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Department of Gastroenterology and Hepatology, Medical School of Hannover, Hannover, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, IL-6, IL-8, IL-16, and IL-18 without
further stimulation. Both IgE-dependent and IgE-independent agonists
(e.g., Gram-negative bacteria) enhanced expression of TNF-. Another
set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was
expressed following activation by IgE receptor cross-linking. If mast
cells were cultured in the presence of IL-4 and SCF, the production and
release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared
with mast cells cultured with SCF alone. By contrast, IL-6 expression
was completely blocked in response to culture with IL-4. In summary,
our data show that mature human mast cells produce proinflammatory
cytokines that may be up-regulated following triggering with
IgE-independent agonists such as bacteria, whereas activation by IgE
receptor cross-linking results in the expression of Th2-type cytokines.
IL-4 enhances the expression of Th2-type cytokines but does not affect
or even down-regulates proinflammatory cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1, IL-3, IL-4, IL-6, and IL-13 (3, 4). Because mast
cells are known to exhibit marked species differences (1),
it has to be examined whether those data can be transferred to the
human system. Little is known about the cytokine profile of
mucosa-derived human mast cells, because the cells can be obtained only
in limited quantities and they are difficult to purify. The few data on
cytokine production by human mast cells are mainly derived from
immunohistochemical studies (5, 6, 7), or studies using
immature bone marrow or cord blood-derived mast cells
(8, 9, 10), or the immature leukemic mast cell line HMC-1
(11, 12, 13, 14). Human lung mast cells were found to express mRNA
for IL-4, IL-5, and GM-CSF and to release the products by IgE-dependent
activation (15, 16, 17). Using recently developed methods for
isolation and culture of human intestinal mast cells (18, 19), we could show that these cells are an important source of
TNF- and IL-5 in human intestinal tissue in vitro as well as in vivo
(20, 21). IL-5 production was only observed if mast cells
were triggered by IgE receptor cross-linking, whereas TNF-
production occurred constitutively but could be enhanced by IgE
receptor-dependent activation (20, 21). Furthermore, we
found recently that IL-4, known to play an important role in B and T
lymphocyte differentiation, strongly enhances the proliferation and
mediator release by human intestinal mast cells and thus may be
involved, apart from stem cell factor
(SCF),3 in human mast
cell regulation (22). In this study, we examined the
expression of a number of different cytokines by purified human
intestinal mast cells with particular respect to the modulatory effect
of IgE receptor cross-linking and IL-4 on the cytokine profile produced
by this cell type. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human intestinal mast cells were isolated from surgery tissue specimens (macroscopically normal border sections, free of tumor cells as determined by histological examination of the tissue) derived from 18 patients (40–77 years of age, 9 men, 9 women), who underwent bowel resection because of cancer. Permission to conduct the study was obtained from the local ethical committee of the Medical School of Hannover. The methods of mechanical and enzymatic tissue dispersion yielding single-cell preparations containing 4 ± 2% (mean ± SD) mast cells has been described in detail elsewhere (18). After overnight incubation in culture medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 25 mM HEPES, 2 mM glutamine, 100 µg/ml streptomycin, 100 µg/ml gentamicin, 100 U/ml penicillin, and 0.5 µg/ml amphotericin; all cell culture reagents were obtained from Life Technologies, Paisley, U.K.), mast cells were enriched by positive selection of c-kit expression using magnetic cell separation (MACS system; Miltenyi Biotech, Bergisch-Gladbach, Germany) and the mAb YB5.B8 (PharMingen, Hamburg, Germany) as described (19, 20, 21). The fraction containing the c-kit-positive cells (mast cell purity 60 ± 25%) was cultured at a density of 2 x 105 MC per ml for up to 21 days in medium supplemented with 25 ng/ml of recombinant human SCF (PeproTech, Rocky Hill, NJ) alone or with 10 ng/ml IL-4 (Novartis, Vienna, Austria) and SCF. Cultured mast cells were fed once a week by exchanging half of the culture medium supplemented with SCF or with SCF and IL-4. During culture for 14–21 days, mast cell purity increased to 98 ± 2%. The endotoxin content of the culture medium was 3 pg endotoxin/ml culture medium measured by LAL COATEST Endotoxin (Chromogenix, Mölntal, Sweden).
