IL-18, a Novel Immunoregulatory Cytokine, Is Up-Regulated

in Crohn’s Disease: Expression and Localization in Intestinal

Mucosal Cells1

Theresa T. Pizarro,2* Miette H. Michie,* Marcia Bentz,* Jan Woraratanadharm,*

Michael F. Smith Jr.,* Eugene Foley,†§ Christopher A. Moskaluk,‡§ Stephen J. Bickston,*§ and
Fabio Cominelli*§
IL-18, a novel immunoregulatory cytokine with potent IFN-g-inducing activities, may play an important role in Th1-mediated
chronic inflammatory disorders. The aim of the present study was to characterize the expression and localization of IL-18 in
colonic specimens and isolated mucosal cell populations from patients with Crohn’s disease (CD), a prototypic Th1-mediated
disorder. Using a semiquantitative RT-PCR protocol, IL-18 mRNA transcripts were found to be increased in freshly isolated
intestinal epithelial cells (IEC) and lamina propria mononuclear cells (LPMC) from CD compared with ulcerative colitis (UC) and
noninflamed control (cont) patients, and were more abundant in IEC compared with LPMC. Immunohistochemical analysis of
surgically resected colonic tissues localized IL-18 to both LPMC (specifically, macrophages and dendritic cells) as well as IEC.
Staining was more intense in CD compared with UC and cont, and in involved (inv) vs noninvolved (n inv) areas. Western blot
analysis revealed that an 18.3-kDa band, consistent with both recombinant and mature human IL-18 protein, was found pre-
dominantly in CD vs UC intestinal mucosal biopsies; a second band of 24 kDa, consistent with the inactive IL-18 precursor, was
detected in n inv areas from both CD and UC biopsies and was the sole form found in noninflamed cont. To our knowledge, this
report is the first describing increased expression of IL-18 in a human Th1-mediated chronic inflammatory disease. In addition,
our studies further support the concept that IEC and dendritic cells may possess important immunoregulatory functions in both
normal, as well as pathological, mucosal immunity. The Journal of Immunology, 1999, 162: 6829 – 6835.

