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Chemokine from Mesothelial Cells1
*
Department of Pathophysiology, University Medical School, Poznan, Poland;
Department of Nephrology and Medical Intensive Care, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt-Universität zu Berlin, Berlin, Germany; and
Department of Visceral and Transplant Surgery, University of Bern, Inselspital, Bern, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(GRO). Pretreatment with
anti-KC Ab significantly reduced the IL-17-driven neutrophil
accumulation. Primary cultures of HPMC expressed IL-17 receptor mRNA.
Exposure of HPMC to IL-17 led to a dose- and time-dependent induction
of GRO mRNA and protein. Combination of IL-17 together with TNF-
resulted in an increased stability of GRO mRNA and synergistic
release of GRO protein. Anti-IL-17 Ab blocked the effects of IL-17
in vitro and in vivo. IL-17 is capable of selectively recruiting
neutrophils into the peritoneal cavity via the release of
neutrophil-specific chemokines from the peritoneal
mesothelium. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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70–90% cells exhibit
the CD45RO+ phenotype of memory cells
(8). The substantial proportion of peritoneal T-cells have
also been found to express either CD8+
isoform or RAG-1 mRNA transcripts (5, 8). The
presence of these traits is believed to reflect thymus-independent T
cell differentiation (9) and may support the concept of
the peritoneal lymphoid tissue as an intestinal thymus (10, 11). A broad spectrum of effects exerted by lymphocyte-derived
mediator IFN-
(12) points to a significant role of
lymphocytes in the inflammatory response. In this respect, increased
levels of IFN- have been detected in the inflamed peritoneum, and
this rise has been clearly attributed to peritoneal lymphocytes
(13).
IL-17 is a newly identified T cell-specific cytokine (14).
The human form of IL-17 is a 20-kDa glycoprotein of 155 aa, the
sequence of which exhibits a close homology to both cytotoxic T
lymphocyte-associated Ag-8 (CTLA-8) and the open reading frame 13 of
T-lymphotropic Herpesvirus saimiri (HVS-13) (15, 16). Expression of IL-17 has been detected almost exclusively in
activated CD4+ and CD8+ T
lymphocytes (predominantly of the memory CD45RO+
subset) (15, 17, 18, 19). In sharp contrast, the specific
IL-17R is widely distributed in most tissues and cell lines (20, 21). Accumulating evidence suggests that IL-17 may be an
important mediator of the hemopoietic system (14, 22). It
has been found to stimulate the production of IL-6, G-CSF, and LIF
(17, 21, 23, 24)- cytokines with a known impact on
hemopoietic progenitors. Moreover, the soluble form of IL-17R protein
has been shown to inhibit mitogen-induced proliferation and IL-2
production in murine T cells (21). In contrast, IL-17 also
appears to be involved in the inflammatory reaction. The expression of
several genes associated with inflammation, including IL-1
, TNF-,
IL-6, IL-8, cyclooxygenase-2, NO synthase, and stromelysin, is
up-regulated after stimulation with IL-17 (17, 23, 25, 26, 27, 28, 29). In addition, increased production of the complement
component C3 in response to IL-17 has been detected in renal proximal
tubule cells (28).
In this study, we set out to investigate the potential role of IL-17 in the peritoneal inflammatory response. We demonstrate that IL-17 selectively recruits neutrophils into the peritoneal cavity and that this effect is likely to be mediated via the release of neutrophil-specific chemokines from the peritoneal mesothelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich Chemie (Deisenhofen, Germany). Tissue culture plastics were from Falcon Becton Dickinson (Heidelberg, Germany). Recombinant human and murine cytokines and anti-cytokine Abs were obtained from R&D Systems (Wiesbaden, Germany). According to the manufacturer, the endotoxin concentration in the above materials was <0.1 ng/µg protein as measured by Limulus amebocyte lysate assay. All cytokine preparations were batched, stored at -70°C, and freshly thawed for each experiment. All media and buffers were of tissue culture grade with an endotoxin concentration of <0.1 ng/ml.
Animal studies
All in vivo experiments were performed using male BALB/Cj inbred mice weighing 25–30 g. BALB/CJ/Han/Imp mice were obtained from the Institute of Occupational Medicine (Lodz, Poland) and housed under 12-h light/dark cycles with free access to standard chow and water. All studies were performed according to the guidelines of the Committee for Animal Studies at University Medical School (Poznan, Poland).
Recombinant mouse IL-17 (rmIL-17) was diluted in sterile endotoxin-free PBS (Dulbecco’s PBS, PAA Laboratories, Linz, Austria) and administered i.p. at a dose of 0.5 µg/mice in a total volume of 500 µl. The concentration of IL-17 to be applied was determined in preliminary dose-response experiments which showed that 0.5 µg IL-17 consistently produced a significant influx of neutrophils (data not shown). Control animals received an equivalent volume of PBS alone. For the procedure the animals were placed under brief ether anesthesia and then allowed to recover. In separate experiments, IL-17 (0.5 µg) was preincubated with monoclonal anti-mouse IL-17 neutralizing Ab (10 µg) at 37°C for 15 min before i.p. administration in 500 µl PBS. In further studies, the animals were first given i.p. injection of either monoclonal anti-mouse KC neutralizing Ab (10 µg/mouse in 200 µl PBS) or PBS alone, and after 15 min they received i.p. either IL-17 (0.5 µg in 300 µl PBS) or PBS alone. In an additional set of experiments, recombinant mouse KC was administered i.p. in 500 µl PBS.
