radioimmunoassays. IL-iaandIL-iI3were

mea-suredusing specific, non-cross-reacting radioimmunoassays (22).

These assays were alsovalidated forIL-1 levelsafter tissue extraction (22).Longitudinalsectionswereimmediatelyfrozeninliquid nitrogen

untilassay.Atthattime, 1 gfrozen tissuewasthawed andlysedin 10

1.Abbreviations usedinthis paper:LTB4,leukotrieneB4; TNF,tumor

necrosis factor.

ml PBS containing a cocktail of protease inhibitors (antipain, apro-tinin,leupeptin, pepstatin A, all at 1

_Ag/ml

and PMSF, 1 mM) (Sigma Chemical Co., St. Louis, MO). Tissue was homogenizedwithan elec-tric homogenizer for 30 s. The homogenate was then centrifuged for 60 min at 100,000 g at 15'C. The supernatant was decanted and assayed directlyforIL- laand IL-13 levels. In brief, 100

gl

of goat anti-rabbit

IL-l, appropriately diluted in assay buffer (0.25% BSA, 0.05% azide, 0.01 M PBS, pH 7.4; anti-IL-la at 1:200,000 and anti-IL-l/3 at 1:6,400) were added to 100Mlof colon tissue sample, followed by 300 Mlofassaybuffer.Thetubeswerevortexed andincubated overnightat roomtemperature. '251-IL-lIaor'25I-IL-l3(10-12,000cpm) was then added,the tubes vortexed again and incubated for an additional 24 h at roomtemperature. 500Mlof 6%polyethyleneglycol (8,000 mol wt), and 2% donkey anti-goat IgG serum (Cambridge Medical Diagnostic, Billerica, MA) diluted in assay buffer were added to precipitate the boundimmunecomplexes from the free '251-IL-1. Thesamples were vortexed, incubated for 1 h, centrifuged at 2,000 g for 20minat4°C,

and the supernatants were decanted; the precipitates were counted in a gamma-counter. All samples were tested in duplicate; standard curves were prepared with rabbit recombinant IL-laandIL-1I3. TheRIAs

have adetection limit of 40 pg/ml and 60 pg/ml for IL-Iaand IL-1fl,

respectively, at the 95% confidence level (22).

mRNAanalysis.Longitudinal sections of distal colon were

imme-diately frozeninliquidnitrogenafterdissection. Approximately 0.25 g frozen tissue was lysed in 10 ml of guanidiumisothiocyanatesolution using anelectric homogenizer for 30 s. Total RNA was extracted by

centrifugation through5.7 M CsCl (prepared by dissolving CsCl in0.1

MEDTA). The RNA pellet was resuspended in 0.25 ml resuspension

buffer(5 mM EDTA, 0.5% sarkosyl, and 5%

_{fl-mercaptoethanol)}

and theconcentration determinedbyultravioletat 260/280 nm ratio spec-trophotometry. For Northern blotanalysis 30 ,ugoftotal RNA was loaded per lane on a 1.2% agarose gel containing 1.6% formaldehyde. After electrophoresis, the gel was soaked in water for 30 min to remove theformaldehydeand the RNA was transferred to nylon membranes (Hybond-N; Amersham Corp., Arlington Heights, IL) by capillary

ac-tion andfixedby ultraviolet radiationasdescribed by the manufac-turer. The blots wereprehybridized for3-12 h withprehybridization buffer followedbyhybridization withthe cDNA probes (31, 32). The rabbitIL-laDNA probe used in these experiments is a 830 bp Eco

RI-BglII fragment from theIL-lacDNA inpUCl9(21). The rabbit

IL-I/3

probe is a 830 bp Eco RI-Eco RIfragment of the full length cDNA(21).Probes werelabeled with[32P]dCTP (DuPont NEN,

Wil-mington, DE) by use ofa random primer DNA labeling kit

(Boehringer Mannheim Biochemicals, Indianapolis, IN)tospecific

ac-tivity of108_-10 cpm/,ug).

Eicosanoid radioimmunoassay.PGE2 and LTB4 were measured in rectaldialysatesbyvalidated radioimmunoassays after lipid extraction

andpurificationbySephadexLH-20 andhighpressure

chromatogra-phy (33, 34).

