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*
Laboratory of Experimental Immunolgy and Departments of
Pathology and
Gastroenterology, University Hospital Gasthuisberg, University of Leuven, Leuven, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Moreover, lamina propria T cells (LP-T) from IBD patients were
more responsive to IL-15 as compared with controls, and IL-15 alone
without a primary T cell stimulus induced IFN- and TNF production by
isolated IBD LP-T cells, especially by LP-T cells from patients with
Crohn’s disease. LP-T cells from IBD patients could induce CD40-CD40
ligand (CD40L) interaction-dependent TNF and IL-12 production by
monocytes in a coculture system. This capacity of LP-T cells was
strongly enhanced by preincubation in IL-15 and was the result of
higher CD40L expression after culture in IL-15. These data indicate
that IL-15 is overexpressed in the inflamed mucosa in IBD and that
IL-15 enhances local T cell activation, proliferation, and
proinflammatory cytokine production by both T cells and macrophages,
the latter via a CD40-CD40L interaction-dependent mechanism. Treatment
directed against IL-15 may have therapeutic potential in
IBD. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains of the IL-2R and its own unique 
-chain
(designated IL-15R) (1, 2, 3). IL-15 mRNA is expressed in
a wide variety of tissues and cells such as skeletal muscle, placenta,
heart, lung, liver, monocytes, epithelium, endothelium, and fibroblast
cells, but its protein is mainly produced by monocytes and macrophages
(1, 4, 5, 6). IL-15R mRNA has also been observed to have a
wide cellular distribution, such as in T cells, B cells, macrophages,
thymic cells, skeletal muscle, lung, and liver (3). This
widespread distribution of the IL-15R system suggests that IL-15
mediates pleiotropic effects on multiple cell types, including cells of
nonlymphoid origin (4). IL-15 induces T cell proliferation
and cytokine production, stimulates locomotion and chemotaxis of normal
T cells (1, 2, 7, 8, 9, 10, 11), and protects them from apoptosis
(12). It enhances NK cell cytotoxicity and Ab-dependent
cell-mediated cytotoxity, up-regulates NK cell survival and production
of NK cell-derived cytokines such as IFN-, GM-CSF, and TNF (5, 13), and induces B cell proliferation and isotype switching
(14).
Recently, IL-15 has been found to be overexpressed in rheumatoid
arthritis (RA)3 and allograft
rejection (15, 16, 17). IL-15, found in the synovial tissue of
RA patients, can recruit and activate T cells in the synovial membrane.
RA synovial fluid T cells activated by IL-15 exhibit proinflammatory
activities in vitro such as cytokine production and CD69 and adhesion
molecule expression, and they up-regulate monocyte TNF
production (15, 16). Of note, treatment targeted
to components of the IL-15R, including IL-15R-chain and the common
-chain, has been shown to effectively prevent development of some
experimental diseases such as collagen-induced arthritis and allograft
rejection (18, 19) and to markedly block Ag-specific
delayed hypersensitivity and leukocyte infiltration within the
delayed-type hypersensitivity sites (20). IL-15 is also
highly expressed at the transcriptional and translational levels in
leprosy skin lesions and by alveolar macrophages from patients with
active sarcoidosis (21, 22). In these patients, IL-15 has
been demonstrated not only to augment the local T cell response in
microbial immunity (21), but also to trigger the sarcoid T
cell growth and to accelerate the development of the T cell aveolitis
(22). Taken as a whole, these observations strongly
indicate that IL-15 plays a role in a variety of pathological
conditions.