At designated time points, the animals were anesthetized, sacrificed by bleeding, and injected i.p. with 2.5 ml PBS containing 3 mM EDTA (PBS/EDTA) (30). The peritoneal cavity was then opened, and the lavage fluid was carefully collected. Cell pellets were resuspended in PBS/EDTA, and total cell counts were determined in a hemocytometer using Türk’s solution. Differential cell counting was performed on cytospin preparations stained with May-Grünwald-Giemsa using a QCA staining kit (Quimica Clinica Aplicada, Amposta, Spain). Aliquots of cell-free peritoneal lavage fluids and sera were stored at -70°C until further analysis for cytokines.
Peritoneal mesothelial cell culture
Human peritoneal mesothelial cells (HPMC)3 were isolated from the specimens of omentum obtained from consenting patients undergoing elective abdominal surgery. Cells were isolated and characterized as described in detail elsewhere (31, 32). Cells were propagated in Earle’s buffered M199 culture medium (Seromed, Biochrom, Berlin, Germany) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), hydrocortisone (0.4 µg/ml), and 10% v/v FCS (Life Technologies, Eggenstein, Germany). Cell cultures were maintained at 37°C in a humidified 5% CO2 atmosphere.
All experiments were performed using cells derived from at least six separate donors, and from the first or second passage to minimize the number of senescent cells which appeared from the third passage onwards (31).
Effect of IL-17 on the production of growth-related oncogene
product (GRO) by human peritoneal mesothelial cells
HPMC were grown to confluence and rendered quiescent by serum
deprivation for 48 h before stimulation. Preliminary experiments
had demonstrated that under these conditions cells could be maintained
for at least up to 120 h without any significant loss of viability
(as assessed by intracellular ATP concentrations). HPMC were exposed to
recombinant human IL-17 at doses ranging from 0.01 to 100 ng/ml. In
some experiments, cells were exposed to IL-17 in the presence or
absence of TNF-. In the inhibition studies, HPMC were pretreated
with transcription (actinomycin D) or translation (cycloheximide)
inhibitors for 45 and 120 min, respectively, and then stimulated with
IL-17. The doses of inhibitors used did not impair cell viability. In
separate experiments, IL-17 preparations were first preincubated with
either anti-human IL-17 polyclonal neutralizing Ab or the
equivalent dose of control IgG of the same class, and then applied to
HPMC cultures.
At designated time intervals, the cell supernatants were removed,
centrifuged at 12,000 x g to remove any cellular
debris, and stored at -70°C until assayed. Cell monolayers were
washed with PBS and solubilized with 0.1 N NaOH. Total cellular protein
was then analyzed using Bradford method with Bio-Rad Protein Assay Dye
Reagent (Bio-Rad Laboratories, Munich, Germany) and BSA as the
standard. Repeated cell counts revealed that 1 µg HPMC protein
corresponded to (mean ± SD) 2.1 ± 1.0 x
103 cells (n = 16). All data for
GRO secretion were expressed as picograms per microgram cellular
protein.
Cytokine measurements
Concentrations of mouse KC and macrophage inflammatory protein-2
(MIP-2) in serum and peritoneal lavage fluid were determined using
Quantikine Mouse Immunoassays (R&D Systems) with sensitivities of 2
pg/ml for KC and 1.5 pg/ml for MIP-2. GRO levels in supernatants
from HPMC cultures were measured using the Quantikine Human GRO
Immunoassay (R&D Systems) with a sensitivity of 5.0 pg/ml.
RNA isolation and analysis
Total RNA from HPMC cultures was extracted with the RNA Isolator
(Genosys Biotechnologies, Cambridge, U.K.) and purified according to
the manufacturer’s protocol. Expression of IL-17R and of IL-17-induced
GRO mRNA was assessed using reverse transcription-PCR or Northern
blot analysis as described below.
Reverse transcription and PCR
One microgram of total RNA was reverse transcribed into cDNA
with random hexamer primers, as previously described (33).
PCR amplification was performed in a total volume of 50 µl consisting
of 2 µl reverse transcription product and 48 µl of the reaction
master mix. The master mix contained 36.25 µl
H2O, 2.5 µl sense and antisense primers (20
µM each), 4 µl dNTPs, 5 µl 10x PCR buffer (100 mM Tris, 500 mM
KCl, 15 mM MgCl2, 0.01% gelatin), and 0.25 µl
Taq polymerase (1.25 U, Amplitaq; Perkin-Elmer Cetus,
Weiterstadt, Germany). The amplification was conducted on the
Perkin-Elmer 480 Thermocycler (Perkin-Elmer Cetus, Applied Biosystems).
Specific oligonucleotide primer pairs were synthesized by TIB MolBiol
SyntheseLabor (Berlin, Germany). The primer sequences were as shown in
Table I
.
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-actin and GRO amplicons began with a 3-min
denaturation step at 94°C and was followed by 27 (GRO), 30
(-actin), or 33 (-actin) cycles of denaturation at 94°C for
40 s, annealing at 55°C for 1 min, and extension at 72°C for 1
min. The final cycle was 94°C for 40 s and 60°C for 10 min.
Preliminary experiments had determined that under these conditions PCR
products were generated during the exponential phase of amplification.
The protocol of PCR amplification with primers for IL-17R was similar
except that annealing temperature was 60°C, samples were amplified
for 35 cycles, and the final extension was at 72°C for 8 min. PCR
products were separated by electrophoresis in 3% agarose gels (FMC
Bioproducts, Biozym Diagnostic, Hess Oldendorf, Germany), stained with
ethidium bromide (1 µg/ml) and visualized under UV transillumination.