Dataanalysis. Results areexpressed asmean±SEM. Statistical analysiswasperformed usingastatistical software (BMDP Inc., Los Angeles, CA). Statistical significancebetween groups ofparametric

data wasevaluatedusingStudent's t testforunpaireddata.

Compari-son betweengroupofnonparametricdataweremadewiththe Mann-Whitney two-sample test.Correlationswere madeusingthePearson'sr

test. Thedifferenceswereconsideredsignificantwhen the P valuewas <0.05.

Results

Table L Time Course ofthe Inflammatory Response inRabbitFormalin-immune Complex Colitis

Time Inflammatory index Edema %Necrosis

h

0 0.3±0.1 0±0 0±0 4 1.6±0.2* 1.6±0.5* 0±0 12 2.8±0.3* 1.9±0.4* 12±6*

24 3.5±0.5* 2.7±0.5* 45±22* 48 4.5±0.7* 3.6±0.3* 89±3*

96 1.3±0.7*$ 1.2±0.5*t 11±10*t

Colitiswasinducedattime0; colontissuewasobtainedat each time pointindicatedaboveandprocessedforlight microscopy.

Inflamma-torycellinfiltrationindex, edemaand percentofmucosal necrosis weremeasured and scored (see Methods). The data are the

mean±SEM of fiverabbitsateachtimepoint.

* P <0.001 vs.0 h,tP<0.01 vs.48 h.

cell

infiltration index,

edema, and percentage of mucosal

ne-crosis,

progressively

increased from

0.3±0.1 (0 h) to 4.5±0.7

(48

h)

(P<

0.001), from 0.3±0.1

to 3.6±0.3 (P< 0.001) and from 0% to 89% (P< 0.001),

respectively.

Asubsequent de-crease inthese parameterswasobserved 96 h

after

the

induc-tion

of colitis (P

<0.01 vs.48

h).

Synthesis of

IL-Iaand IL-I/. IL- laand IL- 13 levelswere

undetectable (< 40 pg/100 mg

of tissue for

IL- 1a and <60 pg/

100

mg

of tissue for

IL-

1,8)

in colon

specimens obtained

from

healthy

rabbits before

the

induction of

colitis (0 h). A

significant increase

in colon

tissue

levels

of

IL-1a and IL-

113

wasobserved at 4 h (P<0.01)with a maximum at 48 h after the

induction of

colitis.

Incontrast, IL-1aandIL-113 concen-trationswere

markedly diminished

at96 h in parallel tothe

decrease

in

inflammation

and

tissue damage (Fig.

1). IL-1a

tissue concentrations

were

approximately five times

greater than IL-

113 levels.

Inflammatory

index, edema,

and percentage

of

mucosal

necrosis

correlated

with

IL-1a and IL-

113

levels. The

correlations

were r =

0.885

(P <

0.0001),

r= 0.789 (P

<

0.0001)

andr =0.752

(P

<

0.0005) for

IL-la

(Fig.2

A)

andr =0.706

(P

<

0.001),

r=0.792

(P

<

0.0001)

andr= 0.604

(P

<

0.005) for

IL-1I

(Fig.

2

B),

respectively.

Both IL-

la

and IL-

113

were absent in

noninflamed

small

intestinal tissues

ob-tained from

thesame

animals.

IL-ia

and

IL-i1 gene expression. The

transcriptional

acti-vation of rabbit

IL- 1a and IL-

113

mRNA

production

in the colon

during

the

induction of

colitis

was

examined by

North-ern

blot

analysis.

The membranewas

first hybridized with

the probe

for rabbit

IL-

11,

stripped,

and

rehybridized

with the

rabbit

IL- 1aprobe. In three separate

experiments,

we

did

not

observe

evidence of

IL-1aor IL-

11

mRNA

before

theonset

of

colitis (0 h).

IL-

113

mRNA was

clearly

observedas

early

as 4 h

after

the

injection of

immune-complexes,

whereas IL-1a mRNA in the same

tissue

sample was weakly

visible.

Peak

mRNA waspresent

for

bothIL-laandIL-

1I3

at12 h. At 96h

therewas no

evidence of continued

presence ofIL-1 mRNA.

Fig.