nterleukin-18 was initially characterized as a novel IFN-g-

I
ple, polarized Th1 responses predominate in autoimmune thyroid-
stimulating factor in mice infected with Propionibacterium itis (9), multiple sclerosis (10), and Helicobacter pylori-induced
acnes and subsequently challenged with LPS (1). Following peptic ulcers (11). In Crohn’s disease (CD)3, one of the idiopathic
the cloning of both the murine and human forms of IL-18 and the chronic inflammatory bowel diseases (IBD), evidence has accu-
initial characterization of their biological activities (2, 3), it has mulated from animal models and human studies to suggest that
been suggested that IL-18 may play a primary role in Th1-medi- Th1 cytokines are involved in the pathogenesis of this condition
ated immune responses (4, 5). In fact, IL-18 acts as a costimulatory (12–14). Taken together, these observations suggest a critical role
factor for the proliferation of, and IFN-g production by, Th1 cells, for Th1 polarized responses in the aforementioned chronic inflam-
and these effects can occur independently of IL-12 (6). To further matory diseases; the specific initiating cytokine(s) driving Th1-medi-
support the concept that IL-18 may be a dominant proinflamma- ated immune responses, however, has not been fully characterized.
tory cytokine involved in Th1-mediated diseases is the recent ob- In the present report, we describe for the first time the expres-
servation that IL-18 has the ability to directly stimulate TNF-a sion of mRNA as well as of pro- and mature IL-18 protein in a
gene expression and synthesis from CD31/CD41 and NK cells typical Th1-mediated disease, i.e., CD. The tissue distribution as
with subsequent production of IL-1b and IL-8 from the CD141 well as the cellular localization of IL-18 in the gut mucosa of
population in peripheral blood (7). patients with IBD, particularly its prevalence in CD, is also reported.
There is increasing evidence that an imbalance of Th1/Th2 po-
larization in favor of Th1 cell subsets may be a key pathogenic Materials and Methods
mechanism in a variety of chronic inflammatory disorders and or- Specimens
gan-specific autoimmune diseases (reviewed in Ref. 8). For exam-
A total of 32 IBD and 16 noninflamed control (cont) (non-IBD) patients
were included in the present study. Colonic surgical specimens from pa-
Departments of *Medicine, †Surgery, and ‡Pathology, and §Digestive Health Center, tients with either ulcerative colitis (UC; n 5 10) or CD (n 5 10), who were
University of Virginia Health Sciences Center, Charlottesville, VA 22908 admitted to the University of Virginia Digestive Health Center for thera-
Received for publication October 22, 1998. Accepted for publication March 18, 1999. peutic bowel resection, as well as cont intestinal tissues obtained from
patients who underwent bowel resection for malignant and nonmalignant
The costs of publication of this article were defrayed in part by the payment of page conditions (n 5 10) were used as a source for mucosal cell populations
charges. This article must therefore be hereby marked advertisement in accordance
(intestinal epithelial cells (IEC) and lamina propria mononuclear cells
with 18 U.S.C. Section 1734 solely to indicate this fact.
(LPMC)) as well as immunohistochemical studies. Endoscopic biopsies
1
This work was supported in part by National Institute of Diabetes and Digestive and from CD, UC, and cont patients undergoing flexible sigmoidoscopy or
Kidney Diseases Grants 42191 and 45740 (to F.C.) and National Institute of Allergy
and Infectious Diseases Grant 40303 (to T.T.P).
2 3
Address correspondence and reprint requests to Dr. Theresa T. Pizarro, Division of Abbreviations used in this paper: CD, Crohn’s disease; IEC, intestinal epithelial
Gastroenterology and Hepatology, University of Virginia Health Sciences Center, cells; LPMC, lamina propria mononuclear cells; UC, ulcerative colitis; cont, nonin-
Building MR4, Box 1002, Room 1124, Charlottesville, VA 22908. E-mail address: flamed control; inv, involved; n inv, noninvolved; IBD, inflammatory bowel disease;
[email protected] h, human.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

6830 IL-18 EXPRESSION AND LOCALIZATION IN IBD

colonoscopy for diagnostic or surveillance purposes (n 5 6/group) were

used for Western blot analysis. All diagnoses were confirmed by clinical,
macroscopic, and histologic criteria. In addition, medical records of all
patients were reviewed and their histories and treatment modalities noted.
None of the patients were on immunomodulators (6-mercaptopurine, aza-
thioprine, and/or methotrexate) at the time of surgery. Their treatment reg-
imens included 5-aminosalicylic acid alone, steroids alone, 5-aminosali-
cylic acid and steroids, or no therapy at all. Informed consent was obtained
from each patient, and the project was approved by the Internal Review
Board of the University of Virginia Health Sciences Center.

