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*
Department of Experimental Biology and Pharmacology, Serono Pharmaceutical Research Institute, Geneva, Switzerland;
Laboratory of Experimental Internal Medicine, Academic Medical Center, Amsterdam, The Netherlands;
InterPharma Laboratories, Nes Ziona, Israel;
Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, CO 80262; and
¶ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Certain cytokines, such as IL-18, originally described as the
IFN-
-inducing factor (10), may play an important role
in the dysregulation of Th1-mediated diseases. IL-18, mainly produced
by APCs, can synergize with IL-12 to increase IFN- secretion,
enhancing NK cell cytotoxicity and stimulating Th1 responses (11, 12). IL-18 also exerts proinflammatory effects by inducing
TNF-
, IL-1
, chemokines such as IL-8, macrophage-inflammatory
protein-1, and monocyte chemoattractant protein-1
(13), NO (14), and adhesion molecules
(15). Recent studies have reported an increase in IL-18
expression by intestinal epithelial cells and macrophages in the
lesions of CD (16, 17, 18, 19) and in the sera of patients with CD
(19). The IL-18-induced cytokines IFN-, TNF-,
IL-1, and IL-8 were also shown to be up-regulated in these lesions
(18). These observations strongly imply that IL-18 plays a
role in the pathogenesis of CD.
IL-18-binding protein (IL-18BP) has been previously described as a
secreted protein that binds and neutralizes IL-18 (20). As
such, it regulates IL-18-induced IFN- production and consequently
influences the Th1 and inflammatory responses. With a single Ig domain,
IL-18BP resembles the extracellular segment of a cytokine receptor.
However, IL-18BP is a novel protein distinct from the IL-1 and IL-18
receptor family members. The human IL-18BP gene encodes at least four
distinct isoforms (IL-18BPa–d), which are derived by alternative
splicing. The isoforms differ primarily in their carboxyl termini and
biological activity. IL-18BP isoforms a and c neutralize the biological
activity of IL-18, whereas b and d do not (21). IL-18BPa
is constitutively expressed in human spleen and, to a lesser extent, in
colon, small intestine, and prostate (20). It is presently
unknown whether the other isoforms are expressed and secreted in vivo.
In addition, little is known about the regulation of IL-18BP
expression. IL-18BP mRNA has been shown to be induced by IFN- in
several cells lines, such as keratinocytes, colon carcinoma/epithelial
cells, and renal mesangial cells (22, 23). However,
IL-18BP expression, cellular localization, and regulation during
disease process remain to be defined. Therefore, in this study we
evaluated the expression of the different isoforms of IL-18BP and
determined the balance between IL-18 and IL-18BP in active CD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgically resected specimens were obtained from patients with active CD undergoing a partial intestinal resection and were immediately snap-frozen. Sixteen CD patients (4 men and 12 women) with a mean age of 42.4 years (range, 27–78 years) and a mean disease duration of 9.5 years (range, 2–21 years) were included. The primary site of involvement was colonic in five patients, ileal in nine patients, and ileocolonic in two patients. The surgically resected specimens contained all layers of the intestinal wall. The diagnosis for the activity of the disease was made by histopathological examination of the surgically resected specimens based on the following criteria: presence of ulcerations, increased numbers of inflammatory cells, and transmural inflammation. At the time of surgery, six patients were on corticosteroids (CS), three on CS plus mesalazine, three on CS plus azathioprine, one on CS plus mesalazine and azathioprine, one on paracetamol, one on CS plus paracetamol, and the remaining one was off treatment. For all patients, indication for surgery was a chronic active course poorly responsive to medical treatment. Control surgically resected specimens (six males and nine females) were obtained from patients undergoing a partial intestinal resection for bowel cancer and were snap-frozen. Control specimens were from macroscopically and microscopically unaffected areas. The mean age of this group was 55.5 years (range: 24–81 years). The surgically resected specimens were used for immunohistochemical studies, real-time PCR, and Western blot analysis. The Medical Ethical Committee of the Academic Medical Center (Amsterdam, The Netherlands) approved the study.
rhIL-18 and rhIL-18BP isoforms
Recombinant human (rh)IL-18 was prepared in E. coli as mutated pro-IL-18 fused to GST, engineered to contain a caspase-8 cleavage site. Following purification, mature human IL-18 was released by caspase-8 cleavage. rhIL-18BP-6His isoforms a–d were transiently expressed in HEK 293 cells as previously described (21). Conditioned media were collected after 72 h and stored at -80°C. rhIL-18BPa-6His was purified to homogeneity from a stable Chinese hamster ovary cell line (21).