Preliminary data have suggested the potential relevance of IL-15 to the immune responses in human inflammatory bowel disease (IBD). Kirman and Nielsen (23) reported that IL-15 could be detected in the sera of patients with ulcerative colitis (UC), but not in patients with Crohn’s disease (CD). Intracellular staining showed that the percentage of IL-15-positive cells was increased in mucosal mononuclear cells in both UC and CD patients. Expression of IL-15 mRNA was also found to be significantly increased in inflamed rectal mucosa of IBD (24). In the present study, we intended to gain further insight concerning the potential role of IL-15 in the pathogenesis of IBD. To this end, we studied local production of IL-15 in inflamed mucosa, as well as the functional effect of IL-15 on mucosal T cells and on T cell-monocyte interactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty-two tissue specimens were obtained from the inflamed ileum of 14 patients with active CD (10 male and 4 female, mean 36 years) and inflamed colon of 8 patients with active UC (six male and two female, mean 45 years). The preoperative diagnosis of IBD was based upon classical clinical, radiological, and endoscopic features and was confirmed by histological examination of the resection specimens. Indications for bowel resection in CD were the presence of fistulae (n = 3) and stenosis with clinical signs of obstruction (n = 11); while in UC indications for surgery were mostly therapy-resistant inflammatory colitis. Among IBD patients, 14 were receiving sulfasalazine or an oral aminosalicylic acid preparation at the time of operation, and 8 patients were on no treatment. None of them received immunosuppressants such as corticosteroids, cyclosporin A, or azathioprine. Control tissues were obtained from 10 patients (7 men and 3 women, mean 58 years) undergoing right hemicolectomy for carcinoma. Ileum was obtained from 7 patients and colon from 10 patients, and all tissues were remote from neoplastic involvement and were macroscopically normal. Additionally, uninvolved ileum (n = 6) from CD and uninvolved colon (n = 5) from UC were also obtained.
Heparinized peripheral blood samples were obtained from outpatients including 10 cases with CD (seven men and three women, mean 34 years) and 10 cases with UC (six men and four women, mean 39 years). The diagnosis was established by conventional clinical features and histological criteria. Only eight patients with CD and four patients with UC were treated with sulfasalazine or an oral aminosalicylic acid preparation. None received any immunosuppressants such as corticosteroids and cyclosporin A. In addition, blood samples from 11 healthy volunteers (seven men and four women, mean 32 years) were also taken for comparison.
Reagents
Purified human rIL-15 was obtained from PeproTech (Rocky Hill,
NJ). Anti-IL-15 mAb M112 (mouse IgG1) was purchased from Genzyme
(Cambridge, MA). Anti-CD40 ligand (CD40L) mAb M90 (mouse IgG1) was a
gift from Immunex (Seattle, WA). FITC- or PE-conjugated anti-CD3
(clone UCHT1, mouse IgG1), FITC-conjugated anti-CD69 (clone FN50,
mouse IgG1), PE-conjugated anti-CD40L (clone TRAP1, mouse IgG1),
and FITC- or PE-conjugated isotype-matched control mouse IgG mAb were
purchased from PharMingen (San Diego, CA). RPMI 1640 medium, PBS, and
penicillin-streptomycin were purchased from BioWhittaker (Heidelberg,
Germany). Bovine calf serum was obtained from HyClone (Logan, UT).
IFN- (2.0 x 107 U/mg) was obtained from
Boehringer Mannheim (Mannheim, Germany). LPS (Escherichia
coli serotype 026:B6), polymyxin B, and amphotericin B were
obtained from Sigma (St. Louis, MO).
Cell preparations and culture conditions
Peripheral blood T cells (PB-T), lamina propria mononuclear cells (LPMC), and lamina propria T cells (LP-T) from all groups were isolated using the method as described (25). Monocytes used in this study were obtained from a single healthy donor by positive selection using the magnetic activated cell sorter (MACS; Miltenyi Biotec, Berdisch Gladbach, Germany) according to the manufacture’s instructions. Briefly, PBMC were magnetically labeled with CD33 MicroBeads (Miltenyi Biotec) at 4°C for 15 min and then passed through a separation column that was placed in the magnetic field of a MACS separator. The magnetically labeled CD33+ cells were retained in the column while the unlabeled CD33- cells run through. After removal of the column from the magnetic field, the magnetically retained CD33+ cells were eluted in PBS supplemented with 5 mM EDTA and 0.5% BSA. The resultant cell population was >90% CD14+, as determined by flow cytometric analysis (FACSort, Becton Dickinson, San Jose, CA). All cell populations were cultured in RPMI 1640 medium supplemented with 0.3 mg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 4 U/ml polymyxin B, 5 µg/ml amphotericin B, and 10% iron-supplemented bovine calf serum at 37°C in 5% CO2-humidified atmosphere.