Isolation of colonic mucosal cell populations and PBMC cell

culture
All colonic surgical specimens were collected immediately following re-
section, opened longitudinally, rinsed, examined for gross morphological
changes, and a representative full-thickness sample obtained. IEC and
LPMC were isolated and purified (separately) as previously reported in
detail (15). Briefly, for LPMC isolation, dissected intestinal mucosa was
freed of mucus and epithelial cells in sequential steps using DTT (Sigma,
St. Louis, MO) and EDTA, and subsequently digested with collagenase and
deoxyribonuclease (both from Worthington Biochemical, Freehold, NJ).
The resulting crude cell suspension was purified using a Ficoll-Hypaque
gradient (lymphocyte separation medium (LSM); Organon Teknika,
Durham, NC) following manufacturer’s protocol, and the preparations
preferentially enriched for intestinal LPMC were subsequently washed
twice with PBS and counted. Macrophages averaged 11% of LPMC, with
no significant differences between control and IBD cells, as previously
reported (15). IEC were isolated from mucosal strips by repeated incuba-
tions in a dispase solution (Boehringer Mannheim, Indianapolis, IN), fol-
lowed by gentle vortexing and filtration through a nylon column. Mono-
nuclear, red blood, and dead cells were removed using a 40% Percoll
gradient (Pharmacia LKB Biotechnology, Piscataway, NJ), where the IEC
equilibrated at the interface; resulting preparations preferentially enriched
for IEC were collected, washed twice in PBS, and subsequently counted. FIGURE 1. Expression of hIL-18 mRNA transcripts in freshly isolated
Using an immunoperoxidase method, all cells with epithelial morphology intestinal mucosal cells. A, Representative Southern blot of multiplex RT-
were stained with the anti-keratin mAb, AE1/AE3 (Boehringer Mann- PCR hybridized with a human full-length IL-18 cDNA probe confirmed
heim), with ,2% contaminating LPMC, as assessed by staining with a
IL-18-specificity of amplified PCR products and showed detectable IL-18
mAb directed against a leukocyte common Ag (CD45RB). PBMC, ob-
tained from a normal volunteer and used as a positive control, were isolated mRNA transcripts in both intestinal EC and LPMC from cont and IBD
through a Ficoll-Hypaque gradient (LSM; Organon Teknika) and cultured patients. Increased IL-18 mRNA steady-state levels were observed in in-
(5.0 3 106 cells/ml) at 37°C in a 5% CO2 atmosphere in the presence or testinal EC compared with LPMC and in mucosal cells from CD compared
absence of LPS (10 mg/ml; Sigma) for 6 h. For experiments analyzing with both cont and UC patients. B, Ratio of IL-18 to control GAPDH
IL-18 mRNA expression, total cellular RNA was prepared from freshly mRNA levels demonstrated a significant increase in both intestinal EC
isolated IEC and LPMC populations, as well as cultured PBMC, using (n 5 6/group) and LPMC (n 5 6/group) from CD, compared with cont and
TRIzol reagent (Life Technologies, Grand Island, NY) (107 cells/ml) ac- UC patients. Negatives of direct positive images were measured on gels,
cording to the manufacturer’s instructions, and processed by RT-PCR and relative quantitation of ethidium-stained bands from multiplex RT-
methodologies as later described.
PCR, representing integrated area under curve of densitometric tracing,
Processing of mucosal biopsies were reported as the ratio of target gene (IL-18) to housekeeping gene