Expression of target mRNAs was assessed by comparison with the
expression of the "housekeeping" genes of - or -actin in the
same sample. The bands corresponding to the intended products were
analyzed using Scanpack 14.1A27 software (Biometra, Göttingen,
Germany). Northern blot analysis
Twenty micrograms total RNA were size-fractionated on 1.2%
agarose, 1.8 M formaldehyde gels and stained with ethidium bromide for
verification of RNA integrity and loading equivalency
(38, 39, 40). The RNA was electrotransferred onto nylon
membranes (Gene Screen, DuPont, Boston, MA) and cross-linked by UV
irradiation. The filters were then prehybridized, hybridized, and
washed under conditions appropriate for the
-32P-labeled GRO and 7S cDNA probes, as
previously described in detail (38, 39, 40). Prehybridization
of the membranes was conducted for 4–8 h at 42°C in a buffer
containing 30% formamide, 1% SDS, 0.75 M NaCl, 5 mM EDTA, 5x
Denhardt’s solution, 100 µg/ml salmon sperm DNA, 10% dextran
sulfate, and 50 mM sodium phosphate buffer, pH 7.4. Hybridization was
conducted at 42°C for 18 h with either 106
cpm/ml of the 32P-labeled GRO probe or
105 cpm/ml of the
32P-labeled 7S cDNA probe. After the
hybridization, the blots were washed under stringent conditions with
two rinses in 2x SSC at 50°C and three washes (20 min each) in 0.2x
SSC/2% SDS at 55°C. Blots were then exposed to Fuji x-ray films with
intensifying screens (DuPont) at -80°C, and the intensity of the
radiographic bands was quantified by video image analysis (Image-Pro
plus, Media Cybernetics, Silver Spring, MD), as previously reported
(38, 41). The ratio between GRO and corresponding 7S
signal was calculated for each sample.
Probe for Northern blot analysis
GRO DNA probe used in Northern blot analysis consisted of a
231-bp fragment corresponding to positions 452–682 of the GRO mRNA.
The probe was designed using nucleotide sequence of the GRO gene as
published by Baker et al. (42). The fragment was cloned by
reverse transcribing human normal pancreas RNA, amplifying the cDNA
obtained by PCR, and ligating the amplicon into the pGEM-T Easy Vector
(Promega, Biotechnology, Madison, WI). The 7S DNA probe consisted of a
212-bp fragment of the 7S RNA which was cloned as described above. This
probe was used to verify equivalent RNA loading in the Northern blot
experiments (38, 40). Authenticity of GRO and 7S
fragments was confirmed by sequencing using the dye terminator method
(ABI 373A, Perkin-Elmer, Rotkreuz, Switzerland). For Northern blot
analysis, the GRO and 7S DNA probes were radiolabeled with
[-32P]dCTP (DuPont International,
Regensdorf, Switzerland) using a random primer labeling system (NEN
Life Science Products, Boston, MA).
Stability of IL-17-induced GRO mRNA
The inherent stability of mesothelial cell GRO mRNA was
assessed by measuring the rate of GRO mRNA degradation in the
presence of the transcription inhibitor actinomycin D. HPMC were
stimulated with IL-17 (50 ng/ml) in the presence or absence of TNF-
(1 ng/ml) for 2 h. After that, cells were washed and pulsed with
actinomycin D (5 µg/ml). At defined time intervals, the total RNA was
extracted, reverse transcribed into cDNA, and PCR amplified for GRO
and -actin as described above.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 3.00 software (GraphPad Software, San Diego, CA). Multiple comparisons of paired data were made with nonparametric repeated measures ANOVA with Friedman modification. Unpaired data derived from animal studies were analyzed using Mann-Whitney U or Kruskal-Wallis tests, when appropriate. A p value of <0.05 was considered significant. All data are presented as means ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Injection of IL-17 i.p. in experimental animals resulted in a
time-dependent increase in the total number of cells in the peritoneal
cavity (Fig. 1). The maximal effect was
observed 4 h after the administration of IL-17 when the
accumulation of cells was 2.6-fold above the numbers detected in
control mice. Differential cell counting revealed that this increase
could be accounted for by a substantial rise in the number of
polymorphonuclear neutrophils (PMN, Table II, Fig. 2). In untreated mice (no i.p.
injections, lavage only, n = 9), PMN constituted merely
1.1 ± 0.4% of the peritoneal cell population. In control
animals, the procedure of i.p. injection and/or PBS itself produced a
small nonspecific increase in the number of PMN. However, the influx of
PMN triggered by IL-17 was significantly above these background levels
(data not shown). During the time frame studied, the absolute number of
cells from other populations did not differ significantly from those
detected in control and untreated animals (Table II). Administration of
IL-17 together with anti-IL-17 neutralizing Ab reduced the specific
IL-17-mediated PMN influx by 61.2 ± 9.5% within 4
h.
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Administration of IL-17 produced a massive increase in the i.p.
concentrations of KC chemokine, a murine analogue of human GRO (Ref.
43 and Fig. 2
). The level of KC increased rapidly within
1 h and by 4 h returned to basal values. Comparison of KC
concentrations in sera and lavage fluids indicated that KC released in
response to IL-17 was of the local i.p. origin. At the 1-h time point,
the mean concentration of KC in the lavage fluid was 3401 ± 592
pg/ml compared with 264 ± 50 pg/ml in serum (n =
6). The mean serum KC level in untreated mice was 150 ± 48 pg/ml
(n = 8) and corresponded to the values detected by the
manufacturer of the mouse KC immunoassay (R&D Systems). In addition,
IL-17 triggered a rapid i.p. release of neutrophil chemoattractant
MIP-2. Within 1 h, MIP-2 concentration in the lavage fluid rose
from 14 ± 5 pg/ml to 1250 ± 116 pg/ml, compared with plasma
levels of 45 ± 6 pg/ml (n = 6–9).