Stimulation of mast cells and coculture experiments
Mast cells were stimulated by IgE receptor cross-linking using
the purified mAb 29C6 (provided by Hoffmann-La Roche, Nutley, NJ)
directed against a non-IgE binding epitope of the high-affinity IgE
receptor -chain. To distinguish between existing and newly
transcribed mRNA, mast cells were incubated with 400 ng/ml actinomycin
D (Sigma, St. Louis, MO) before stimulation for 14 h as described
(21). For coculture experiments, eosinophils (purity
89–96%, n = 3) were isolated from venous blood of
informed healthy volunteers as described previously (23)
and cultured directly after isolation with purified mast cells in
24-well plates separated by a transwell membrane (
= 0.4 µm, Nunc,
Roskilde, Denmark) using the medium described for mast cell culture. As
a positive control, IL-5 (PeproTech) instead of mast cells was added to
the medium at a final concentration of 2 ng/ml.
RNA preparation and semiquantitative RT-PCR
Total RNA was prepared from human intestinal cell preparations
containing 98–100% mast cells using the RNeasy Mini Kit (Qiagen,
Hilden, Germany). For RT-PCR, 200 ng of total RNA was treated for 15
min at 37°C with 10 U RNase-free DNase (Promega, Madison, WI) to
remove genomic DNA. After denaturation for 10 min at 70°C,
cDNA was synthesized for 1 h at 37°C by adding
Superscript reverse transcriptase (Life Technologies, Eggenstein,
Germany) and 20 pmol oligo(dT) primers (Pharmacia, Uppsala, Sweden). A
one-tenth volume of the cDNA was used for one PCR. Thirty-five cycles
(60 s at 94°C, 80 s at 60°C, 70 s at 72°C) were
performed with 2.5 U Taq DNA polymerase (Life Technologies)
and 20 pmol of the specific sense and antisense primers for the cDNAs
of IL-1ß (5'-CGATCACTGAACTGCACGCTCCG-3';
5'-GGTGAAGTCAGTTATATCCTGGCCG-3'), IL-2 (5'-CAAGAATCCCAAACTCACCAGG-3';
5'-CAATGGTTGCTGTCTCATCAGC-3'), IL-3 (5'-CCTTGAAGACAAGCTGGGTT-3';
5'-ATTCATTCCAGTCACCGTCC-3'), IL-4 (5'-CGGACACAAGTGCGATATCACC-3';
5'-CCAACGTACTCTGGTTGGCTTCC-3'), IL-5 (5'-CGAACTCTGCTGATAGCCAATG-3';
5'-CCACTCGGTGTTCATTACACCAAG-3'), IL-6 (5'-CACACAGACAGCCACTCACCTC-3';
5'-CTCAGGCTGGACTGCAGGAAC-3'), IL-7 (5'-GGACTTCCTCCCCTGATCCTTG-3';
5'-CTTTGTTGGTTGGGCTTCACCCAG-3'), IL-8
(5'-GCAGCTCTGTGTGAAGGTGCAG-3'; 5'-GCATCTGGCAACCCTACAACAG-3'), IL-9
(5'-GATCCTGGACATCAACTTCCTC-3'; 5'-CTTGCCTCTCATCCCTCTCATC-3'), IL-10
(5'-CCGTGGAGCAGGTGAAGAATGC-3'; 5'-CAGCTAGAAAGCGTGGTCAGGC-3'), IL-11
(5'-ATGAACTGTGTTTGCCGCCTGGTC-3'; 5'-CGTCAGCTGGGAATTTGTCCTTC-3',
IL-12 (5'-CCAAGAACTTGCAGCTGAAG-3'; 5'-TGGGTCTATTCCGTTGTGTC-3', IL-13
(5'-ATGCATCCGCTCCTCAATCC-3'; 5'-TTCAGTTGAACCGTCCCTCG-3'), IL-15
(5'-GTATTGTAGGAGGCATCGTGG-3'; 5'-GTTCATCTGATCCAAGGTCTG-3'), IL-16
(5'-GCCCGACCTCAACTCCTCCACT-3'; 5'-GTCTCCAGCAGCTGTGGTTTCC-3'), IL-17