Immunohistochemistry
Tissue samples (n = 10 in CD, n = 7 in UC, n = 7 in normal ileum, and n = 10 in normal colon) were immediately embedded in OCT compound (Tissue-Tek; Miles, Elkhart, IN), frozen in liquid nitrogen, and stored at -70°C. Cryostat sections (5 µm) of intestinal tissue were fixed and stained as previously described (25). Endogenous peroxidase activity in colonic tissue was blocked with 0.3% H2O2 supplemented with 0.1% saponin (Sigma) for 30 min. Serial sections were first incubated with anti-IL-15 mAb M112 (10 µg/ml) for 30 min. After washing, the sections were incubated for 30 min with biotin-labeled rabbit anti-mouse IgG (Dako, Glostrup, Denmark) at 1:400. After additional washes, the sections were incubated for 30 min with avidin-biotin-peroxidase complex (Dako). All procedures were conducted at room temperature. The color reaction developed with 3-amino-9-ethylcarbazole (Janssen, Beerse, Belgium). The slides were counterstained with hematoxylin. Cells were counted as described (25). Negative control experiments were performed by incubating sections with irrelevant isotype-matched mouse IgG1 or by omitting the primary Ab.
For double staining for IL-15 and CD68 (activated macrophages) expression, a combination of the peroxidase technique and the alkaline phosphatase antialkaline phosphatase technique was used (25). Endogenous alkaline phosphatase was blocked by levamisole. An anti-CD68 mAb KP1 (Dako) was used for the identification of activated macrophages, and M112 was applied for the identification of IL-15.
IL-15 production by LPMC
LPMC from IBD patients and controls were cultured at the
concentration of 1.0 x 106/ml in the
absence or presence of LPS (5 µg/ml) or IFN- (1000 U/ml). After
48 h of culture, supernatants were aspirated and stored at
-70°C for IL-15 assay.
T cell activation by IL-15
For proliferation studies, PB-T or LP-T cells (1.0 x 106/ml) were plated in 96-well U-bottom plates (Nunc, Roskilde, Denmark) in triplicate in a total volume of 200 µl per well in the presence of IL-15 (1, 10, 100 ng/ml). After 72 h of culture, 1 µCi [3H]thymidine (ICN, Costa Mesa, CA) was added to the wells for the last 8 h to determine DNA synthesis. [3H]thymidine incorporation was measured using a beta scintillation counter (Packard, Meriden, CT). Values were calculated as stimulation index of mean cpm in the presence of IL-15 over the mean cpm of unstimulated cultures.
We also assayed for the production of IFN- and TNF by IBD PB-T or
LP-T cells after activation by IL-15. To this end, PB-T and LP-T cells
(1 x 106/ml) were cultured in a 1-ml volume
in 24-well plastic plates (Nunc) in the absence or presence of IL-15
(100 ng/ml). After 72 h of culture, supernatants were harvested
and assayed for IFN- and TNF.
T cell activation markers such as CD69 and CD40L were measured at two time intervals, i.e., 24 and 48 h. For this purpose, purified PB-T or LP-T cells (1.0 x 106/ml) from IBD patients and controls were incubated in the presence of 100 ng/ml of IL-15. Cells were harvested at the indicated time and assessed for the expression of CD69 and CD40L by staining with the following markers: CD3 (FITC and PE), CD69 (FITC), CD40L (PE), or isotype-matched control mAbs for 30 min at 4°C. After washes, the cells were fixed with 0.5 ml 1% paraformadehyde in saline and analyzed on a FACSort.