Neutralization of KC reduces IL-17-stimulated neutrophil recruitment
The observation that IL-17-induced PMN influx was preceded by a
rapid increase in KC levels (Fig. 2) suggested that the effect could
have been mediated by KC, a powerful neutrophil chemoattractant
(43). Treatment i.p. with recombinant KC resulted in a
dose-dependent and selective increase in PMN recruitment (data not
shown). The dose of 0.5 µg KC/animal produced a 7.5 ± 0.7-fold
increase in PMN accumulation within 4 h. Furthermore,
administration of anti-KC neutralizing Ab before IL-17 injection
reduced the IL-17-specific PMN infiltration within 4 h by
67.2 ± 7.9%, although it had no significant effect on basal PMN
influx triggered by the injection of PBS vehicle (Fig. 3).
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RT-PCR analysis using primers specific for either the
extracellular or intracellular domain of IL-17R revealed that HPMC
expressed IL-17R mRNA. Constitutive expression of IL-17R gene
transcripts was detected in all primary cultures of HPMC examined (Fig. 4).
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production by peritoneal mesothelial cells
HPMC released GRO constitutively. Exposure of HPMC to a
recombinant form of human IL-17 resulted in a time- and dose-dependent
increase in GRO generation. With IL-17 at a dose of 50 ng/ml, this
increase became significant above control levels after 6 h of
incubation and was followed by large increments during the next 18
h and a plateau by 24 h (Fig. 5).
Statistically significant increase in GRO secretion was achieved
with IL-17 at a dose of 1 ng/ml and above (Fig. 6). Administration of IL-17 in the
presence of anti-IL-17 neutralizing Ab reduced IL-17-stimulated
GRO production from 825.9 ± 97.3 to 131.8 ± 35.9 pg/µg
cell protein (n = 7, p < 0.05),
approximately to the baseline release of GRO (107.6 ± 33.9
pg/µg cell protein). In contrast, control Ab of the same class did
not affect the stimulatory activity of IL-17 (882.0 ± 115.0
pg/µg cell protein).
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release
Preexposure of HPMC to actinomycin D for 45 min at 37°C resulted
in a dose-dependent decrease in IL-17-stimulated but not in
constitutive GRO secretion. Maximal inhibition was achieved with the
dose of 1 µg/ml, which reduced IL-17-driven GRO release by
90.1 ± 6.8% (687.3 ± 116.8 vs 68.1 ± 46.8 pg/µg
cell protein) to the level detected in unstimulated cells
(n = 5, p < 0.01). Generation of
GRO in HPMC stimulated with IL-17 could also be inhibited by
cycloheximide. At the highest nontoxic dose of cycloheximide tested (50
µg/ml) GRO release was reduced by 51.4 ± 6.1% (747.3
± 105.4 vs 363.1 ± 45.5 pg/µg cell protein, n
= 7, p < 0.01).
Exposure to IL-17 induces GRO mRNA in peritoneal mesothelial
cells
Stimulation of HPMC with IL-17 induced a time- and dose-dependent
accumulation of GRO mRNA as demonstrated by Northern blot analysis
(Fig. 7). Unstimulated cells expressed
very faint signals for GRO mRNA. After treatment with IL-17, the
GRO mRNA expression was rapidly up-regulated within 1 h.
Increased expression of GRO mRNA was detected in cells stimulated
with a dose of IL-17 as low as 0.1 ng/ml.
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superinduces IL-17-driven GRO synthesis by stabilizing
GRO mRNA
Exposure of HPMC to TNF- increased GRO release in a time-
and dose-dependent manner (data not shown). Combination of TNF-
together with IL-17 triggered GRO production above the levels
generated by each stimulus alone and significantly above the calculated
additive value (Fig. 8). This synergistic
effect became evident when TNF- (1 ng/ml) was combined with 0.1
ng/ml IL-17, and maximal synergy was obtained at the highest dose of
IL-17 tested (100 ng/ml). GRO release under these conditions was
2.2 ± 0.2-fold above the predicted additive value. Actinomycin D
chase experiments revealed that in HPMC treated with a combination of
IL-17 and TNF- the degradation of GRO mRNA was delayed compared
with cells treated with IL-17 alone (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Administration of IL-17 i.p. resulted in a massive and selective influx
of neutrophils. Neutrophil population appeared to have been targeted
specifically because the trafficking of other leukocyte subsets was not
significantly affected. Similar effects of IL-17 were observed in the
rat airways where intratracheal IL-17 instillation induced selective
accumulation of neutrophils in the bronchoalveolar lavage fluid
(45, 46). The specificity of these effects was confirmed
by the inhibition with anti-IL-17 neutralizing Ab. The fact that
this inhibition was incomplete (60%) could be explained by the
limited neutralizing capacity of the Ab under in vivo conditions in
comparison with the dose of IL-17 used.
Because IL-17 has been shown to have no direct effect on neutrophil
chemotaxis in vitro (45), we hypothesized that PMN
accumulation in response to IL-17 could have been mediated by the
induction of chemokines. Analysis of the peritoneal fluid revealed that
IL-17 induced a rapid rise in i.p. levels of KC which preceded the
influx of PMN. The similar time course of KC induction has been
observed in various models of murine peritonitis (30, 47).