(GAPDH). All samples were normalized to an internal standard (PBMC 1
Colonic mucosal biopsies were obtained and processed as previously de- LPS) processed with each experiment to account for interassay variability.
scribed (16). IBD specimens were derived from areas of active disease Each bar represents the mean 6 SEM. Asterisks indicate significant dif-
(involved (inv)) as well as macroscopically noninvolved (n inv) areas from
ferences between experimental groups (p, p , 0.02; pp, p , 0.03). Sta-
the same patient. In brief, tissue samples were rinsed twice in saline so-
lution, placed in Eppendorf tubes containing 500 ml of RIPA buffer (150 tistical analysis was performed using the Mann-Whitney two-sample test.
mM NaCl, 1% Igepal CA-630, 50 mM Tris (pH 8.0) containing aprotinin 279 bp, hIL-18.
(1.0 mg/ml), 0.5 mM PMSF, 0.1 mM sodium orthovanadate; all from
Sigma) and homogenized for 30 s using a Brinkmann (Westbury, NY)
hand-held polytron. Tissue homogenates were briefly centrifuged and im-
mediately frozen at 220°C for later assays.
only, and sterile double distilled H2O only, served as negative PCR con-
Semiquantitative RT-PCR for human (h)IL-18 trols. Singleplex PCR, using primer pairs specific for either hIL-18 or
hGAPDH only, was conducted in identical conditions as detailed above to
cDNA was synthesized by RT from 0.50 mg total cellular RNA using the verify multiplex PCR results and rule out the possibility of primer pair-
GeneAmp RNA PCR kit (Perkin-Elmer, Branchburg, NJ), according to primer pair interactions. Resulting amplified fragments were resolved on
manufacturer’s protocol. The aforementioned reaction in the absence of 3% NuSieve GTG (FMC BioProducts, Rockland, ME): 1.5% agarose gels
murine leukemia virus RT served as a negative RT control. Resulting stained with ethidium bromide and visualized through a UV light digital
cDNA (1.0 ml of RT reaction) was amplified in a 25-ml reaction volume imaging system. Negatives of direct positive images were measured using
containing primer pairs (0.4 mM/primer) of both the target gene (hIL-18) an analytical software (Scion Image 1.59; Scion, Frederick, MD), and rel-
and housekeeping gene (human GAPDH), according to manufacturer’s ative quantitation of ethidium-stained bands, representing integrated area
protocol (GeneAmp RNA PCR kit) using AmpliTaq Gold DNA polymer- under curve of densitometric tracing, were reported as the ratio of target
ase (both from Perkin-Elmer). PCR reaction mixture was overlaid with 50 gene (hIL-18) to housekeeping gene (hGAPDH). All samples were nor-
ml mineral oil, and multiplex PCR amplification was conducted in a DNA malized to the same internal standard (PBMC 1 LPS) processed with each
thermal cycler (Omnigene Thermocycler; Hybaid, Teddington, U.K.) un- experiment (n 5 6) and run on each gel to account for interassay variabil-
der the following conditions: 1 cycle of 94°C, 12 min; 30 cycles of 96°C, ity. The primer pairs used to amplify hIL-18 were: 59-GATCGCTTC
30 s; 55°C, 1 min; 72°C, 30 s; and 1 cycle of 72°C, 10 min. The same RT CTCTCGCAACAAACTA-39 (upstream) and 59-GTCCGGGGTGCAT
sample derived from PBMC stimulated with LPS was used as a positive TATCTCTACAGT-39 (downstream), resulting in a 279-bp fragment; and
PCR control/internal standard, and RT negative plus PCR mixture, mixture for the housekeeping gene, hGAPDH: 59-GGAAGGTGAAGGTCG
The Journal of Immunology 6831