Monocyte cytokine production induced by IL-15-activated LP-T cells
Monocytes from a single healthy donor were isolated as described above. Isolated LP-T cells (2.0 x 106/ml) from either involved mucosa of IBD patients or uninvolved mucosa of IBD patients and controls were incubated in the presence of either culture medium alone or IL-15 (100 ng/ml) for 12 h. These LP-T cells were collected, washed with PBS three times, and then fixed in 1.0% paraformaldehyde in PBS for 30 min at 4°C. Following an additional three washes, fixed T cells (5.0 x 105/ml) were cocultured with monocytes (2.5 x 105/ml) for 48 h. Supernatants were harvested and assayed for IL-12 and TNF production. For blocking studies, anti-CD40L mAb M90 (10 µg/ml), which blocks the CD40-CD40L interactions (26), was added to the cocultures to examine the effects of CD40L signaling on monocyte cytokine production. In parallel to these experiments, LP-T cells from all groups were also fixed immediately after isolation and cocultured with healthy monocytes using the same protocol. Supernatants were collected and assessed for TNF and IL-12.
Measurement of cytokines
TNF and IL-12 p70 were assayed by sandwich ELISA as described
(25). IL-15 was measured by sandwich ELISA using matched
Ab pairs and following the manufacturer’s instructions (R&D Systems,
Minneapolis, MN). IFN- was measured by ELISA using matched Ab pairs
according to the manufacture’s instructions (BioSource International,
Nivelles, Belgium). The sensitivity of each assay was 10 pg/ml.
Statistical analysis
Data were expressed as mean ± SEM. Statistical analysis was performed using the Wilcoxon test for paired samples and Mann-Whitney U test for unpaired data. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Fig. 1
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immunohistological analysis demonstrated the presence of numerous
IL-15-positive cells with strong cytoplasmic staining in inflamed
mucosa from IBD patients. The percentage of IL-15-positive cells in
lamina propria of inflamed ileum from CD and of inflamed colon from UC
was significantly higher than that in the ileum and colon from control
patients (29.5 ± 4.2 in CD, 27.6 ± 4.5 in UC, 5.2 ±
1.7 in normal ileum, and 3.9 ± 1.1 in normal colon,
p < 0.001). In two patients with infectious colitis
(Samonella species), the number of IL-15-positive cells was
similar to controls. The majority of IL-15-positive cells in both CD
and UC were found in the lamina propria. In CD, IL-15-positive cells
were also found in the submucosa, muscularis externa, and serosa in
diseased areas. Occasional IL-15-positive cells were also found in the
mantle zones around lymphoid follicles in the inflamed bowel wall,
while germinal centers were always negative. In UC, IL-15-positive
cells were rare in the submucosa and absent from the deeper layers in
the diseased areas. In sections from normal ileum and colon, only a few
positive cells were seen. Epithelial cells were negative for IL-15 in
all sections. Staining with an isotype-matched control Ab was negative
(data not shown).
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D). Double positive
cells were mainly seen in the mucosa, and to a lesser extent also in
the submucosa, muscularis propria, and serosa. IL-15 production by LPMC
IL-15 is mainly induced by microbial Ags such as LPS,
Mycobacterium leprae, and Staphylococcus aureus
Cowan strain 1 (5, 6, 8, 21, 27). We studied IL-15
secretion by lamina propria macrophages in vitro. LPMC were isolated
and cultured with LPS (5 µg/ml) or IFN- (1000 U/ml) for
48 h. As shown in Fig. 2, LPMC from
inflamed areas of IBD patients, when stimulated with either LPS or
IFN-, indeed released IL-15. The levels in the supernatants of IBD
LPMC were significantly higher compared with those in the supernatants
of control LPMC (p < 0.005). Additionally,
LPMC were isolated from uninvolved ileum of six patients with CD,
uninvolved colon from five patients with UC, and colon of one patient
with diverticulitis and four patients with infectious colitis
(Samonella species). These LPMC, similar to control LPMC,
could produce only low levels of IL-15 when stimulated with LPS
(31 ± 6.5 pg/ml) or IFN- (25.4 ± 5 pg/ml), and these
levels were significantly lower than those produced by LPMC from
inflamed areas of IBD patients (data not shown). We further cocultured
LPMC from involved mucosa of eight patients with CD and six patients
with UC with CD40L-transfected 3T6 cells, but no IL-15 protein was
found in the supernatants. These results, consistent with earlier
report (27), indicate that CD40L expressed on LP-T cells
(25) does not trigger monocytes/macrophages to produce
IL-15.