KC is a chemokine with a powerful chemotactic activity toward
neutrophils (48) and, indeed, the i.p. injection of
recombinant KC triggered a massive accumulation of PMN in the
peritoneal cavity. Furthermore, when IL-17-receiving animals were
pretreated with anti-KC neutralizing Ab, the IL-17-mediated PMN
recruitment was reduced by >60%. Again, the magnitude of inhibition
observed under these conditions could be influenced by the neutralizing
capacity of the Ab used. However, it is also possible that the
remaining chemotactic activity could be attributed to other chemokines
induced by IL-17. Indeed, we found that IL-17 also produced a
significant rise in i.p. MIP-2 levels. In this respect, Laan et al.
(45) have convincingly documented the role of MIP-2 as a
mediator of IL-17-induced leukocyte trafficking in the rat, and Walley
et al. (49) have demonstrated the importance of MIP-2 in
the pathogenesis of cecal ligation and puncture model of peritonitis.
MIP-2 is a chemokine with no exact human homologue but closely related
to either IL-8 (50) or GRO (43, 51).
The observation that the levels of KC in the peritoneum were much
higher than those detected in serum suggested the local origin of KC.
One possible source could be the peritoneal macrophage; however, in a
recent study Ajuebor et al. (47) have demonstrated that in
an LPS model of peritonitis in mice the removal of peritoneal
macrophages and monocytes did not diminish the peritoneal generation of
KC. We have therefore concentrated on peritoneal mesothelial cells. The
peritoneal mesothelium is a recognized source of chemotactic activity
in the peritoneum (32, 52, 53, 54), and it has been
demonstrated that on appropriate stimulation human mesothelial cells
are capable of generating GRO, a homologue of KC (55).
The presence of IL-17R mRNA in HPMC was demonstrated by RT-PCR and
confirmed the ubiquitous nature of IL-17R distribution (20, 21). Exposure of quiescent HPMC to IL-17 led to a significant
time- and dose-dependent increase in the secretion of GRO. The range
of IL-17 doses that triggered this effect corresponded to those that
had been shown to stimulate the release of cytokines in other in vitro
systems (17, 25, 26, 28, 29, 35, 45, 56). The IL-17-driven
GRO release could be inhibited in a dose-dependent manner by the
pretreatment of HPMC with both transcription and translation inhibitors
which suggested that IL-17 stimulated de novo GRO synthesis.
Northern blot analysis confirmed that exposure of HPMC to IL-17
resulted in a rapid up-regulation of GRO mRNA. Using a rat
intestinal epithelial cell line, Awane et al. (29) have
recently analyzed the IL-17-activated signal transduction pathway
leading to the induction of CINC, a C-X-C chemokine related to human
GRO. In this system, IL-17 has been shown to induce the activity of
NF-
B-dependent CINC promoter via the pathway regulated by
TNFR-associated factor-6 and NF-B-inducing kinase. IL-17-induced
NF-B activity has also been demonstrated in macrophages
(25), chondrocytes (26), and fibroblasts
(21).
We have found that IL-17-induced generation of GRO could be
synergistically augmented in the presence of TNF-. This effect was
at least partially related to the stabilization of GRO mRNA. Because
IL-17 has been demonstrated to stimulate the release of TNF- from
macrophages (25), one may imagine that by acting
simultaneously on mesothelial cell and macrophages in the peritoneal
cavity in vivo lymphocyte-derived IL-17 amplifies the generation of
GRO and increases the transperitoneal chemotactic gradient for
neutrophils. In this respect, it has been demonstrated that truncated
form of GRO, which acts as a C-X-C chemokine receptor antagonist, is
capable of inhibiting leukocyte recruitment into the peritoneal cavity
(57). In other cell systems, IL-17 has been shown to
synergize with TNF- in the production of IL-8 (35, 45),
IL-6, and GM-CSF (17). The potential synergy between IL-17
and TNF- under in vivo conditions is currently being
investigated.
The exact role of IL-17 in human physiology and pathology remains to be
determined. Available data suggest that IL-17 may promote cartilage
destruction in various forms of arthritis (23, 26, 56, 58, 59) and mediate alloimmune reactivity and organ allograft
rejection (28, 60). Our findings demonstrate that IL-17
may act as a potent and selective inducer of neutrophil chemotaxis. In
the setting of the peritoneal cavity, this effect appears to be
mediated through the stimulation of GRO release from peritoneal
mesothelial cells. These properties of IL-17 provide further evidence
that it can be classified as a proinflammatory cytokine
(22). Because IL-17-producing
CD45RO+ cells predominate among i.p. lymphocytes,
IL-17 may have a significant role to play in the peritoneal
inflammatory response. Our findings also add to the understanding of
mesothelial cell biology and point to the importance of
lymphocyte-mesothelial interactions in peritonitis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Achim Jörres, Department of Nephrology and Medical Intensive Care, Universitätsklinikum Charité, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany.
3 Abbreviations used in this paper: HPMC, human peritoneal mesothelial cells; GRO, growth-related oncogene ; MIP-2, macrophage inflammatory protein-2; PMN, polymorphonuclear leukocyte.
Received for publication February 23, 2000. Accepted for publication August 21, 2000.