Abs to hIL-18BP
Anti-human (h)IL-18BP mAbs 582.10 and 657.27 were used for Western blot analysis, ELISA, and FACS analysis (24). Anti-hIL-18BP mouse polyclonal was generated by injecting BALB/c mice s.c. with rhIL-18BPa-6His and used for Western blot analysis.
Anti-hIL-18BP mAb 95-H20 was generated by injecting BALB/c mice s.c. into the four limbs as well as intranuchally, with 50 µg per injection of rhIL-18BPa-6His in PBS with adjuvant (monophosphoryl lipid A plus trehalose dicorynomycolate Emulsion; RIBI adjuvant system (Corixa, Hamilton, MT)) on days 0, 7, and 28. Four days after the third immunization, the lymph nodes were removed and digested with 2.4 µg/ml collagenase IV (Worthington Biochemical, Lakewood, NJ) and 0.1% DNase (Sigma-Aldrich, St. Louis, MO). Isolated cells were then fused with Sp2/0 myeloma cells using polyethylene glycol 1000 (Fluka, Buchs, Switzerland). Hybridomas were resuspended in DMEM-F12, 10% FCS (Life Technologies, Rockville, MD) in the presence of hypoxanthine, aminopterin, and thymidine and plated in 96-well plates at a concentration of 5 x 104 cells/ml. Hybridoma culture supernatants were screened for anti-IL-18BP-reactive Abs by ELISA. Briefly, ELISA plates were coated with goat anti-mouse F(ab')2 Abs (Jackson ImmunoResearch Laboratories, West Grove, PA). Hybridoma culture supernatants were added followed by biotinylated rhIL-18BP-6His. To select Abs directed against free IL-18BP and IL-18BP bound to IL-18, the screening was performed with or without rhIL-18. Non-neutralizing Abs recognizing free and bound IL-18BP were selected and subcloned. mAb 95-H20, a mouse IgG1 mAb, was used for immunohistochemical studies.
Immunohistochemical studies
Serial cryosections (10 µm) from control or active CD surgically resected specimens were mounted on poly-L-lysine-coated Superfrost/Plus glass slides (Polylabo, Geneva, Switzerland) and fixed in ice-cold acetone. After a brief rehydration in PBS, sections were blocked for 30 min in PBS supplemented with 2% FCS (Cansera, Rexdale, Canada), 1% human serum (AB+ serum; Transfusion Center, Annemasse, France) and 0.5% BSA (Sigma-Aldrich). Endogenous peroxidase activity was quenched with 1% hydrogen peroxide (H2O2; Fluka) in PBS, 2% FCS, 1% human serum, and 0.5% BSA for 1 h. IL-18BP-producing cells were immunostained overnight with neat culture supernatant containing anti-hIL-18BP mAb 95-H20, followed by biotinylated goat anti-mouse Ab (5 µg/ml; Jackson ImmunoResearch Laboratories) in PBS containing 0.5% BSA for 1 h and then avidin/biotinylated HRP complex (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) for 30 min. Slides were developed using 3-amino-1-ethylcarbazole (Sigma-Aldrich) according to the manufacturer’s protocol, counterstained with hematoxylin (Sigma-Aldrich), and mounted.