FIGURE 2. Immunohistochemical stain-

ing for hIL-18 in paraffin-embedded sections
from noninflamed control and UC intesti-
nal tissue resections. A, Using an affinity-
purified goat anti-human IL-18 polyclonal
Ab, cont colonic mucosa obtained from a
resection specimen without evidence of
IBD revealed weak to moderate staining of
superficial IEC, as well as scattered inflam-
matory cells within the lamina propria. C,
In colonic mucosa not involved by inflam-
matory changes from a UC patient (UC n
inv), more diffuse and qualitatively in-
creased staining of the epithelium was ob-
served when compared with cont. E, In in-
flamed and eroded mucosa from a UC
specimen (UC inv), increased epithelial
staining compared with cont was again ob-
served; in addition, granulation tissue ad-
jacent to the epithelium demonstrated
weak to moderate staining of scattered in-
flammatory cells. B, D, and F, Control im-
munohistochemical reactions using an iso-
type goat IgG that correspond to specimens
in A, C, and E, respectively, are shown.
A–D, 3100 original magnification; E and
F, 3150 original magnification.

GAGTC-39 (upstream) and 59-AAGGTGGAGGAGTGGGTGTC-39 well as macroscopically n inv areas. Tissue samples were fixed by immer-
(downstream), resulting in an 880-bp amplified fragment. sion in 10% buffered formalin phosphate (Fisher Scientific, Pittsburgh, PA)
for 2–7 days at 4°C, sequentially dehydrated in 50%, 70%, 95%, and ab-
Southern blot analysis solute ethanol (30 min/each) with agitation, and finally cleared in xylene
Southern blot analysis was performed on resolved multiplex PCR products (2 3 30 min). Samples were then embedded in Paraplast1 embedding
to verify the specificity of hIL-18 PCR-amplified cDNA fragments. Spe- media (Oxford Labware, St. Louis, MO), and resulting blocks were stored
cifically, resulting gels were denatured using a 1.5 M NaCl/0.5 M NaOH at room temperature for later tissue sectioning. Five-micron-thick serial
solution (45 min), rinsed briefly with double distilled H20, and neutralized sections were obtained, mounted on poly-L-lysine-coated Superfrost/Plus
with a 1.0 M Tris/1.5 M NaCl solution (2 3 30 min). Gels were then glass slides (Fischer Scientific), deparaffinized in xylene (2 3 10 min), and
transferred to Magnagraph nylon membranes (Schleicher & Schuell, dehydrated in absolute ethanol (2 3 2 min). Colonic tissue sections were
Keene, NH) using 203 SSC buffer, and transferred DNA fragments were then blocked with normal rabbit serum (Vectastain Elite ABC kit; Vector
UV-cross-linked (Fb-UVXL-1000 X-linker; Stratagene, La Jolla, CA) to Laboratories, Burlingame, CA) for 20 min, permeabilized for 15 min with
membranes. Resulting filters were prehybridized for 4 h at 65°C in a so- saponin buffer (1% FCS, 0.1% sodium azide, 0.1% saponin in PBS; all
lution containing 7% SDS, 1% polyethylene glycol, 23 standard saline from Sigma), and blocked again for endogenous peroxidase using 1% H202
citrate phosphate/EDTA, and 5% BSA/nonfat milk; an IL-18 probe, syn- in saponin buffer for 45 min away from light. IL-18-producing cells were
thesized from a full length 481-bp hIL-18 cDNA sequence and radiola- immunostained, according to manufacturer’s protocol (Vectastain Elite
beled with [a-32P]dCTP using the Prime-a-Gene labeling system (Pro- ABC kit), for 12–16 h using an affinity-purified goat anti-human IL-18
mega, Madison, WI), was subsequently added (at 4 3 106 cpm/ml) and polyclonal Ab (1.0 mg/ml) (a kind gift from M. Tsang, R&D Systems,
filters hybridized overnight at 65°C. Hybridized filters were washed (2 3 Minneapolis, MN) as the primary detecting Ab. The following day, slides
15 min) at 65°C in 1% SDS/50 mM NaCl/1.0 mM EDTA, air-dried, ex- were washed in saponin buffer, incubated with a biotinylated anti-goat IgG
posed to X-OMAT autoradiography film (Eastman Kodak, Rochester, NY) for 45 min, washed again in saponin buffer, and incubated with an avidin-
with intensifying screens at 280°C, and film was subsequently developed. biotin complex (ABC) for 30 min away from light. Immunoreactive cells
were visualized by addition of diaminobenzidine substrate and lightly
Immunohistochemical studies
counterstained with hematoxylin. All incubations were conducted at room
As mentioned earlier, all colonic surgical specimens were collected imme- temperature, unless otherwise noted. Isotype control sections were pre-
diately following resection, opened longitudinally, rinsed, examined for pared under identical immunohistochemical conditions, as described
gross morphological changes, and a representative full-thickness sample above, replacing the primary IL-18-detecting Ab with a purified, normal
obtained. IBD specimens were derived from areas of active disease (inv) as goat IgG control Ab (R&D Systems).
6832 IL-18 EXPRESSION AND LOCALIZATION IN IBD

FIGURE 3. Immunohistochemical stain-

ing for hIL-18 in paraffin-embedded sections
from CD intestinal tissue resections. A, Us-
ing an affinity-purified goat anti-human
IL-18 polyclonal Ab, colonic mucosa not
involved by inflammatory changes in a pa-
tient with CD (CD n inv) revealed diffuse
and strong staining of both the colonic ep-
ithelium, as well as scattered inflammatory
cells within the lamina propria. C, In the
earliest manifestations of CD (i.e., “aph-
thoid” lesion: focal neutrophilic cryptitis
overlying a normal lymphoid aggregate),
intense staining is again observed in the
epithelium with an increased number of in-
flammatory cells in the lamina propria also
showing strong staining. E, In contrast to
the findings in UC, inflamed and eroded
mucosa from a colonic resection severely
involved with CD showed intense staining
of inflammatory cells present in granula-
tion tissue and within the lamina propria.
B, D, and F, Control immunohistochemical
reactions using an isotype goat IgG that
correspond to specimens in A, C, and E,
respectively, are shown. A and B, 3100
original magnification; C–F, 3150 original
magnification.