(5'-GGACTGTGATGGTCAACCTG-3'; 5'-CGGACACCAGTATCTTCTCC-3'), IL-18
(5'-GCTGCTGAACCAGTAGAAGA-3'; 5'-GTTCTCACAGGAGAGAGTTG-3'), TNF-
(5'-GAGCTGAGAGATAACCAGCTGGTG-3'; 5'-CAGATAGATGGGCTCATACCAGGG-3'),
GAPDH (5'-ACCACAGTCCATGCCATCAC-3'; 5'-TCCACCACCCTGTTGCTGTA-3'),
c-kit (5'-GGAGATCTGTGAGAATAGGCTC-3';
5'-CCCATAGGACCAGACGTCACTTTC-3'), CD3
(5'-AGTTGGCGTTTGGGGGCAAGATGGTAATGAAGAAA-3';
5'-CCCAGGAAACAGGGAGTCGCAGGGGGACTGGAGAG-3' (synthesized by Life
Technologies). A one-fifth volume of the PCR products was separated on
1% agarose gel containing ethidium bromide (500 ng/ml) and
photographed. To exclude false positive results by contaminating
lymphocytes, negative controls were conducted using the
specific primer pair for CD3. Positive controls for mast cell mRNA were
performed using the primer pair for c-kit. To observe
relative changes in mRNA expression, duplex PCR according to the
"primer-dropping" method was performed as described
(20). Duplex PCRs were started with the primer pair for
the cytokine of interest, and after a determined number of cycles the
primer set of GAPDH was added. In each case three samples of the
reactions were taken in the course of exponential DNA increase,
separated on 1% agarose gels containing ethidium bromide and analyzed
using an automated bioimaging analyzer (Fuji BAS-1000; Raytest,
Germany). To ensure that specific cDNAs were amplified, PCR fragments
were sequenced by the dideoxy method using the T7 SequencingTM kit
(Pharmacia).
Immunocytochemistry
Cultured cells were harvested, cytocentrifugated on slides and
fixed in acetone for 10 min. Immunostaining was performed using the
primary Abs mouse anti-human IL-1ß mAb (R&D Systems, Wiesbaden,
Germany), rat anti-human IL-3 Ab (PharMingen, San Diego, CA) at 50
µg/ml, rat anti-human IL-4 Ab (PharMingen) at 50 µg/ml, rat
anti-human IL-5 Ab (PharMingen) at 50 µg/ml, rat anti-human
IL-6 Ab (PharMingen) at 50 µg/ml, mouse anti-human IL-8 mAb
(LeukoSite, Cambridge, MA) at 10 µg/ml, goat anti-human IL-9 Ab
(Genzyme, Cambridge, MA) at 20 µg/ml, rat anti-human IL-13 Ab
(PharMingen) at 50 µg/ml, mouse anti-human IL-16 mAb
(ImmunoDiagnostics, Bedford, MA) at 10 µg/ml, rabbit anti-human
IL-18 Ab (BioSource, Camarillo, CA) at 1 µg/ml, and rabbit
anti-human TNF- Ab (Genzyme) at 0.28 µg/ml. Slides were
incubated with the primary Abs for 16 h at 4°C. The primary Abs
were linked by a biotinylated secondary Ab to a streptavidin-peroxidase
conjugate, and the substrate/chromogen (hydrogen peroxide/AEC) was
converted to a red deposit (LAB-SA System; Zymed, South San Francisco,
CA). Controls were conducted with appropriate nonimmune control sera
diluted corresponding to the primary Ab concentration (Immunotech,
Marseille, France). Slides were counterstained with Mayer’s hemalam
(Merck, Darmstadt, Germany).