Double immunofluorescence labeling was performed to identify cell types expressing IL-18BP. After rehydration and blocking (as above), sections were incubated overnight with anti-hIL-18BP biotinylated mAb 95-H20 (20 µg/ml) mixed with either the endothelial cell marker, FITC-conjugated mouse anti-human CD31 (1/50; BD PharMingen, San Diego, CA) or the macrophage/monocyte marker, FITC-conjugated anti-human CD68 (1/25; DAKO, Carpinteria, CA), in PBS 0.5% BSA. Following a PBS wash, IL-18BP labeling was visualized using streptavidin-Texas Red (Southern Biotechnology Associates, Birmingham, AL). All isotype-matched controls were negative.
RNA, cDNA, and protein preparation
RNA and protein were extracted from control or active CD surgically resected specimens using TRIzol (Life Technologies) according to the manufacturer’s instructions, which allowed total RNA and protein recovery from the same tissue sample. RNA integrity was assessed by electrophoresis on 1% agarose gels. cDNA was synthesized from 1 µg of total RNA using a reverse transcription system (Promega, Madison, WI) according to the manufacturer’s protocol. After RNA recovery, proteins were extracted by sequential precipitation. Total protein was quantified using the bicinchoninic acid protein assay (Pierce, Rockford, IL). Protein extraction from TRIzol was validated by comparison of proteins directly lysed in Laemmli sample buffer and proteins extracted from TRIzol. Both methods gave identical protein profiles by SDS-PAGE and Coomassie blue staining. Western blotting of both protein preparations also gave identical specificity and intensity.
Real-time PCR
SYBR green real-time PCR primers for human IL-18BP, IL-18, and
GAPDH (housekeeping control) were designed using Primer Express
software from PE Applied Biosystems (Foster City, CA) (Table I
). The specificity and optimal primer
concentrations were tested. The IL-18BP primers did not distinguish
among the various isoforms. Potential genomic DNA contamination was
excluded by performing PCR with specific intron-GAPDH primers. The
absence of nonspecific amplification was confirmed by analyzing the PCR
products by agarose gel electrophoresis. Real-time PCR was performed
with 5 µl/well of reverse transcription products (0.5 ng total RNA),
25 µl/well SYBR Green PCR master mix (PE Applied Biosystems) with 0.5
U/well of AmpErase (PE Applied Biosystems) uracil
N-glycosylase and 300 nM primers. PCR was performed at
50°C for 2 min and 95°C for 10 min, and then run for 40 cycles at
95°C for 15 s and 60°C for 1 min on the ABI PRISM 7700
Detection System (PE Applied Biosystems). The reverse-transcribed cDNA
samples were thus amplified and their cycle threshold values were
determined. All cycle threshold values were normalized to the
housekeeping gene GAPDH.
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Semiquantitative PCR primers for human IL-18BP and -actin
(housekeeping control) were designed (Table I
). PCR were performed in a
total volume of 50 µl in the presence of 1.25 U of AmpliTaq DNA
Polymerase (PerkinElmer, Wellesley, MA), 2.5 mM dNTPs (Amersham,
Arlington Heights, IL), and 50 pmol of forward and reverse PCR primers.
Reactions were incubated in a PTC-200 Peltier Effect Thermal Cycler (MJ
Research, Cambridge, MA) under the following conditions: denaturation
for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1
min at 72°C. Absence of contaminating genomic DNA was confirmed by
performing PCR on the nontranscribed RNA. PCR products were analyzed by
agarose gel electrophoresis and the band size was verified by
comparison with a 1-kb ladder (Life Technologies).
ELISA
Maxisorp microtiter 96-well ELISA plates (Labsystems, Chicago, IL) were coated with 100 µl/well anti-hIL-18BP mAb 582.10 (2 µg/ml) overnight at room temperature. Soluble hIL-18BP was then detected using biotinylated anti-hIL-18BP mAb 657.27 (0.5 µg/ml) followed by incubation with peroxidase-conjugated extravidin (1/10,000; Sigma-Aldrich). The sensitivity of the ELISA was 100 pg/ml. Conditioned media from HEK 293 cells transfected with the different hIL-18BP isoforms illustrated that the ELISA recognized only isoforms a and c. IL-18/IL-18BP complexes were then formed using rhIL-18 with conditioned media containing either isoform a or c and tested. This experiment demonstrated that the ELISA detected only free IL-18BP (data not shown).