Western blot analysis were analyzed using a statistical program (BMPD, Los Angeles, CA). The
methods used included the Kruskal-Wallis test for nonparametric data and
Total protein levels of tissue homogenates derived from colonic mucosal the Mann-Whitney two-sample test for parametric data. Differences were
biopsies (n 5 6/experimental group) were quantitated using a modification considered significant when p , 0.05.
of the Bradford colorimetric procedure (Bio-Rad Protein Assay; Bio-Rad
Laboratories, Hercules, CA). Biopsy homogenates (standardized to 20 mg
of total protein/lane) were then resolved by 15% denaturing SDS-PAGE
and transferred for 1 h at 100 V in transfer buffer (20% methanol, 192 mM Results
glycine, 25 mM Tris (pH 8.0); all from Sigma) onto polyvinylidene diflu- Expression of IL-18 mRNA transcripts in isolated mucosal cell
oride membrane (Bio-Rad) using an electrophorectic transfer unit (Mini
populations
Trans-Blot Electrophorectic transfer cell; Bio-Rad). Recombinant hIL-18
(35 ng) (a kind gift from Dr. Monica Tsang) was used as a positive control. The expression of mRNA transcripts in freshly isolated CD, UC,
Following transfer, membranes were blocked overnight at 4°C in 20 ml of and noninflamed controls is represented in Fig. 1. IL-18 mRNA
5% nonfat dry milk in PBS, washed in PBS containing 0.1% Tween (Sig-
ma) (3 3 10 min), and incubated with an affinity-purified goat anti-human transcripts were found to be more abundant in both IEC and LPMC
IL-18 polyclonal Ab (1.0 mg/ml) (a kind gift from M. Tsang) for 3 h at obtained from CD compared with UC and controls. Southern blot
room temperature on an orbital shaker. Blots were subsequently washed analysis (Fig. 1A) confirmed the specificity of the amplified prod-
(3 3 10 min), as above, and incubated with an anti-goat IgG conjugated ucts to hIL-18. Fig. 1B shows the relative levels of IL-18 mRNA
HRP (1:100) (Sigma) for 1.5 h at room temperature, also on an orbital
shaker. Finally, blots were washed (2 3 10 min), as above, and in PBS only
transcripts in IEC and LPMC obtained from CD, UC, and nonin-
(1 3 10 min), incubated with 6.5 ml/membrane of enhanced chemilumi- flamed controls (n 5 6/group). IL-18 mRNA transcripts were sig-
nescence detection reagent (Amersham Life Science, Arlington Heights, nificantly increased in CD (2.68 6 0.36 for IEC and 1.25 6 0.17
IL) for 1 min at room temperature, and exposed to X-OMAT autoradiog- for LPMC) compared with cont (1.29 6 0.31 for IEC, p . 0.02
raphy film with intensifying screens for 5–15 s. and 0.55 6 0.19 for LPMC, p , 0.03) as well as UC (1.89 6 0.26
for IEC, p , 0.04 and 0.72 6 0.12 for LPMC, p , 0.04). Although
Statistical analyses a trend in increased IL-18 mRNA levels was detected in intestinal
For IL-18 mRNA levels, all samples were normalized to the internal stan- mucosal cell populations from UC compared with cont, these ob-
dard and results (IL-18/GAPDH) are expressed as mean 6 SEM. The data served differences did not reach statistical significance.
The Journal of Immunology 6833

FIGURE 4. Cellular localization of

hIL-18 expression in paraffin-embedded sec-
tions from CD intestinal tissue resections,
as detected by immunohistochemistry. A,
Epithelium uninvolved by inflammation
(CD n inv) showed intense staining of IEC
and scattered inflammatory cells within the
lamina propria (3400 original magnifica-
tion). B, Positively staining mononuclear
cells located within the lamina propria
possess abundant cytoplasm, vesicular
retiform nuclei, and are morphologically
consistent with tissue macrophages (histio-
cytes) (3450 original magnification). C,
Submucosal lymphoid aggregate demon-
strated hIL-18-positive cells (3100 origi-
nal magnification), and upon higher power
(D) showed that the positively staining
cells have abundant cytoplasm, extended
cytoplasmic processes, and are morpholog-
ically consistent with dendritic cells (3450
original magnification).