Measurement of histamine and cytokines in supernatants
Histamine was measured by RIA (Coulter-Immunotech, Krefeld,
Germany). Cytokines were quantified by ELISA according to the
manufacturer’s instructions. The kits from R&D Systems were used to
measure TNF-, IL-3, IL-4, IL-5, and IL-6, and the kits from
BioSource were used to measure IL-10 and IL-13. The detection limits
were 5 pg/106 cells (TNF-), 31.2
pg/106 cells (IL-3), 23.4
pg/106 cells (IL-4), 7.8
pg/106 cells (IL-5, IL-6), 1
pg/106 cells (IL-10), or 19.5
pg/106 cells (IL-13), respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mature human mast cells were isolated from intestinal tissue and
cultured in the presence of SCF. As demonstrated in Fig. 1
, the mRNA for IL-1ß IL-6, IL-8,
IL-16, and IL-18 was clearly detectable in mast cells that were not
further stimulated. As shown recently (21), IL-5 mRNA was
hardly detectable under such conditions but clearly present after
stimulation of the cells by IgE receptor cross-linking with 100 ng/ml
mAb 29C6 for 6 h. The mRNA for IL-3, IL-9, and IL-13 was not
expressed in SCF-cultured cells without further stimulation but was
detected following stimulation for 6 h. Inconsistently, in four of
eight experiments IL-10 mRNA was expressed with further induction
following IgE receptor cross-linking. IL-4 mRNA was not
(n = 5) or only hardly detectable (n =
3) after stimulation with mAb 29C6. The mRNA expression for IL-16 and
IL-18 was moderately down-regulated following IgE receptor
cross-linking. Neither in resting nor in stimulated mast cells was mRNA
found for IL-2, IL-7, IL-11, IL-12, IL-15, and IL-17. To distinguish
between stabilized existing and newly transcribed mRNA in response to
IgE receptor cross-linking, mast cells were incubated for 14 h
with actinomycin D, known to inhibit mRNA transcription, before
stimulation (21). After pretreatment with actinomycin D
and subsequent mast cell stimulation, mRNA levels for IL-6, IL-8,
IL-16, and IL-18 remained unaffected. In contrast, mRNA expression for
IL-3, IL-5, IL-9, IL-10, and IL-13 was absent or only hardly
detectable, indicating a regulation of cytokine expression in response
to IgE-receptor cross-linking by induction of transcription
(n = 3).
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A shows the time
kinetics of mRNA expression by mast cells in response to stimulation
with high-affinity IgE receptor cross-linking using 100 ng/ml mAb 29C6.
mRNA expression for IL-5 started 15 min after activation. The peak
expression of IL-5 mRNA was at 90 min and could be detected until
6 h after stimulation. We found similar time curves for IL-3 and
IL-13 mRNA, whereas IL-9 mRNA expression peaked only after stimulation
for 6 h. In contrast, TNF- mRNA expression peaked already
following stimulation for 30 min, and then the expression decreased. To
analyze the dose dependency of cytokine mRNA expression, mast cells
were stimulated using 1, 10, or 100 ng/ml mAb 29C6. In this set of
experiments, mRNA for IL-3, IL-5, IL-9, and IL-13 was absent in samples
of SCF-cultured cells without further stimulation. mRNA expression
occurred after activation with a minimal concentration of 1 ng/ml mAb
29C6 for 6 h or 10 ng/ml mAb 29C6 for 1 h, respectively (Fig. 2B). The up-regulation of TNF- mRNA expression occurred
on the same conditions.
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Intracytoplasmic cytokine expression in human intestinal mast
cells cultured for 2 wk in the presence of SCF (98–100% pure,
n = 3) was studied by immunocytochemistry and is
summarized in Table I. In accordance with
the mRNA data, we found clear immunoreactivity for IL-3, IL-5, IL-9,
and IL-13 only if mast cells were treated with 100 ng/ml mAb 29C6 for
6 h. The release of cytokines by human intestinal mast cells
(96–100% pure) was measured by ELISA. We tested IL-3, IL-4, IL-5,
IL-6, IL-10, and IL-13. Only IL-6 was released spontaneously in 6
h (mean: 36 pg IL-6/106 mast cells) with an
increase following stimulation by IgE receptor cross-linking (mean: 108
pg IL-6/106 mast cells). TNF- (mean: 181 pg
TNF-/106 mast cells), IL-3 (mean: 23 pg
IL-3/106 mast cells), IL-5 (mean: 367 pg
IL-5/106 mast cells), and IL-13 (mean: 16 pg
IL-13/106 mast cells) were released following
stimulation. In lysates of mast cells not stimulated by IgE receptor
cross-linking, small amounts of TNF- (8.4 ± 3.2 pg
TNF-/106 mast cells) and IL-6 (4.0 ± 3.8
pg IL-6/106 mast cells) were measured, whereas
IL-3, IL-5, and IL-13 were not detected. Following stimulation with mAb
29C6, we measured 50 ± 32 pg IL-5/106 mast
cells, 27 ± 27 pg IL-6/106 mast cells, and