3

illustrates

theNorthern blot of one of the three

experi-ments. IL-1a and IL-

113

mRNA was notobserved in

nonin-flamed

control

samples obtained from the

small

intestine

of thesame

animals.

Production

ofcolonic PGE2

and LTB4.

The time course

of

PGE2 and

LTB4

production

during

the

induction of colitis is

shown in Fig. 4. No changes in these eicosanoids were ob-served at4 and 12 h. A significant increase in colonic PGE2 and

LTB4

levelswasobserved onlyat48 and 24 h,

respectively.

At 96 h, colonic PGE2 and LTB4 production diminished to levelssimilartothoseobserved in control rabbits (0 h).

Effects

oftreatmentwithIL-i ra.Treatment of rabbits with human recombinant IL- 1 ra significantly reduced inflamma-tory index from 3.2±0.4 to 1.4±0.3 (P < 0.02), edema from 2.2±0.4 to 0.6±0.3 (P<0.01) andnecrosis from 43±10% to 6.6±3.2% (P < 0.03) compared to vehicle-treated colitis ani-mals(Fig. 5). Representative macroscopic appearance and his-tologic sections of distal colons obtained from the two experi-mental groups are shown in Fig. 6.

Discussion

Thereisincreasing evidence that IL- 1 is one of the key media-tors of the inflammatory response after microbial invasion,

immunological reactions,

and

tissue

injury.

In this report we show that IL- 1aand IL- 113synthesisoccursearly in the rabbit model of

formalin-immune

complex colitis. The rapid

tran-50

A *p 0.01 vs0-hrm

# p(0.02va48-hr. 40.

o09;

rn~rmvrrwT1rq

r~zrwTr1-rT

0 4 12 24 48 96

TIME (hour.)

10

B

* p(0.01 vs0-hr.

8-

*p(O .01

vs

48-hr.

0

10~

0 111"-=:=-:=-==-:F111 -F1F1-F1F1F 1

O4 12 24 48 96

TIME(hour.)

Figure1. Time course ofIL-1production in colonic tissue after ad-ministration of intravenous immunocomplexes.

IL-i

a(A) and IL-

1,BI

(B) weremeasured by specific radioimmunoassays after tissue extrac-tion (see text fordetails). Colonic tissue IL- 1 levels were below the

detection limit (dashed line) at time 0 and increased up to 48 h (P

A 70

- _50.

I 0 40

s 30Q

a

z 20

10-ol

0

70

2

50-80

1

40-3 4

INFLMMATORYINDEX

t

~A

30 .

20

A

10A iA

i

0 c . .

60

50-40

2

EDEMA

3 1

A

30 .

20 A A

10

_A~~~

A A

I _I

50

%NECROSIS

75

25

B 18

8

0I

W-14

12

104

84

42

2-. p(O.001

r-0.70

A

*

A

AA AA A

0 1 2 3 4 5 6 u

INFRAMMAYT0ItINDl

16R.

14

12

c

8

CID

%-O 104

84

6

4-

2-ur I I

0 1 2 3 4

EDEM

16 .

i

₈

I

14*

12

10+

8+

25 50

X NECROSIS

75 100

Figure 2.Relationshipof colonic tissue IL-I levelstoinflammationandtissue injury. Inflammatoryindex, edemaand mucosalnecrosiswere

microscopicallyassessedoncoded samples. Correlations coefficientsweredeterminedfrom all data points ofIL-Iandhistologicscores.(A)

Correlation with IL-la.(B)Correlation withIL-1If.

scriptional activation and appearance ofIL-I in thecolonic tissueafter theinitiation ofcolitiswereclearly demonstrated in

these studies. Tissue levels of IL-l rapidly increased at 4 h, reachedmaximalor nearmaximal levelsat24-48 h and then decreased to control values by 96 h after the induction of colitis. In parallel, IL-la and IL-1I3 mRNA levels, undetect-able before the induction of colitis,werefoundat4h, peaked

at 12 h, but wereundetectable by 96 h. Moreover, the

rela-tionship of IL-1 synthesistotheincrease ininflammation and tissuedamage is of particular importance. Therewere highly

significant correlations between IL-1a tissue levels and the inflammatory cell infiltration index, edema and necrosis (r =0.89, 0.79,and0.75, respectively). Rachmilevitzetal. have

recently shown increased colonic IL-1 production in a rat

model ofcolonic inflammation (35). However, in the latter study, IL-1 activity was measured by bioassays which are

known to detect IL-2, IL-4, IL-6, and IL-7. In combination theseresultssuggest that IL-l playsanimportant role in

ini-tiating colonic inflammation. The marked reduction in

in-flammatory cell infiltration, edema and necrosisseenafterthe administration of the IL-lraprovides additional strong sup-portforthishypothesis.