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Experiments on synovial fluid T cells in RA patients have
demonstrated that these cells exhibit enhanced responsiveness to IL-15
and that IL-15 in the absence of a primary stimulus can enhance several
T cell activities (15, 16). Therefore, the
proliferation-inducing effect of IL-15 on highly purified T cells from
both IBD patients and controls was compared. IBD LP-T cells, when
stimulated with 1, 10, and 100 ng/ml of IL-15, demonstrated a
significantly higher proliferative stimulation index than those from
controls (Fig. 3A,
p < 0.005). Of note, PB-T cells from IBD patients also
proliferated more strongly in response to 10 and 100 ng/ml of IL-15
(Fig. 3B, p < 0.005). These data indicate
that IL-15, in the absence of a primary stimulus, is a strong inducer
of proliferation of T cells in IBD patients, and suggest that these
cells are primed to become more responsive to IL-15.
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and TNF have been
considered as important proinflammatory cytokines in IBD, especially in
CD (29, 30). Therefore, we studied the effect of IL-15 on
proinflammatory cytokine production by IBD T cells. Data in Fig. 4, A and B
demonstrate that the production of IFN- and TNF was significantly
increased in the IBD LP-T cell cultures in the presence of IL-15,
especially in CD. Moreover, IFN- but not TNF was also significantly
induced in the supernatants of IBD PB-T cells incubated with IL-15
(Fig. 4, C and D). These data indicate that IL-15
can directly induce IBD T cells to produce proinflammatory mediators
that participate in tissue damage.
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Expression of T cell activation molecules after IL-15 challenge
CD69 is a marker of activation on T cells (32). We
studied the kinetics of CD69 expression on IBD T cells after IL-15
priming. Very low to absent levels of CD69 expression were found on
freshly isolated PB-T cells from all groups (Fig. 5A). After IL-15 stimulation,
its expression was found to be enhanced in all groups, but it was
significantly higher on IBD PB-T cells as compared with healthy
controls (p < 0.005 at 24 or 48 h of
culture). Using two-color immunofluorescence, we analyzed CD69
expression on PB-CD4+ and
PB-CD8+ T cells from one patient with active CD
and one patient with active UC in the presence of IL-15 (100 ng/ml).
After 24 h of culture, 14 and 15% of the
CD4+ T cells and 41 and 33% of the
CD8+ T cells were CD69+.
Thus, PB-CD8+ T cells from IBD patients are more
responsive than CD4+ T cells (data not shown). In
contrast, high levels of CD69 expression were found on freshly isolated
LP-T cells from both IBD patients and controls, with no difference
between the groups (p > 0.05). CD69 expression
on LP-T cells did not change by >5% in the presence of IL-15 (Fig. 5B).
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In a previous study, we have shown that LP-T cells from IBD
patients induce cytokine secretion by normal monocytes
(25). Moreover, IL-15 has been shown to regulate synovial
T cell-dependent cytokine secretion by synovial macrophages in RA
(16). After demonstrating that IL-15 plays an important
role in inducing IBD LP-T cell activation, we wanted to investigate
whether IL-15 could enhance T cell-dependent IL-12 and TNF production
by monocytes in vitro by an effect on T cells. LP-T cells from all
groups were either used immediately after isolation or cultured with or
without IL-15 for 12 h. Fixation of T cells was accomplished by
incubation in 1% paraformaldehyde for 30 min at 4°C. This procedure
is known to prevent T cell cytokine secretion but to maintain
cell-membrane integrity, providing an experimental system for study of
cell contact-mediated effects. Paraformaldehyde-fixed LP-T cells were
then cocultured with healthy monocytes repeatedly isolated from a
single donor. Fig. 6 shows that elevated
levels of IL-12 and TNF were found in the supernatants of monocytes
cocultured with LP-T cells from IBD patients. When control LP-T cells
were cultured in IL-15, they were also able to induce IL-12 and TNF
production by monocytes, similar to untreated IBD LP-T cells.