48 ± 43 pg TNF-/106 mast cells. IL-3 or
IL-13 were not detected in cell lysates. This finding demonstrates
that, in contrast to mast cell mediators such as histamine, a larger
part of mast cell-derived cytokines has to be synthesized de novo
before release. In all cases, neither IL-4 nor IL-10 were detectable in
supernatants derived from mast cells (detection limit: 23.4 pg
IL-4/106 mast cells, 1 pg
IL-10/106 mast cells). To analyze in vitro
effects of mast cell-derived cytokines, we performed coculture
experiments with purified human intestinal mast cells (98–100% pure,
n = 3) and purified blood eosinophils (89–96% pure,
n = 3). Mast cells were either challenged with 10 ng/ml
mAb 29C6 or buffer control, and eosinophil survival was recorded after
1 wk of culture. In another set of experiments, eosinophils were
cultured with or without supernatants derived from mast cells triggered
with mAb 29C6. These experiments showed that coculture of eosinophils
with mast cells triggered with mAb 29C6 or addition of supernatant
derived from stimulated mast cells enhanced eosinophil survival by
about 159 ± 19% or 160 ± 27%, respectively, compared with
buffer control. Similar effects could be achieved by addition of 2
ng/ml IL-5 (181 ± 34%).
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In a recent study, we showed that IL-4 strongly enhances
proliferation and mediator release of human intestinal mast cells
(22). To analyze the effect of IL-4 on cytokine
expression, mast cells were cultured in the presence or absence of
IL-4, and the expression of TNF-, IL-3, IL-5, IL-6, and IL-13 was
tested. If the cells were cultured with SCF and IL-4, we found mRNA for
IL-3, IL-5, and IL-13 already in cells without further stimulation.
Following stimulation with mAb 29C6, mRNA expression for IL-3, IL-5,
and IL-13 was further enhanced. Using semiquantitative RT-PCR, we found
an IL-4-induced 4-fold increase of mRNA expression for IL-3 and IL-5 in
mast cells stimulated by IgE receptor cross-linking (Fig. 3). The data obtained for mRNA expression
were confirmed on the protein level by measurement of cytokine content
in supernatants of mast cells with or without stimulation by IgE
receptor cross-linking. As shown in Fig. 4, the release of histamine in response
to IgE receptor cross-linking was about 2-fold increased if mast cells
were cultured with IL-4. The release of IL-3, IL-5, and IL-13 in
response to IgE receptor cross-linking was also strongly enhanced, the
release of IL-3 and IL-13 was about 4-fold higher, and the release of
IL-5 was about 2-fold higher than in cells cultured without IL-4.
Moreover, IL-5 was released spontaneously by mast cells cultured with
IL-4 without further stimulation by IgE receptor cross-linking. In
contrast, IL-4 completely inhibited the production of IL-6 in mast
cells. Neither mast cells stimulated by IgE receptor cross-linking nor
mast cells without further stimulation released detectable amounts of
IL-6 if cultured in the presence of IL-4 (Fig. 4). Correspondingly, we
found no mRNA for IL-6 in mast cells cultured with IL-4. The expression
of mRNA for IL-1ß, IL-8, IL-16, IL-18, and TNF- as well as the
release of TNF- (Fig. 4) were not affected by IL-4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, IL-6, and IL-18, as well as IL-8 and IL-16,
without further stimulation. Moreover, upon activation by IgE receptor
cross-linking, mast cells produce the Th2 cytokines IL-3, IL-5, IL-9,
and IL-13. As reported for the immature human mast cell line HMC-1
(11, 14), human intestinal mast cells do not produce the
Th1-type cytokines IL-2, IL-12, and IFN-
(data on IFN- not
shown). In accordance to our data, HMC-1 cells were found to express
IL-1ß and IL-8 constitutively (11, 14). In contrast,
HMC-1 express IL-3 constitutively and IL-6 following stimulation with
ionophore (14), whereas human intestinal mast cells
express IL-6 without further stimulation, but IL-3 only after
activation. Furthermore, IL-5 production was not found in HMC-1 cells
(14). The differences in cytokine expression by HMC-1 and
mature intestinal mast cells may be due to the fact that HMC-1 had to
be stimulated with phorbol myristate acetate or ionophore instead of
IgE receptor cross-linking because HMC-1 lack a functional IgE receptor
(24). This may also be a reason for the failure to detect
IL-5 in HMC-1 cells, because IL-5 expression in isolated human mast
cells is induced only following stimulation by IgE receptor
cross-linking, as shown here and in previous studies using human bone
marrow or cord blood-derived mast cells (8, 25) or human
lung mast cells (16).