Levels of IL-18 were quantified by using a commercial ELISA (Medical and Biological Laboratories, Nagoya, Japan; Immunotech, Westbrook, ME) whose sensitivity was 12.5 pg/ml. Experiments, also conducted in our laboratory, showed that this ELISA recognized total IL-18 and thus cannot discriminate between free and IL-18BP-bound IL-18 (data not shown).
Western blot analysis
For the detection of hIL-18BP and hIL-18, 10 µg of protein from control and active CD surgically resected specimens was separated by SDS-PAGE and transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Membranes were blocked for 2 h at 37°C with 5% PBS-defatted milk powder and then incubated for 1 h at room temperature with mouse anti-hIL-18BP mAb 657.27 (1 µg/ml), mouse polyclonal anti-hIL-18BP, or goat polyclonal anti-hIL-18 (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were then washed extensively before incubation with an anti-mouse or anti-goat HRP-conjugated secondary Ab (Sigma-Aldrich), respectively, and visualized by chemiluminescence (ECL reagents, Hyperfilm ECL; Amersham). To confirm the identity of the different isoforms, conditioned media from transiently transfected HEK 293 cells expressing the different isoforms were used as positive controls. For the detection of IL-18/IL-18BP complexes, the molar equivalent of rhIL-18 was incubated with conditioned media from HEK 293 cells transfected with either isoform a or c for 30 min at room temperature before electrophoresis and Western blotting.
Cell culture
Primary HUVECs (Clonetics, San Diego, CA) were cultured
according to the manufacturer’s instructions. All experiments were
performed using the HUVEC at passage 3. HUVEC were treated with a
mixture of cytokines, IL-1 (10 ng/ml), TNF- (10 ng/ml), and
IFN- (20 ng/ml) (R&D Systems, Minneapolis, MN), for 24 h. Cells
were then harvested, and the RNA was isolated and subjected to RT-PCR
for analysis of IL-18BP mRNA levels. Supernatants were collected and
analyzed for IL-18BP production by ELISA.
Human PBMC were isolated from heparinized blood from healthy donors by
Ficoll density gradient centrifugation (Pharmacia, Peapack, NJ).
Monocytes/macrophages were isolated from whole PBMC preparations by
depletion of non-monocytes using MACS Monocyte Isolation kit (Miltenyi
Biotec, Auburn, CA) according to the manufacturer’s instructions. The
purity was assessed by FACS analysis (> 93% of
CD64+ cells). Isolated cells were seeded in
48-well plates at 2 x 107 per ml (6 x
106 per well) and cultured in RPMI 1640
supplemented with 10% human serum type AB (Sigma-Aldrich), 2 mM
L-glutamine, and 10 U/ml penicillin/streptomycin. Total
PBMC were stimulated with the mixture of cytokines supplemented with
LPS (100 ng/ml; Calbiochem, La Jolla, CA) or with IL-18 (20 ng/ml;
Promega), IL-1 (10 ng/ml), TNF- (10 ng/ml), IFN- (20 ng/ml)
(R&D Systems) and LPS alone. Purified monocytes/macrophages were
stimulated with IFN- (20 ng/ml) and TNF- (10 ng/ml). Supernatants
were analyzed for IL-18BP by ELISA 48 h after stimulation.
Flow cytometry analysis of IL-18BP expression
Intracellular IL-18BP was detected in PBMC by flow cytometry
using a FACSCalibur (BD Biosciences, Mountain View, CA). Brefeldin A (5
µg/ml; Sigma-Aldrich) was added 4 h before assay. FITC-labeled
anti-CD64 mAb (BD PharMingen) was used as a marker to identify
monocytes/macrophages. The proportion of CD64+
cells in PBMC preparations was 12%. Intracellular IL-18BP was detected
using biotinylated mouse anti-IL-18BP mAb 582.10 followed by
streptavidin-PE conjugate. All isotype-matched controls were negative.
Data are presented as the difference (
) between the mean
fluorescence intensity (MFI) of the positive label (i.e.,
anti-IL-18BP) and the MFI of the negative control (i.e., mouse
IgG1).