Several ofthebiological properties of IL-I place this

cyto-kine inastrategicposition during the cascade of inflammatory responsesinthis model.IL-1 triggersgeneexpressionfor

sev-eralinflammatorycytokines (1). Of these, the abilityof IL-1to

p(0.0001

r-0.79

ai

Is

ra

p(0.0001

r-0.79

A~~~~~~

A

AA

A AA

A

-

*

_{*-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~}

Ip

p(0.0005

r-0.75

A

A

*1.

p(0.005

r-0.60

A

6-B A A

4

_4AI

&A AA

2 A A

0 i

-1

nx

time (h)

0

4

12

48

96

A

IL-1:

B

IL-lcx

Figure3.Northernblotanalysisof colonicextractusi rabbit IL-1.Colonictissuewasobtainedatthe timesi induction of colitis forisolation of total RNA.(A) Hy withan830-bpIL-1l probe. (B) Hybridizationwitha tothesameNorthern blot afterstrippingtheprobe.

induce neutrophil-activating

protein-l

(IL-8) (36-38)

and other monocyte-derived chemotactic factors (39) is

particu-larly important

since these

factors

mayaccountfor the emigra-tion of neutrophils andmonocytesinto the injured colon. In

addition to IL-8, IL-1 stimulates gene expression of

itself,

tumor necrosis

factor, colony stimulating

proteins and IL-2

through

IL-6 (1).

Therefore,

IL- Imaybea

primary

initiator of inflammationaswellasbe involved in the prolongation of the

lesions associated with this model. The

ability

of the IL-lrato

reduce

significantly

colonic

inflammation

speaks for the

im-portanceofIL- I inboth phases of the colitis, but doesnotrule

outthe

participation

ofother mediators.

Colon IL- a concentrations were five timesgreater than IL- I levels. Incontrast,IL-IB mRNA levelsweregreater at4

h and similartothose of IL- aat12 and 48 h. These resultsare

in accord with other studies in human mononuclear cells where

IL-1B

mRNA has been

found

tobe

predominant

(40), whereas

cell-associated

IL- a

is

produced ingreateramounts

inresponsetoendotoxin

stimulation

(4, 41, 42). These

obser-vations

suggest

differences

in the regulation of IL-1aandIL-I

at _{the transcriptional and translational levels. We have}

re-cently shown that IL- a is more potent and more

effective

than

IL-lI#

in

inducing eicosanoid production

in rabbit colon ( 18). Taken together these datasuggestthat IL- amaybe the

predominant form

of IL-I produced by the rabbit colon. Pref-erential

production

of IL-la has

been

noted in other

tissues

and cell types; for example

human keratinocytes

appear to

produce

predominantly

IL-la(43).

Inflammation

and necrosiswere

decreased

_by

96 h

after

the

induction

of

colitis consistent

with the

decreases

in

IL-I

-_--

18

S

mRNA levels

and

IL-l

proteins. We

have

recently

shown that

pretreatment of rabbits with a

low-dose

of IL-ll 24 h

before

the

induction

of colitis protects

against

the

subsequent

devel-opment of

inflammation

through

a

mechanism that

requires

enhanced

prostaglandin

synthesis (44).

The

increased

synthe-sis of

_PGE2

is not

unexpected

as

IL-1

is

known

to

stimulate

gene

expression

for

cyclooxygenase (16)

as

well

as

phospholi-pase

A2

(45). Thus,

the marked

increased

in

PGE2

observedat

48 hmaybea

modulating factor responsible,

inpart, for the

decrease

in

inflammation observed

at 96 h.

Prostaglandins

may act

through

inhibition

of

cytokine

production

(4,

46,

47),

inhibition

of

leukotriene synthesis (48)

or

immune

suppres-sion

(49).