Nasal mast cells derived from patients with perennial allergic
rhinitics and human lung mast cells and were reported to express IL-4
(5, 7, 15, 26). We could not confirm these observations
for human intestinal mast. This discrepancy could be due to the
heterogeneity of mast cells derived from different tissues (1, 7). Moreover, the mast cells used in our study were isolated
from nonallergic donors. Therefore, we cannot exclude the possibility
that intestinal mast cells in allergic individuals are capable of
producing IL-4 as shown previously for nasal mast cells
(26). It was reported that HMC-1 (14) but not
tissue mast cells produce IL-10. We found IL-10 mRNA expression by
intestinal mast cells in four of eight experiments, but did not detect
IL-10 protein in mast cell-derived supernatants, which could be due to
a late appearance of maximal IL-10 expression following stimulation, as
described for HMC-1 (14). Extending previous studies
performed with human bone marrow-derived mast cells, we found
expression of the CD4+ T cell chemoattractant
cytokine IL-16 by intestinal mast cells, which may provide one
mechanism for T cell recruitment in mast cell-dependent intestinal
inflammation (9). Finally, we report for the first time
that mature human mast cells are capable of producing IL-18. IL-18,
first described as IFN--inducing factor, ranks with other
proinflammatory cytokines as a contributor to systemic and local
inflammation because of its ability to induce expression of TNF-,
IL-1ß, and both CXC and CC chemokines in human monocytes
(27).
The occurrence of IL-3, IL-5, IL-9, and IL-13, which are thought to be produced by lymphocytes of the Th2 phenotype, is a characteristic feature of IgE-mediated allergic inflammation as well as parasitic infection with important pathophysiological implications (28). Here we show that the production of this set of cytokines may occur in a lymphocyte-independent fashion or is at least supported by mast cells, which, by their capacity to store the cytokines to some extent (1), may provide them more rapidly compared with Th2-type cells. IL-3 and IL-5 are known to promote activation and proliferation of eosinophils and basophils (23, 29, 30). In a recent study (21), we reported that in vivo IL-5 expression by mast cells was detectable in patients with inflammatory bowel disease and food allergy, but not in healthy controls, suggesting that IL-5 production by mast cells is a typical feature of inflammatory conditions. Possibly, the same is true for other mast cell-derived Th2-type cytokines such as IL-3, IL-9, and IL-13.
IL-4 is known to regulate IgE production in B lymphocytes and development of IL-4- and IL-5-producing Th2-type lymphocytes (31, 32). Recombinant IL-4 was found to down-regulate IL-1ß mRNA and to enhance IL-3 and IL-8 mRNA expression in HMC-1 cells (12, 13). Recently, Toru et al. (10) reported an enhanced production of IL-13 following IL-4 priming in immature cord blood-derived cultured mast cells. We could show that IL-4 enhances the release of histamine, leukotriene C4, and IL-5 in mature human mast cells triggered by IgE receptor cross-linking (22). In this study, we demonstrate that IL-4 modulates the cytokine profile produced by mature human mast cells. IL-4 enhances the release of IL-3, IL-5, and IL-13 induced by IgE receptor cross-linking and, most interestingly, renders mast cells capable of producing and releasing IL-5 even without IgE receptor cross-linking. In contrast, we found that the expression of IL-6 in mast cells was completely inhibited by IL-4. The mechanism of IL-4 effects on cytokine production by human intestinal mast cells is unclear at present. Inhibition studies using actinomycin D indicated that both the up- and the down-regulation of cytokine expression by IL-4 is regulated at the transcriptional level. Because we did not find any IL-4 production by mast cells themselves, IL-4 that may regulate cytokine production in mast cells seems to be produced by other cells such as Th2 lymphocytes or basophils (22, 30, 32).