Statistical analysis
For IL-18BP and IL-18 mRNA and protein levels, results are expressed as mean ± SEM. Results were analyzed using the Wilcoxon rank test by the statistical program S-Plus 2000 Professional (Release 3; Insightful, Seattle, WA). Differences were considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the expression and localization of IL-18BP,
immunohistochemistry was performed on cryosections of surgically
resected specimens from patients with active CD or from control
patients. Evaluating the control sections, vessels and a few scattered
cells expressed IL-18BP (Fig. 1, A and B). IL-18BP was also observed associated
with the vessels of active CD patients (Fig. 2, A and B).
Interestingly, an increased number of IL-18BP-positive cells were
scattered in the submucosa (Fig. 2, A–C) and
overlying lymphoid aggregates (Fig. 2C). Thus, the
endothelial cells within the vessels and the cells infiltrating the
submucosa appeared to be the major source for IL-18BP during
active CD.
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A), CD31-positive cells (Fig. 3D), and IL-18BP-positive cells (Fig. 3, B and
E) were detected in the submucosa of active CD.
Superimposing the two fluorescent images confirmed that the macrophages
and endothelial cells were indeed positive for IL-18BP (Fig. 3, C and F, respectively). Other cell specific
markers, e.g. anti-epithelial Ag clone Ber-EP4 (epithelial cells),
anti-CD94 (NK cells), anti-CD3 (T cells), anti-CD19, and
anti-CD20 (B cells) were analyzed but did not colocalize with the
IL-18BP staining (data not shown). Thus, macrophages and endothelial
cells appear to be the major source of IL-18BP within intestinal
tissues obtained from active CD patients.
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Analysis of IL-18BP and IL-18 expression was performed by
real-time PCR on the surgically resected specimens obtained from
patients with active CD and from control patients (Fig. 4). IL-18BP mRNA was detectable in
control patients (mean, 1.74; SD, 0.1) and the levels were
significantly increased in active CD patients (mean, 8.92; SD, 2.4,
p < 0.001). Similarly, IL-18 mRNA was present in
control patients (mean, 2.83; SD, 0.2), again with increased levels in
active CD patients (mean, 4.40; SD, 0.5, p <
0.05).
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A). Total
IL-18 in the specimens from active CD patients was also significantly
increased compared with control patients (1576 ± 246.3 vs
410.4 ± 75.1 pg/ml, respectively; p < 0.001)
(Fig. 5B). Analysis of IL-18BP and IL-18 levels of
individual specimens demonstrated a significant positive correlation
between the two proteins (r = 0.87; p
< 0.0001), revealing that the increase in IL-18BP parallels the IL-18
up-regulation known to be associated with active CD
(16, 17, 18, 19). Unbound IL-18BP isoform a/c was in excess
compared with total IL-18 (49.5 ng/ml vs 1570 pg/ml, respectively) in
active CD specimens.
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A), as well as the complexes
IL-18/IL-18BPa and IL-18/IL-18BPc with an approximate molecular mass of
58 kDa (Fig. 6B). Bands of 42, 40, and 58 kDa, comigrating
with the free and complexed recombinant proteins, were also observed in
the specimens, demonstrating that free IL-18BP isoform a and c as well
as the IL-18/IL-18BP complex were present in intestinal tissue
(Fig. 6C). Using an anti-IL-18 polyclonal Ab further
confirmed the presence of the IL-18/IL-18BP complex in the active CD
and control specimens (Fig. 6D), suggesting that a
proportion of IL-18 is bound to IL-18BP. Free mature IL-18 and
pro-IL-18 were also present (Fig. 6D).
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E. Bands of
42, 40, and 35 kDa (isoforms a, c, and d, respectively) were observed
in the active CD and control specimens, whereas isoform b was not
detected (Fig. 6F). These results further confirm the
presence of the IL-18/IL-18BP complex (Fig. 6F). An
additional band of 
19 kDa revealed by the anti-IL-18BP mouse
polyclonal Ab was analyzed by mass spectrometry and found to be the
actin binding protein (data not shown). IL-18BP production by endothelial cells and macrophages
To further investigate whether the IL-18BP production by
intestinal macrophages and endothelial cells was relevant to other
types of macrophages and endothelial cells, experiments were
performed with HUVEC and peripheral blood monocytes/macrophages.