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A. Freitas, J. C. Alves-Filho, T. Victoni, T. Secher, H. P. Lemos, F. Sonego, F. Q. Cunha, and B. Ryffel IL-17 Receptor Signaling Is Required to Control Polymicrobial Sepsis J. Immunol., June 15, 2009; 182(12): 7846 - 7854. [Abstract] [Full Text] [PDF]
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J. R. Nichols, A. L. Aldrich, M. M. Mariani, D. Vidlak, N. Esen, and T. Kielian TLR2 Deficiency Leads to Increased Th17 Infiltrates in Experimental Brain Abscesses J. Immunol., June 1, 2009; 182(11): 7119 - 7130. [Abstract] [Full Text] [PDF]
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 H. P. Lemos, R. Grespan, S. M. Vieira, T. M. Cunha, W. A. Verri Jr., K. S. S. Fernandes, F. O. Souto, I. B. McInnes, S. H. Ferreira, F. Y. Liew, et al. Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFN{gamma} production PNAS, April 7, 2009; 106(14): 5954 - 5959. [Abstract] [Full Text] [PDF]
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Y. Ke, K. Liu, G.-Q. Huang, Y. Cui, H. J. Kaplan, H. Shao, and D. Sun Anti-Inflammatory Role of IL-17 in Experimental Autoimmune Uveitis J. Immunol., March 1, 2009; 182(5): 3183 - 3190. [Abstract] [Full Text] [PDF]
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Y. Liu, Y. Yuan, Y. Li, J. Zhang, G. Xiao, Y. Vodovotz, T. R. Billiar, M. A. Wilson, and J. Fan Interacting Neuroendocrine and Innate and Acquired Immune Pathways Regulate Neutrophil Mobilization from Bone Marrow following Hemorrhagic Shock J. Immunol., January 1, 2009; 182(1): 572 - 580. [Abstract] [Full Text] [PDF]
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 J. J. Yu, M. J. Ruddy, H. R. Conti, K. Boonanantanasarn, and S. L. Gaffen The Interleukin-17 Receptor Plays a Gender-Dependent Role in Host Protection against Porphyromonas gingivalis-Induced Periodontal Bone Loss Infect. Immun., September 1, 2008; 76(9): 4206 - 4213. [Abstract] [Full Text] [PDF]
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 R. C. Furze and S. M. Rankin The role of the bone marrow in neutrophil clearance under homeostatic conditions in the mouse FASEB J, September 1, 2008; 22(9): 3111 - 3119. [Abstract] [Full Text] [PDF]
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R. M. McLoughlin, J. C. Lee, D. L. Kasper, and A. O. Tzianabos IFN-{gamma} Regulated Chemokine Production Determines the Outcome of Staphylococcus aureus Infection J. Immunol., July 15, 2008; 181(2): 1323 - 1332. [Abstract] [Full Text] [PDF]
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P. D. Doodes, Y. Cao, K. M. Hamel, Y. Wang, B. Farkas, Y. Iwakura, and A. Finnegan Development of Proteoglycan-Induced Arthritis Is Independent of IL-17 J. Immunol., July 1, 2008; 181(1): 329 - 337. [Abstract] [Full Text] [PDF]
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C. A. Cox, G. Shi, H. Yin, B. P. Vistica, E. F. Wawrousek, C.-C. Chan, and I. Gery Both Th1 and Th17 Are Immunopathogenic but Differ in Other Key Biological Activities J. Immunol., June 1, 2008; 180(11): 7414 - 7422. [Abstract] [Full Text] [PDF]
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K. Wu, S. Katiyar, A. Li, M. Liu, X. Ju, V. M. Popov, X. Jiao, M. P. Lisanti, A. Casola, and R. G. Pestell Dachshund inhibits oncogene-induced breast cancer cellular migration and invasion through suppression of interleukin-8 PNAS, May 13, 2008; 105(19): 6924 - 6929. [Abstract] [Full Text] [PDF]
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L. I. Rutitzky, L. Bazzone, M. G. Shainheit, B. Joyce-Shaikh, D. J. Cua, and M. J. Stadecker IL-23 Is Required for the Development of Severe Egg-Induced Immunopathology in Schistosomiasis and for Lesional Expression of IL-17 J. Immunol., February 15, 2008; 180(4): 2486 - 2495. [Abstract] [Full Text] [PDF]
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A. Valaperti, R. R. Marty, G. Kania, D. Germano, N. Mauermann, S. Dirnhofer, B. Leimenstoll, P. Blyszczuk, C. Dong, C. Mueller, et al. CD11b+ Monocytes Abrogate Th17 CD4+ T Cell-Mediated Experimental Autoimmune Myocarditis J. Immunol., February 15, 2008; 180(4): 2686 - 2695. [Abstract] [Full Text] [PDF]
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F. Huang, C.-Y. Kao, S. Wachi, P. Thai, J. Ryu, and R. Wu Requirement for Both JAK-Mediated PI3K Signaling and ACT1/TRAF6/TAK1-Dependent NF-{kappa}B Activation by IL-17A in Enhancing Cytokine Expression in Human Airway Epithelial Cells J. Immunol., November 15, 2007; 179(10): 6504 - 6513. [Abstract] [Full Text] [PDF]
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 C. C. Caldwell, J. Tschoep, and A. B. Lentsch Lymphocyte function during hepatic ischemia/reperfusion injury J. Leukoc. Biol., September 1, 2007; 82(3): 457 - 464. [Abstract] [Full Text] [PDF]
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J.-H. Park, Y.-G. Kim, M. Shaw, T.-D. Kanneganti, Y. Fujimoto, K. Fukase, N. Inohara, and G. Nunez Nod1/RICK and TLR Signaling Regulate Chemokine and Antimicrobial Innate Immune Responses in Mesothelial Cells J. Immunol., July 1, 2007; 179(1): 514 - 521. [Abstract] [Full Text] [PDF]