Furthermore, we could show recently that not only IgE receptor
cross-linking and IL-4 but also bacteria effected cytokine production
in human intestinal mast cells. We found that Gram-negative bacteria
are capable of enhancing TNF- production in these cells by an
IgE-independent means (20). The first evidence for the
induction of TNF- synthesis in mast cells by bacteria came from mice
models. In these studies, mast cell-derived TNF- was found to have a
protective role in bacterial infection (33, 34). In
contrast, we found no effect of bacteria on the synthesis of Th2
cytokines (data not shown).
Fig. 5 summarizes our findings about the
differential cytokine expression by human intestinal mast cells. All
experiments were performed with mast cells cultured in the presence of
SCF, because SCF is the only cytokine that provides human mast cell
survival and is permanently present in tissues containing mast cells,
thus presenting some kind of "physiological condition" as suggested
earlier (18, 35). Our data suggest that the expression of
proinflammatory cytokines occurs constitutively in human intestinal
mast cells and may be up-regulated following triggering of the cells
with IgE-independent agonists such as bacteria (20).
Activation of mast cells by IgE receptor cross-linking also results in
an up-regulation of proinflammatory cytokines but in addition induces
the expression of Th2-type cytokines. IL-4 further enhances the
expression of Th2-type cytokines but does not affect or even
down-regulates proinflammatory cytokines such as IL-6.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephan C. Bischoff, Department of Gastroenterology and Hepatology, Medical School of Hannover, D-30623 Hannover, Germany. E-mail address:
3 Abbreviation used in this paper: SCF, stem cell factor.
Received for publication July 6, 1999. Accepted for publication October 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am. J. Respir. Cell Mol. Biol. 10:471.[Abstract]
and interleukin-1ß mRNA expression in HMC-1 cells: differential regulation of gene product expression by recombinant interleukin-4. Exp. Hematol. 21:1271.[Medline]
(TNF-) in human intestinal tissue. Gut 44:643.
RI, CD40L, IL-4, and IL-13 and can induce IgE synthesis in B cells. J. Clin. Invest. 99:1492.[Medline]
-inducing factor) induces IL-1ß and IL-8 via TNF- production from non-CD4+ human blood mononuclear cells. J. Clin. Invest. 101:711.[Medline]
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 A. C. DA SILVA, M. F. S. SILVA, C. BIANCHI, R. C. RIBEIRO, A. NOBREGA, and O. SANT'ANNA Is Oral Tolerance Correlated with IgE Levels and Mast Cell Numbers? Ann. N.Y. Acad. Sci., December 1, 2004; 1029(1): 394 - 397. [Abstract] [Full Text] [PDF]
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T. R. Hundley, A. M. Gilfillan, C. Tkaczyk, M. V. Andrade, D. D. Metcalfe, and M. A. Beaven Kit and Fc{epsilon}RI mediate unique and convergent signals for release of inflammatory mediators from human mast cells Blood, October 15, 2004; 104(8): 2410 - 2417. [Abstract] [Full Text] [PDF]
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 K. C. Roy, G. Bandyopadhyay, S. Rakshit, M. Ray, and S. Bandyopadhyay IL-4 alone without the involvement of GM-CSF transforms human peripheral blood monocytes to a CD1adim, CD83+ myeloid dendritic cell subset J. Cell Sci., July 15, 2004; 117(16): 3435 - 3445. [Abstract] [Full Text] [PDF]
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 M. Babina, S. Guhl, A. Starke, L. Kirchhof, T. Zuberbier, and B. M. Henz Comparative cytokine profile of human skin mast cells from two compartments--strong resemblance with monocytes at baseline but induction of IL-5 by IL-4 priming J. Leukoc. Biol., February 1, 2004; 75(2): 244 - 252. [Abstract] [Full Text] [PDF]
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G. Sellge, A. Lorentz, T. Gebhardt, F. Levi-Schaffer, H. Bektas, M. P. Manns, D. Schuppan, and S. C. Bischoff Human Intestinal Fibroblasts Prevent Apoptosis in Human Intestinal Mast Cells by a Mechanism Independent of Stem Cell Factor, IL-3, IL-4, and Nerve Growth Factor J. Immunol., January 1, 2004; 172(1): 260 - 267. [Abstract] [Full Text] [PDF]