Immunohistochemical localization of IL-18 in surgically resected Fig. 3 demonstrates immunohistochemical staining for hIL-18 in
colonic specimens CD. In n inv colonic mucosa obtained from patients with CD,
In colonic mucosa obtained from surgical noninflamed controls, diffuse and strong staining of the colonic epithelium, as well as
weak to moderate staining of superficial IEC and scattered inflam- scattered inflammatory cells in the lamina propria, was observed
matory cells within the lamina propria was observed (Fig. 2A). By (Fig. 3A). In apthoid lesions (focal neutrophilic cryptitis overlying
comparison, more diffuse and qualitatively increased IL-18 stain- a normal lymphoid aggregate), which are hallmark features of the
ing of the epithelium was observed in n inv colonic mucosa of UC earliest manifestations of CD, intense staining of IL-18 was ob-
patients (Fig. 2C). In severely inflamed UC tissues, increased epi- served in the epithelium as well as in the inflammatory cells in-
thelial staining, as well as weak to moderate staining of scattered filtrating the lamina propria (Fig. 3C). In contrast to the results
inflammatory cells, was detected (Fig. 2E). Corresponding control obtained from UC patients, severely affected CD colonic tissue
sections using an isotype goat IgG are shown in Fig. 2, B, D, and F. resections, particularly advanced lesions, revealed intense staining
of inflammatory cells in granulation tissue and within the lamina
propria (Fig. 3E). Corresponding control sections using an isotype
goat IgG are shown in Fig. 3, B, D, and F.
The cellular localization of IL-18 expression in CD by immu-
nohistochemistry (Fig. 4) showed that IEC, as well as scattered
inflammatory cells within the lamina propria, were a major source
of IL-18 (Fig. 4A). Mononuclear cells located within the lamina
propria that possessed abundant cytoplasm, vesicular retiform nu-
clei, and were morphologically consistent with tissue macrophages
(histiocytes) also stained positively for hIL-18 (Fig. 4B). In addi-
tion, submucosal lymphoid aggregates from CD intestinal tissues
FIGURE 5. Western blot analysis of hIL-18 expression in colonic mu- demonstrated positive IL-18 expression (Fig. 4C); specific IL-18
cosal biopsies obtained from IBD and noninflamed control patients. An staining cells located within these aggregates possessed abundant
amount of 20 mg of total protein from tissue homogenates (n 5 6/exper- cytoplasm, extended cytoplasmic processes, and were identified as
imental group) was resolved by 15% denaturing SDS-PAGE and trans-
being morphologically consistent with dendritic cells (Fig. 4D).
ferred onto polyvinylidene difluoride membrane. Membranes were blocked
with 5% nonfat dry milk in PBS, subsequently washed in PBS containing
0.1% Tween, and incubated with an affinity-purified goat anti-human IL-18
Expression of pro- and mature IL-18 protein in mucosal
polyclonal Ab (1.0 mg/ml). Blots were finally incubated with an anti-goat biopsies
IgG conjugated HRP Ab (1:100), and hIL-18 was detected by enhanced Western blot analysis of colonic mucosal biopsies demonstrated
chemiluminescence. Representative blot of six separate experiments is the presence of both the pro- and mature forms of hIL-18. A blot
shown and demonstrates that the 18.3-kDa mature form of IL-18 (lower representative of six separate experiments (Fig. 5) demonstrates
arrow) is more abundant in CD compared with UC intestinal mucosal bi-
that the 18.3-kDa mature form of hIL-18 is more abundant in in-
opsies and is absent in cont. The 24-kDa inactive precursor form (middle
arrow) was detected in nonaffected areas from both CD and UC biopsies
testinal mucosal biopsies from CD compared with UC patients and
and was the sole form found in normal noninflamed controls. A third band is absent in noninflamed controls. The 24-kDa inactive precursor
of ;31 kDa was observed in n inv CD and UC colonic mucosal biopsies form of IL-18 was detected in nonaffected areas from both CD and
and was absent in cont as well as involved areas of IBD specimens (upper UC biopsies and was the sole form found in normal noninflamed
arrow). Recombinant hIL-18 (35 ng) was used as a positive control. controls. A third band of ;31 kDa was observed in n inv CD and
6834 IL-18 EXPRESSION AND LOCALIZATION IN IBD