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 J. J. Yu, M. J. Ruddy, G. C. Wong, C. Sfintescu, P. J. Baker, J. B. Smith, R. T. Evans, and S. L. Gaffen An essential role for IL-17 in preventing pathogen-initiated bone destruction: recruitment of neutrophils to inflamed bone requires IL-17 receptor-dependent signals Blood, May 1, 2007; 109(9): 3794 - 3802. [Abstract] [Full Text] [PDF]
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 K. L. Smiley, M. M. McNeal, M. Basu, A. H.-C. Choi, J. D. Clements, and R. L. Ward Association of Gamma Interferon and Interleukin-17 Production in Intestinal CD4+ T Cells with Protection against Rotavirus Shedding in Mice Intranasally Immunized with VP6 and the Adjuvant LT(R192G) J. Virol., April 15, 2007; 81(8): 3740 - 3748. [Abstract] [Full Text] [PDF]
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K. Shibata, H. Yamada, H. Hara, K. Kishihara, and Y. Yoshikai Resident V{delta}1+ {gamma}{delta} T Cells Control Early Infiltration of Neutrophils after Escherichia coli Infection via IL-17 Production J. Immunol., April 1, 2007; 178(7): 4466 - 4472. [Abstract] [Full Text] [PDF]
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M. Umemura, A. Yahagi, S. Hamada, M. D. Begum, H. Watanabe, K. Kawakami, T. Suda, K. Sudo, S. Nakae, Y. Iwakura, et al. IL-17-Mediated Regulation of Innate and Acquired Immune Response against Pulmonary Mycobacterium bovis Bacille Calmette-Guerin Infection J. Immunol., March 15, 2007; 178(6): 3786 - 3796. [Abstract] [Full Text] [PDF]
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 M. Frink, A. Lu, B. M. Thobe, Y.-C. Hsieh, M. A. Choudhry, M. G. Schwacha, S. L. Kunkel, and I. H. Chaudry Monocyte chemoattractant protein-1 influences trauma-hemorrhage-induced distal organ damage via regulation of keratinocyte-derived chemokine production Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1110 - R1116. [Abstract] [Full Text] [PDF]
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 F. Shen, Z. Hu, J. Goswami, and S. L. Gaffen Identification of Common Transcriptional Regulatory Elements in Interleukin-17 Target Genes J. Biol. Chem., August 25, 2006; 281(34): 24138 - 24148. [Abstract] [Full Text] [PDF]
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J. L. VanCott, A. E. Prada, M. M. McNeal, S. C. Stone, M. Basu, B. Huffer Jr., K. L. Smiley, M. Shao, J. A. Bean, J. D. Clements, et al. Mice Develop Effective but Delayed Protective Immune Responses When Immunized as Neonates either Intranasally with Nonliving VP6/LT(R192G) or Orally with Live Rhesus Rotavirus Vaccine Candidates. J. Virol., May 1, 2006; 80(10): 4949 - 4961. [Abstract] [Full Text] [PDF]
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M. A. Ingersoll and A. Zychlinsky ShiA Abrogates the Innate T-Cell Response to Shigella flexneri Infection Infect. Immun., April 1, 2006; 74(4): 2317 - 2327. [Abstract] [Full Text] [PDF]
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T. Kielian, A. Haney, P. M. Mayes, S. Garg, and N. Esen Toll-Like Receptor 2 Modulates the Proinflammatory Milieu in Staphylococcus aureus-Induced Brain Abscess Infect. Immun., November 1, 2005; 73(11): 7428 - 7435. [Abstract] [Full Text] [PDF]
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 C. C. Caldwell, T. Okaya, A. Martignoni, T. Husted, R. Schuster, and A. B. Lentsch Divergent functions of CD4+ T lymphocytes in acute liver inflammation and injury after ischemia-reperfusion Am J Physiol Gastrointest Liver Physiol, November 1, 2005; 289(5): G969 - G976. [Abstract] [Full Text] [PDF]
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 K. I. Happel, P. J. Dubin, M. Zheng, N. Ghilardi, C. Lockhart, L. J. Quinton, A. R. Odden, J. E. Shellito, G. J. Bagby, S. Nelson, et al. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae J. Exp. Med., September 19, 2005; 202(6): 761 - 769. [Abstract] [Full Text] [PDF]
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F. Shen, M. J. Ruddy, P. Plamondon, and S. L. Gaffen Cytokines link osteoblasts and inflammation: microarray analysis of interleukin-17- and TNF-{alpha}-induced genes in bone cells J. Leukoc. Biol., March 1, 2005; 77(3): 388 - 399. [Abstract] [Full Text] [PDF]
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S. Yamazaki, T. Muta, S. Matsuo, and K. Takeshige Stimulus-specific Induction of a Novel Nuclear Factor-{kappa}B Regulator, I{kappa}B-{zeta}, via Toll/Interleukin-1 Receptor Is Mediated by mRNA Stabilization J. Biol. Chem., January 14, 2005; 280(2): 1678 - 1687. [Abstract] [Full Text] [PDF]
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 M. Umemura, T. Kawabe, K. Shudo, H. Kidoya, M. Fukui, M. Asano, Y. Iwakura, G. Matsuzaki, R. Imamura, and T. Suda Involvement of IL-17 in Fas ligand-induced inflammation Int. Immunol., August 1, 2004; 16(8): 1099 - 1108. [Abstract] [Full Text] [PDF]
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M. J. Ruddy, F. Shen, J. B. Smith, A. Sharma, and S. L. Gaffen Interleukin-17 regulates expression of the CXC chemokine LIX/CXCL5 in osteoblasts: implications for inflammation and neutrophil recruitment J. Leukoc. Biol., July 1, 2004; 76(1): 135 - 144. [Abstract] [Full Text] [PDF]