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C. A. Oskeritzian, W. Zhao, A. L. Pozez, N. M. Cohen, M. Grimes, and L. B. Schwartz Neutralizing Endogenous IL-6 Renders Mast Cells of the MCT Type from Lung, but Not the MCTC Type from Skin and Lung, Susceptible to Human Recombinant IL-4-Induced Apoptosis J. Immunol., January 1, 2004; 172(1): 593 - 600. [Abstract] [Full Text] [PDF]
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C.-K. Yu and C.-L. Chen Activation of Mast Cells Is Essential for Development of House Dust Mite Dermatophagoides farinae-Induced Allergic Airway Inflammation in Mice J. Immunol., October 1, 2003; 171(7): 3808 - 3815. [Abstract] [Full Text] [PDF]
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B. Lamkhioued, S. G. Abdelilah, Q. Hamid, N. Mansour, G. Delespesse, and P. M. Renzi The CCR3 Receptor Is Involved in Eosinophil Differentiation and Is Up-Regulated by Th2 Cytokines in CD34+ Progenitor Cells J. Immunol., January 1, 2003; 170(1): 537 - 547. [Abstract] [Full Text] [PDF]
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T.-J. Lin, R. Garduno, R. T. M. Boudreau, and A. C. Issekutz Pseudomonas aeruginosa Activates Human Mast Cells to Induce Neutrophil Transendothelial Migration Via Mast Cell-Derived IL-1{alpha} and {beta} J. Immunol., October 15, 2002; 169(8): 4522 - 4530. [Abstract] [Full Text] [PDF]
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H. Helmby and R. K. Grencis IL-18 Regulates Intestinal Mastocytosis and Th2 Cytokine Production Independently of IFN-{gamma} During Trichinella spiralis Infection J. Immunol., September 1, 2002; 169(5): 2553 - 2560. [Abstract] [Full Text] [PDF]
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 H. Konishi, H. Tsutsui, T. Murakami, S. Yumikura-Futatsugi, K.-i. Yamanaka, M. Tanaka, Y. Iwakura, N. Suzuki, K. Takeda, S. Akira, et al. IL-18 contributes to the spontaneous development of atopic dermatitis-like inflammatory skin lesion independently of IgE/stat6 under specific pathogen-free conditions PNAS, August 20, 2002; 99(17): 11340 - 11345. [Abstract] [Full Text] [PDF]
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A. Lorentz, D. Schuppan, A. Gebert, M. P. Manns, and S. C. Bischoff Regulatory effects of stem cell factor and interleukin-4 on adhesion of human mast cells to extracellular matrix proteins Blood, February 1, 2002; 99(3): 966 - 972. [Abstract] [Full Text] [PDF]
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K. Blease, C. Jakubzick, J. M. Schuh, B. H. Joshi, R. K. Puri, and C. M. Hogaboam IL-13 Fusion Cytotoxin Ameliorates Chronic Fungal-Induced Allergic Airway Disease in Mice J. Immunol., December 1, 2001; 167(11): 6583 - 6592. [Abstract] [Full Text] [PDF]
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 T. Biedermann, M. Kneilling, R. Mailhammer, K. Maier, C. A. Sander, G. Kollias, S. L. Kunkel, L. Hultner, and M. Rocken Mast Cells Control Neutrophil Recruitment during T Cell-mediated Delayed-type Hypersensitivity Reactions through Tumor Necrosis Factor and Macrophage Inflammatory Protein 2 J. Exp. Med., November 13, 2000; 192(10): 1441 - 1452. [Abstract] [Full Text] [PDF]
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C. T. Mierke, M. Ballmaier, U. Werner, M. P. Manns, K. Welte, and S. C. Bischoff Human Endothelial Cells Regulate Survival and Proliferation of Human Mast Cells J. Exp. Med., September 11, 2000; 192(6): 801 - 812. [Abstract] [Full Text] [PDF]
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H. Ochi, N. H. De Jesus, F. H. Hsieh, K. F. Austen, and J. A. Boyce IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production PNAS, September 12, 2000; 97(19): 10509 - 10513. [Abstract] [Full Text] [PDF]
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98%. Total RNA was prepared from mast cells challenged with 100
ng/ml mAb 29C6 for 6 h or with buffer control. RT-PCR was
conducted using specific primer pairs. DNA fragments obtained after 35
cycles were separated on an 1% agarose gel. One representative of
eight experiments is shown.