UC colonic mucosal biopsies and was absent in noninflamed con- of IL-18 to this pathogenic model. In fact, IL-18 is not only pro-
trols as well as inv areas of IBD specimens (Fig. 5). In addition, the duced by APC (i.e., tissue histiocytes and dendritic cells) to mount
control lane contains a band ;36 kDa in size, and most likely a Th1 polarized response in synergy with, and independently of,
represents a dimer product of the recombinant hIL-18 protein. IL-12 through IFN-g production (Th1-inductive phase), but it also
stimulates TNF-a gene expression and secretion from activated T
cells (effector phase) (7). In addition, IL-18 is capable of stimu-
Discussion lating IL-1b as well as IL-8 from activated macrophages, thus
There is increasing evidence that IL-18 may be a key proinflam- affecting the final common pathway of CD immunopathogenesis.
matory cytokine as well as an important mediator of Th1 polarized Therefore, it is conceivable that IL-18 may, in fact, fulfill the req-
immune responses (reviewed in Ref. 17). In the present study, we uisite as a primary initiating cytokine in Th1-mediated diseases,
investigated the expression and cellular localization of IL-18 in such as CD. Recent animal studies using mAb neutralization
CD, a prototypic Th1-mediated disease. Our results show that the against IL-18 in organ-specific autoimmune diseases have sup-
mature form of IL-18 is indeed markedly overexpressed in intes- ported this concept (17).
tinal lesions of CD patients, but not in UC, another form of IBD in In summary, our studies provide conclusive evidence that IL-18
which polarized Th2 responses are believed to play a dominant may play a key pathogenic role in Th1-mediated disorders, such as
role (18). To our knowledge, this is the first report of a putative CD, and provide a rationale for anti-IL-18-based treatment in these
role of IL-18 in mediating human organ-specific autoimmunity. conditions. Differently from current anti-cytokine strategies, such
IL-18 transcripts were detected in colonic mucosal cells from as blockade of TNF-a in CD and rheumatoid arthritis, which pri-
both CD and UC, as well as noninflamed control patients. How- marily target the Th1 effector phase, this approach would possess
ever, IL-18 mRNA levels were significantly increased in both the theoretical advantage of affecting the inductive phase of Th1 T
LPMC and IEC populations from CD patients only when com- cell activation. Future animal, as well as human, studies will test
pared with the aforementioned experimental groups. By compar- the validity of this hypothesis.
ison, although the inactive pro-form (24 kDa) was the sole form
detected in noninflamed controls and in macroscopically n inv IBD Acknowledgments
colonic biopsies, the mature form was highly expressed in only We thank Monica Tsang for supplying human rIL-18 and the polyclonal
affected CD tissues. The increased expression of mature IL-18 pro- anti-IL-18 Ab, Michael F. Smith Jr. for his collaborative efforts in synthe-
tein appears to be specific for CD, since UC tissues with compa- sizing the hIL-18 cDNA probe, Kristen Arseneau and Jeannie Shifflet for
rable severity of inflammation displayed only minimally detectable their assistance with the IBD database, and Mitchell Guanzon for excellent
levels of IL-18. Thus, the activation of IL-18 in CD is unlikely technical assistance.
secondary to the inflammatory responses, but may represent a pri-
mary abnormality within this patient population. However, since References
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