Importantly, IL-12 and TNF production was significantly higher in the
supernatants of monocytes cocultured with IL-15-activated IBD LP-T
cells, demonstrating that IL-15 increases the ability of IBD LP-T cells
to induce monocyte cytokine production. We know from our previous study
that CD40L on IBD LP-T cells is responsible for monocyte cytokine
production in these cocultures. CD40L is a 33-kDa glycoprotein that is
transiently expressed on the surface of activated T cells,
predominantly the CD4+ T cells (33, 34). Expression of CD40L is induced by Th cell activation.
Interestingly, inclusion of anti-CD40L mAb in this coculture system
of IL-15-activated LP-T cells and normal monocytes significantly
down-regulated monocyte IL-12 and TNF production (Fig. 6). Fixed T
cells from all groups or monocytes cultured alone or incubated with
IL-15 did not produce measurable amounts of cytokines (data not
shown).
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demonstrates that IL-15 induced low
expression of CD40L on control LP-T cells, while its expression was
more strongly induced and also maintained at higher levels on IBD LP-T
cells after 24 and 48 h of IL-15 stimulation
(p < 0.005 vs controls at 24 or 48 h of
culture). On the contrary, its expression was not significantly changed
on PB-T cells from all groups (data not shown). From these data, it is
proposed that IL-15 alone without a primary T cell stimulus triggers
IBD LP-T cell CD40L expression and that IL-15-activated LP-T
cells further promote monocyte IL-12 and TNF secretion following
engagement by CD40 on monocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Finally, our results also show that IL-15 facilitates monocyte
IL-12 and TNF production in a T cell-macrophage contact-dependent way,
in which CD40-CD40L interactions play an essential role.
IL-15 mRNA is expressed in a variety of tissues and cells (1, 4), while its protein is mainly produced by activated
monocytes/macrophages and monocyte-derived dendritic cells, especially
on stimulation with microbial products such as LPS, M.
leprae, and S. aureus Cowan strain 1
(5, 6, 8, 15, 21, 27). Findings on expression of IL-15
mRNA without protein suggest that production of IL-15 mRNA and IL-15
protein are differentially regulated. Our results show local production
of IL-15 in the inflamed mucosa in IBD as evidenced by
immunohistochemistry but also by culture of LPMC with LPS or IFN-,
allowing us to demonstrate the actual secretion of this cytokine.
Similar findings have been reported in patients with RA and leprosy
(15, 21). Although CD40 ligation of monocytes or
macrophages strongly induces proinflammatory cytokine production such
as IL-6, IL-8, IL-12, and TNF (33, 34), our present
results demonstrate that IL-15 is not secreted by monocytes when
stimulated with IBD LP-T cells, previously shown to express functional
CD40L (25). Moreover, CD40L transfectants were unable to
induce IL-15 production by lamina propia macrophages, although some
IL-12 and TNF were found in the same coculture system. We also
cocultured purified monocytes from four healthy donors with
CD40L-transfected 3T6 cells for 48 h. IL-15 protein was not
measured in the supernatants (data not shown). These findings suggest
that IL-15 secretion by mucosal macrophages is not induced by T
cell-dependent CD40 engagement but that it may rather be induced by
microbial Ags. Our results are in accordance with earlier reports
(27, 35). IL-15 protein is not detected in the
supernatants of PBMC when stimulated with irradiated CD40L-transfected
L cells, but it can be measured in S. aureus Cowan
strain-stimulated cell cultures (27). Monocyte-derived
dendritic cells produce IL-15 when infected by influenza virus, but not
when stimulated with CD40L or LPS (35). To date, luminal
bacteria and their products have been proposed to be involved in the
initiation and perpetuation of IBD (29, 30). One mechanism
potentially involved might be through induction of IL-15. A
leaky intestinal barrier with increased intestinal permeability in IBD
intensifies luminal Ag absorption (29, 30), which, in
turn, may lead to an exaggerated local IL-15 production in the
mucosa.