Secondary

inflammatory

cytokines

maybe

involved

in the

pathogenesis

of

inflammation

in this model.

These

would in-clude the

_ability

of IL- to

initiate transcription

of

IL-8 (50,

5

1)

and

GM-CSF

(52).

The IL-8 gene is

rapidly expressed

upon

< 18 S exposuretoIL- and IL-8

is chemotactic for

neutrophils and

1000

A *p(p 0.05va 0-hrs

0p(0.05va48-hra

800-

600-ing

acDNAto

indicated after ?

bridization

400-/

830-bh IL-Ia

TIME

(hours)

04 12 24 48 96

TIME(hour.)

Figure4.Timecourseof colonic PGE2 and LTB4productionafter administration of immune complexes. PGE2 and LTB4were mea-suredin rectal dialysatesatthe time points indicated. PGE2

produc-tionwassignificantlyincreased only 48 h after the induction of co-litis(P<0.05).LTB4 levelsweresignificantly increasedat24(P

<0.05)and48 h(P<0.02).At 96 h, colonic PGE2 and LTB4

pro-duction diminishedtolevels similartothose observed incontrol rab-bits(P<0.05). Each pointrepresentsthemean±SEMoffiverabbits. (A) PGE2. (B) LTB4.

a

z

0

3+

- vehicle EM IL-1ra

" p( 0.01

T

2+

1+

60

+

_ vehicle EM IL-Ira *" _{p (0.03}

T

40+

20+

oI

Figure 5. Effects oftreatmentwith IL-Ira oninflammation and tis-suedamageinrabbit immune colitis. IL-ira(5 mg/kg;n=8)was

administeredintravenously2 hbefore and 1,9, 17, 25, 33 h after the

immunocomplex administration. Control rabbits (n= 10)were

treated withvehicle(0.5mlPBS).Eachbarrepresentsthe mean±SEM.Asterisks indicatesignificant differences between the

twogroups(*P<0.02, **P<0.01,***P<0.03). Statistical analysis

wasperformed using the Mann-Whitney two-sampletest.

lymphocytes (53). Related molecules in this family also in-clude macrophage inflammatory proteins someof which are

structurallyrelatedtoIL-8, while othersproducedifferent

in-flammatoryeffectssuchasedema andcapillary leakage. GM-CSF increasesneutrophil superoxide production, whichmay

beinvolved in tissuedamage (54, 55).IL-l isalsoaninducer of TNF(56)and IL-6(57). AlthoughIL-6 hasnointrinsic inflam-matoryproperties, its abilitytostimulate BcellsaswellasT cellsmaycontributetotheprolongationof tissueresponseto

certainantigens. Finally,thesynergismbetween IL-1 andTNF

in

inducing

capillary-leak syndromes (58), PGE2 synthesis (59)

and cell

death

(60)

are well

established.

Thus, the early

expres-sion

of

IL- I

in

this

modelsuggests a

mechanism

whereby IL-l

initiates

acascadeof other cytokines acting independently or

synergistically with

IL- 1.

Arole

for lipid

mediators such as LTB4 and platelet

acti-vating factor

(PAF) in the

induction

and

enhancement

of

colon

inflammation in experimental colitis

has beenproposed by

several

studies

(18, 61-63). In

this

study, LTB4 synthesis was documented only 24 h after the induction of colitis and

did

not correlate

with

the development

of inflammation

or necrosis.

This

suggests that LTB4 production may be a second-ary event,

possibly

a consequence

of neutrophil

infiltration.

One

possible

sequence

of

events would be initial synthesis of

IL-l

followed

by

expression of

IL-8, IL-8 chemotaxis of

neu-trophils with subsequent production of

LTB4 (64).These data are

consistent with

our

preliminary studies

(unpublished

ob-servations) in which administration of specific

LTB4 receptor

blockers

or PAF

antagonists

to

rabbits

with formalin-immune

complex colitis failed

to

reduce

inflammation

or

tissue

dam-age. However,

LTB4 is

apotent

chemotactic

agent

for

neutro-phils

and monocytes and could be involved in amplifying the

initial inflammatory stimulus.