Interestingly, IL-18BP mRNA was found to be constitutively expressed in
HUVEC, which then increased following 24 h of stimulation with
inflammatory cytokines (IL-1, TNF-, IFN-) (Fig. 7A). Similar to our in situ
observations, a significant increase of IL-18BP production was observed
(30-fold) (Fig. 7B).
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, TNF-, IFN-)
combined with LPS also induced an significant increase in IL-18BP
secretion (p < 0.05) (Fig. 8A). A similar induction was
observed after 48 h of stimulation with LPS, IFN-, and TNF-
alone, demonstrating their individual potential to induce IL-18BP
production (Fig. 8A). IL-1 and IL-18 alone had no
significant effect (Fig. 8A).
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B). IL-18BP was observed primarily in
CD64+ monocytes/macrophages. Thus, CD64-positive
cells appear to be the major source of IL-18BP in PBMC. The ability of
these cells to produce IL-18BP was further confirmed using
culture supernatants of purified monocytes/macrophages from total PBMC
with or without stimulation. A 4-fold increase in IL-18BP levels was
observed in 48-h supernatants of TNF-- or IFN--stimulated
monocytes/macrophages (Fig. 8C). A similar induction of
IL-18BP was observed using differentiated THP-1 cells (data not
shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intestinal endothelial cells and macrophages were found to be the major source of IL-18BP in the submucosa, and an increased number of IL-18BP-expressing macrophages were present within CD specimens. Our results document for the first time a significant increase in IL-18BP transcripts in specimens from active CD patients compared with control patients. Unbound IL-18BP isoform a/c protein levels were quantified and found to be significantly higher in active CD specimens compared with control tissue and, interestingly, higher than the amount of total IL-18 (49.5 ± 6 ng/ml vs 1570 ± 246 pg/ml, respectively). Thus, IL-18BP up-regulation correlated with the increase of IL-18 previously shown to be associated with CD. IL-18BP and IL-18 expression were also analyzed in surgically resected specimens from patients with ulcerative colitis, another form of inflammatory bowel disease in which polarized Th2 responses are believed to play a dominant role. These experiments revealed that IL-18BP and IL-18 transcript and protein levels in ulcerative colitis specimens were comparable to the ones in control specimens (data not shown).
The presence of IL-18BP in CD lesions would suggest neutralization of IL-18 activity, unless patients with active CD preferentially undergo differential splicing to produce more of the inactive isoforms (b and d) than the active ones (a and c). These patients would then have a reduced ability to regulate the activity of IL-18 during the course of the disease. In fact, our results demonstrated for the first time that, although two of seven patients appeared to have higher levels of IL-18BP isoform d, free isoform a was always present at comparable or elevated levels to that of b, c, or d. In the future, a quantitative comparison will be needed, because reagents are currently unavailable to obtain an exact quantification of the inactive isoforms. Furthermore, Western blot analysis demonstrated that, while IL-18 was complexed with IL-18BP, free mature IL-18 was also observed. These observations suggest that, despite the presence of IL-18BP, some IL-18 activity may be available for perpetuating the pathogenesis of CD.
The presence of free mature IL-18 in active CD specimens which also
express an excess of free IL-18BP isoform a was astonishing and may
indicate another function of IL-18BP in addition to its ability to bind
IL-18. Like most cytokine receptors, the IL-18 receptor is composed of
a ligand binding chain (IL-18R), originally described as
IL-1R-related protein, and an accessory chain (IL-18R), named
accessory protein-like. Recently, IL-1H4, an IL-1-related protein, has
been shown to bind IL-18R but not the IL-1R. IL-1H4 has a high degree
of homology to IL-18 (25, 26). It is therefore possible
that IL-1H4 could bind IL-18BP. Although to date there is no reported
biological effect of IL-1H4, binding of IL-1H4 to IL-18BP may account
for the reduced capability of IL-18BP to bind IL-18. Another possible
hypothesis to explain the presence of free IL-18 found by Western
analysis together with an excess of active free IL-18BP isoform a would
be that, in the tissue, IL-18 was not accessible to endogenous
IL-18BP.