Several studies have demonstrated effects of IL-15 on resting T cells.
IL-15 signals its receptor on the surface of T cells and induces the
activation of Janus kinase 1 and 2 as well as the tyrosine
phosphorylation and nuclear translocation of STAT3 and STAT5
(36). IL-15 selectively induces memory
CD4+, CD8+ T cell, and
naive CD8+ T cell proliferation, but not naive
CD4+ T cell proliferation (9). Thus,
it is considered as important for T cell activation in a TCR/CD3
complex-independent fashion, and it may preferentially facilitate the
activation of memory T cells which in inflammatory tissues represent
the majority of T cells (37). IL-15 has also been shown to
induce cytokine secretion by T cells (10, 16, 28). The
present studies demonstrate that IBD LP-T cells are more sensitive to
IL-15 than resting control T cells. IL-15 strongly induces IBD LP-T
cell proliferation and triggers IBD LP-T cells, particularly CD LP-T
cells, to produce IFN- and TNF. IFN- and TNF are important
proinflammatory mediators in the immunopathogenesis of IBD (29, 30). Administration of anti-TNF mAb effectively prevents
mucosal inflammation in CD (38). Therefore, our findings
on IL-15 effects might be relevant to the pathogenesis of IBD, as IL-15
stimulates mucosal T cell activation and proinflammatory cytokine
production. Moreover, IL-15 may be an effector cytokine for strong
bystander stimulation of T cells and up-regulating their survival
(12, 31).
In a previous study, we have demonstrated that IBD LP-T cells express CD40L and that they can induce IL-12 and TNF production by monocytes in a coculture system. This monocyte cytokine production is initiated by CD40 ligation (25). In contrast, cell contact between monocytes/macrophages and IL-15-activated T cells has been shown to lead to enhanced production of TNF and IL-12 by monocytes/macrophages (16, 31, 39). Inclusion of anti-CD40L neutralizing mAb in these cocultures was shown by Avice et al. to down-regulate IL-12 production, indicating that CD40-CD40L interactions play a critical role in the IL-15 effects (31). Our results demonstrated that IL-15 could stimulate IBD LP-T cells to express CD40L and that IL-15-activated IBD LP-T cells more strongly (as compared with freshly isolated IBD LP-T cells) induced monocyte TNF and IL-12 secretion in a CD40-CD40L interaction-dependent manner. Because the monocytes in our coculture system have not been in contact with IL-15, we think that the effect of IL-15 on monocyte cytokine secretion can be predominantly attributed to the enhanced CD40L expression on T cells after incubation with IL-15. This finding was different from an earlier report (31) in which IL-15 was unable to induce CD40L expression on resting CD4+ T cells from healthy controls. In fact, we also did not observe an effect of IL-15 on PB-T cell CD40L expression, but only on LP-T cells. Thus, IL-15 seems to enhance CD40L expression on activated T cells only. To further substantiate this point, we have stimulated PB-T cells from IBD patients and normal controls with immobilized anti-CD3. CD40L expression was found to be markedly induced on these T cells after 12–16 h of culture. Importantly, in the presence of IL-15, CD40L expression was further enhanced (our unpublished results).