Although some

of

thenaturally occurring inhibitors of IL-I

activity

are

non-specific (for

example, lipoproteins and

uro-modulin),

the

existence of

a

specific

IL-l

inhibitor

was

demon-strated

by its

ability

to

block

IL-l

but not

mitogen

or IL-2

induced

thymocyte

proliferation.

Theinitial observation of a

specific

IL-l

inhibitor

was made in the sera from human vol-unteers

receiving

endotoxin (65) and this

was

followed

by re-ports

of specific

IL- 1

inhibitory activity in

the

urine of febrile

patients (66) and patients with

monocyte

leukemia

(67). The

urinary inhibitor

was

partially purified

as a 23-kD protein (68) and the

mechanism of its action

was shown to be at the level of

blocking

the

binding

of

IL-1 to T

cells and fibroblasts

(68) and

reduction in biological

responses to IL- 1 (69). The production

of

IL-l

inhibitory activity

was also shown by human mono-cytes

stimulated with immune complexes (70)

and was shown tobe

specific. This latter

report

has

particular significance for

the model of colitis

we use.

The

purification,

cloning, and

expression

of recombinant

IL-l

inhibitor (23)

have

provided

material

toshow

that it is

apure receptor

antagonist with

no

agonist

activity

and termed the

IL-

lra.

This

protein

has

al-lowed

usto

examine the

effects of

a

specific blockade of

IL- 1 receptors in

the colitis

model.

There are two

distinct

IL- 1 receptors in murine

cells;

an

80-kD

glycoprotein

foundonT

cells, fibroblasts, hepatocytes,

and natural

killer cells

anda

68-kD

protein

found

onB

cells,

macrophages,

and

neutrophils (71-73).

Initial studies

have shown that

the recombinant

nonglycosylated

IL- lra

(a)

binds to

cells

expressing

primarily

the 80-kD

IL-1

receptor

with

nearly

the same

affinity

as

IL-1;

and

(b)

competes

with either

IL- 1 aor IL-

1I3

onthese cells

(24).

The IL-lradoesnot

bind

to

murine cell lines

expressing

the 68-kD receptor. This may suggest that the receptors in

rabbit colonic tissue

are

of

the

80-kD

type.

However,

since the IL- Ira shares 26 identical and

40% amino acid

homology

with

IL-Ill

and

19 identical and 26%

conserved

homology

with

IL-la,

the

IL-lra may

also

block

the

binding

of

IL-I tothe

68-kD rabbit

IL-I receptor.

Although the

nature

of the cell

targets

for

the IL- I

in the colitis

model remains unknown, the present studies

establish the

effi-(I)

On

0

w

z

VEHICLE

IL-Ira

A

~

1~itS .r vt;k

zo f

Figure6.Representative colonicgross andmicroscopicappearanceafterIL-Iraorvehicletreatment.(A)Thevehicle-treatedcolonisthicken

withlarge areasoferythemaandfocal necrosis (arrows).The IL- ra-treatedcolon hasonly few foci oferythema(arrowheads). (B)

Vehicle-treated colonshowingmanyneutrophilsin thelaminapropria(arrows)andepithelialcellmucusdepletion(arrowhead). Hematoxylinand

eosin X300. (C) Colon treated withIL- 1rademonstratingfewinflammatorycells(arrow)and intactepithelium. Hematoxylinandeosin X>300.

cacyofthe IL- I ra toblock theinvivoactivityof IL- 1 produced

in the colon. Other pro-inflammatory cytokines induced by

the immunecomplexesincolitisareeitherplayingasecondary role or are expressed subsequent toIL- 1 synthesis.

Acknowledgments

The authors thank MarjorieLee andJennieJing fortechnical

assis-tance.WeacknowledgeDr. Alan R.Shaw and Dr. PaulWingfield of

the Glaxo Institute ofMolecularBiology, Geneva, Switzerland and

Joseph G. Cannon of Tufts University for theirvaluablecontribution

tothis work regardingthecloningandexpression of rabbit interleu-kin-l. Wealsothank Robert D. Zipser for helpful discussion of the manuscript.

Thiswork was support in part by grants DK-36869 andAI-15614

fromtheNational Institutes ofHealth. Dr. Cominelliisarecipientofa

Career DevelopmentAwardfromtheBlinderFoundation forCrohn's

disease.

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