Interestingly, using the murine model of TNBS-induced colitis, we found that administration of rhIL-18BPa significantly ameliorated the disease and reduced parameters of colonic inflammation (27). Similar findings using the murine model of dextran sulfate sodium-induced colitis and neutralizing IL-18 Abs were also reported (28, 29). Although the IL-18BP excess found in CD lesions is not understood, the results obtained with experimental animal models suggest that neutralizing IL-18 may be beneficial and that administration of rIL-18BP may be considered as a possible therapy.
Our results thus demonstrate that IL-18BP transcript and protein are
up-regulated in active CD intestinal specimens by endothelial cells and
macrophages. As observed in our in vitro studies, this increase of
IL-18BP production was also found in other types of endothelial cells
(HUVEC) and monocytes/macrophages (purified from total PBMC) after
stimulation with proinflammatory cytokines (TNF-, IFN-). Because
these proinflammatory cytokines are reported to be increased in the
intestinal tissue during CD (18), one could hypothesize
that they significantly contribute to the up-regulation of IL-18BP
observed in active CD specimens. Interestingly, gene induction of
IL-18BPa by IFN- has been recently observed using cultures of
colonic biopsy specimens (23). However, direct isolation
of intestinal endothelial cells and macrophages would be further needed
to verify this hypothesis and to study the IL-18BP regulation in the
intestinal tissue. Moreover, the production of IL-18BP by macrophages
is a surprising finding, because activated macrophages are the main
source of IL-18 (30). One needs also to further
investigate whether macrophages found in inflamed intestinal tissue
produce both IL-18 and IL-18BP as well as how this expression is
regulated.
In summary, our results show that endothelial cells and macrophages were the major source of IL-18BP within the intestinal submucosa. In addition, levels of IL-18BP transcript and protein are significantly up-regulated in active CD specimens compared with control specimens, with isoform a, c, and d being detected. Despite the presence of the appropriate neutralizing isoforms, free IL-18 is still observed in specimens from active CD and highlights the complexity of the regulation of IL-18 activity. Further studies are needed to elucidate the role of IL-18BP in intestinal tissue during CD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yolande Chvatchko, Department of Experimental Biology and Pharmacology, Serono Pharmaceutical Research Institute, 14 chemin des Aulx, 1228, Plan-les-Ouates, Geneva, Switzerland. E-mail address: yolande.chvatchko{at}serono.com
3 Abbreviations used in this paper: CD, Crohn’s disease; IL-18BP, IL-18-binding protein; rh, recombinant human; MFI, mean fluorescence intensity; CS, corticosteroid; h, human.
Received for publication November 8, 2001. Accepted for publication January 25, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 B. Moller, J. Paulukat, M. Nold, M. Behrens, N. Kukoc-Zivojnov, J. P. Kaltwasser, J. Pfeilschifter, and H. Muhl Interferon-{gamma} induces expression of interleukin-18 binding protein in fibroblast-like synoviocytes Rheumatology, March 1, 2003; 42(3): 442 - 445. [Abstract] [Full Text] [PDF]
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N. K. Banda, A. Vondracek, D. Kraus, C. A. Dinarello, S.-H. Kim, A. Bendele, G. Senaldi, and W. P. Arend Mechanisms of Inhibition of Collagen-Induced Arthritis by Murine IL-18 Binding Protein J. Immunol., February 15, 2003; 170(4): 2100 - 2105. [Abstract] [Full Text] [PDF]
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 J. A. Gracie, S. E. Robertson, and I. B. McInnes Interleukin-18 J. Leukoc. Biol., February 1, 2003; 73(2): 213 - 224. [Abstract] [Full Text] [PDF]
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