The observations made in the present studies provide a potential
mechanism of T cell responses in inflamed mucosa of IBD. A model system
can be envisaged in which exposure to inflammatory stimuli in vivo such
as LPS or mycobacterial heat-shock proteins causes mucosal macrophages
to secrete proinflammatory mediators such as IL-15 and TNF. Both
function to recruit leukocytes into inflamed mucosa. IL-15 induces T
cell proliferation, favors T cell/macrophage interactions and
proinflammatory cytokine production, and promotes B cell isotype
swiching in the lamina propria. IL-15 not only directly triggers T cell
activation and proinflammatory cytokine production, but it also may
synergize with other cytokines (e.g., IL-12) to mediate immune injury
in the mucosa (40). IL-15 indeed enhances expression of
the IL-12Rß1-chain on CD4+ T cells and
synergizes with IL-12 to strongly induce CD4+ T
cell IFN- production (31). Additionally, IL-15 may also
induce intraepithelial lymphocyte proliferation and enhance their
cytotoxic activity against intestinal epithelial cells
(41). All these events, which can potentially be initiated
and/or maintained by IL-15, may result in chronic inflammation, an
abnormal immune response, and tissue damage. Therefore, treatment
targeted against IL-15 may be beneficial for these patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jan L. Ceuppens, Laboratory of Experimental Immunology, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; IBD, inflammatory bowel disease; UC, ulcerative colitis; CD, Crohn’s disease; CD40L, CD40 ligand; PB-T, peripheral blood T cells; LPMC, lamina propria mononuclear cells; LP-T, lamina propria T cells.
Received for publication September 24, 1999. Accepted for publication January 14, 2000.
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  H. P. Carroll, V. Paunovic, and M. Gadina Signalling, inflammation and arthritis: Crossed signals: the role of interleukin-15 and -18 in autoimmunity Rheumatology, September 1, 2008; 47(9): 1269 - 1277. [Abstract] [Full Text] [PDF]
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 C. L. Combe, M. M. Moretto, J. D. Schwartzman, J. P. Gigley, D. J. Bzik, and I. A. Khan Lack of IL-15 results in the suboptimal priming of CD4+ T cell response against an intracellular parasite PNAS, April 25, 2006; 103(17): 6635 - 6640. [Abstract] [Full Text] [PDF]
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A Di Sabatino, R Ciccocioppo, F Cupelli, B Cinque, D Millimaggi, M M Clarkson, M Paulli, M G Cifone, and G R Corazza Epithelium derived interleukin 15 regulates intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac disease Gut, April 1, 2006; 55(4): 469 - 477. [Abstract] [Full Text] [PDF]
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E. Mortier, J. Bernard, A. Plet, and Y. Jacques Natural, Proteolytic Release of a Soluble Form of Human IL-15 Receptor {alpha}-Chain That Behaves as a Specific, High Affinity IL-15 Antagonist J. Immunol., August 1, 2004; 173(3): 1681 - 1688. [Abstract] [Full Text] [PDF]
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I N Farstad and K E A Lundin Gastrointestinal intraepithelial lymphocytes and T cell lymphomas Gut, February 1, 2003; 52(2): 163 - 164. [Full Text] [PDF]
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N. Ohta, T. Hiroi, M.-N. Kweon, N. Kinoshita, M. H. Jang, T. Mashimo, J.-I. Miyazaki, and H. Kiyono IL-15-Dependent Activation-Induced Cell Death-Resistant Th1 Type CD8{alpha}{beta}+NK1.1+ T Cells for the Development of Small Intestinal Inflammation J. Immunol., July 1, 2002; 169(1): 460 - 468. [Abstract] [Full Text] [PDF]
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I Monteleone, P Vavassori, L Biancone, G Monteleone, and F Pallone Immunoregulation in the gut: success and failures in human disease Gut, May 1, 2002; 50(90003): iii60 - 64. [Abstract] [Full Text] [PDF]
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R. Hoontrakoon, H. W. Chu, S. J. Gardai, S. E. Wenzel, P. McDonald, V. A. Fadok, P. M. Henson, and D. L. Bratton Interleukin-15 Inhibits Spontaneous Apoptosis in Human Eosinophils via Autocrine Production of Granulocyte Macrophage-Colony Stimulating Factor and Nuclear Factor-kappa B Activation Am. J. Respir. Cell Mol. Biol., April 1, 2002; 2 |