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M. J. Ruddy, G. C. Wong, X. K. Liu, H. Yamamoto, S. Kasayama, K. L. Kirkwood, and S. L. Gaffen Functional Cooperation between Interleukin-17 and Tumor Necrosis Factor-{alpha} Is Mediated by CCAAT/Enhancer-binding Protein Family Members J. Biol. Chem., January 23, 2004; 279(4): 2559 - 2567. [Abstract] [Full Text] [PDF]
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W. H. Faour, A. Mancini, Q. W. He, and J. A. Di Battista T-cell-derived Interleukin-17 Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of the p38 Mitogen-activated Protein Kinase Cascade: ROLE OF DISTAL SEQUENCES IN THE 3'-UNTRANSLATED REGION OF COX-2 mRNA J. Biol. Chem., July 11, 2003; 278(29): 26897 - 26907. [Abstract] [Full Text] [PDF]
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Y. Chen, P. Thai, Y.-H. Zhao, Y.-S. Ho, M. M. DeSouza, and R. Wu Stimulation of Airway Mucin Gene Expression by Interleukin (IL)-17 through IL-6 Paracrine/Autocrine Loop J. Biol. Chem., May 2, 2003; 278(19): 17036 - 17043. [Abstract] [Full Text] [PDF]
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K. I. Happel, M. Zheng, E. Young, L. J. Quinton, E. Lockhart, A. J. Ramsay, J. E. Shellito, J. R. Schurr, G. J. Bagby, S. Nelson, et al. Cutting Edge: Roles of Toll-Like Receptor 4 and IL-23 in IL-17 Expression in Response to Klebsiella pneumoniae Infection J. Immunol., May 1, 2003; 170(9): 4432 - 4436. [Abstract] [Full Text] [PDF]
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D. R. Chung, D. L. Kasper, R. J. Panzo, T. Chtinis, M. J. Grusby, M. H. Sayegh, and A. O. Tzianabos CD4+ T Cells Mediate Abscess Formation in Intra-abdominal Sepsis by an IL-17-Dependent Mechanism J. Immunol., February 15, 2003; 170(4): 1958 - 1963. [Abstract] [Full Text] [PDF]
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S. Ferretti, O. Bonneau, G. R. Dubois, C. E. Jones, and A. Trifilieff IL-17, Produced by Lymphocytes and Neutrophils, Is Necessary for Lipopolysaccharide-Induced Airway Neutrophilia: IL-15 as a Possible Trigger J. Immunol., February 15, 2003; 170(4): 2106 - 2112. [Abstract] [Full Text] [PDF]
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 P. W. Hellings, A. Kasran, Z. Liu, P. Vandekerckhove, A. Wuyts, L. Overbergh, C. Mathieu, and J. L. Ceuppens Interleukin-17 Orchestrates the Granulocyte Influx into Airways after Allergen Inhalation in a Mouse Model of Allergic Asthma Am. J. Respir. Cell Mol. Biol., January 1, 2003; 28(1): 42 - 50. [Abstract] [Full Text] [PDF]
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J. Maertzdorf, A. D. M. E. Osterhaus, and G. M. G. M. Verjans IL-17 Expression in Human Herpetic Stromal Keratitis: Modulatory Effects on Chemokine Production by Corneal Fibroblasts J. Immunol., November 15, 2002; 169(10): 5897 - 5903. [Abstract] [Full Text] [PDF]
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S. B. Forlow, E. J. White, K. L. Thomas, G. J. Bagby, P. L. Foley, and K. Ley T Cell Requirement for Development of Chronic Ulcerative Dermatitis in E- and P-Selectin-Deficient Mice J. Immunol., November 1, 2002; 169(9): 4797 - 4804. [Abstract] [Full Text] [PDF]
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D. R. Chung, T. Chitnis, R. J. Panzo, D. L. Kasper, M. H. Sayegh, and A. O. Tzianabos CD4+ T Cells Regulate Surgical and Postinfectious Adhesion Formation J. Exp. Med., June 3, 2002; 195(11): 1471 - 1478. [Abstract] [Full Text] [PDF]
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C. E. Jones and K. Chan Interleukin-17 Stimulates the Expression of Interleukin-8, Growth-Related Oncogene-alpha , and Granulocyte-Colony-Stimulating Factor by Human Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., June 1, 2002; 26(6): 748 - 753. [Abstract] [Full Text] [PDF]
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F. Benchetrit, A. Ciree, V. Vives, G. Warnier, A. Gey, C. Sautes-Fridman, F. Fossiez, N. Haicheur, W. H. Fridman, and E. Tartour Interleukin-17 inhibits tumor cell growth by means of a T-cell-dependent mechanism Blood, March 15, 2002; 99(6): 2114 - 2121. [Abstract] [Full Text] [PDF]
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S. Aggarwal and A. L. Gurney IL-17: prototype member of an emerging cytokine family J. Leukoc. Biol., January 1, 2002; 71(1): 1 - 8. [Abstract] [Full Text] [PDF]
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G. Pan, D. French, W. Mao, M. Maruoka, P. Risser, J. Lee, J. Foster, S. Aggarwal, K. Nicholes, S. Guillet, et al. Forced Expression of Murine IL-17E Induces Growth Retardation, Jaundice, a Th2-Biased Response, and Multiorgan Inflammation in Mice J. Immunol., December 1, 2001; 167(11): 6559 - 6567. [Abstract] [Full Text] [PDF]
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) or an equivalent volume of PBS vehicle (controls,
). At each time point, the peritoneal lavage fluid was
obtained from four to nine animals. *, Statistically significant
difference compared with the values obtained from the controls at the
same time point.
) in the peritoneal cavity was determined in mice injected with 0.5
µg rmIL-17. Data were obtained from six to nine animals per time
point. *, Significant difference compared with values obtained before
the injection of IL-17.
, TNF-
, IL-17; 