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Controls Neutrophil Migration Across the Mesothelium In Vitro and In Vivo1
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Institute of Nephrology, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom;
Theodor Kocher Institute, University of Bern, Bern, Switzerland; and
Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the process of leukocyte recruitment into the peritoneal cavity.
Stimulation of cultured human peritoneal mesothelial cells with IFN-
(1–100 U/ml) alone or in combination with IL-1
(100 pg/ml) or
TNF-
(1000 pg/ml) resulted in significant up-regulation of monocyte
chemoattractant protein-1 and RANTES protein secretion. In contrast,
IFN- inhibited basal and IL-1-, and TNF--induced production of
IL-8. The modulating effects of IFN- on chemokine production
occurred at the level of gene expression, and the degree of regulation
observed was dependent on the doses of IL-1 and TNF- used.
Analysis of the functional effects of IFN- on IL-1-induced
transmesothelial PMN migration with an in vitro human transmigration
system and an in vivo murine model of peritoneal inflammation
demonstrated that IFN- was able to down-regulate PMN migration
induced by optimal doses of IL-1. These effects were mediated in
vivo via down-regulation of CXC chemokine synthesis. These findings
suggest that IFN- may play a role in controlling the phenotype of
infiltrating leukocyte during the course of an inflammatory response,
in part via regulation of resident cell chemokine
synthesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, cytokine-activated HPMC have been shown to secrete both
neutrophil-specific CXC (IL-8 and growth-related oncogene (GRO) )
and mononuclear-specific CC chemokines (monocyte chemoattractant
protein (MCP)-1, RANTES and inflammatory protein-10; Ref.
8). Studies that use a transwell culture systems, in which
polarized cultures of HPMC are established on porous inserts, have
demonstrated that chemokine secretion by IL-1- or TNF--activated
HPMC is directional, with >80% of the chemokine secreted apically,
resulting in the formation of a chemotactic gradient across the cell
monolayer. Furthermore, specific Ab blocking studies have demonstrated
a direct role for HPMC-derived IL-8 and MCP-1 in mediating increased
transmesothelial polymorphonuclear leukocyte (PMN) and MNC migration,
respectively (7). Evidence for similar mechanisms
occurring in vivo arises from studies showing elevation of i.p. levels
of these same chemokines during peritoneal inflammation, which
correlated directly with the number and phenotype of the recruited cell
populations (9, 10, 11, 12).
Although recent studies have established an important role for
HPMC-derived chemokine and adhesion molecule expression in mediating
PMN and MNC influx into the peritoneal cavity, little is known about
the mechanisms that control the switch in the phenotype of cell
infiltrate from PMN to MNC (13). Measurement of i.p.
cytokine levels during peritoneal dialysis-associated peritonitis
suggests that i.p. elevation of the T cell-derived cytokines IFN-,
IL-2, and IL-10 occur during infection (14, 15). Recent in
vitro studies indicate that IFN- is capable of modulating both CXC
and CC chemokine secretion in a number of cell types, data that suggest
that it plays a central role in controlling leukocyte influx
(16, 17, 18). However, the role of IFN- in the control of
peritoneal leukocyte recruitment has not been determined.
In the present study, we have examined the effects of IFN- on
neutrophil and mononuclear-specific chemokine production by cytokine
(IL-1 or TNF-)-activated HPMC, and investigated the potential of
IFN- to modulate the transmesothelial migration of PMN both in vitro
and in vivo. Our results indicate that IFN- has differential effects
on MCP-1, RANTES, and IL-8 production. These changes in chemokine
secretion correlated directly with the ability of IFN- to
down-regulate IL-1- or TNF--induced PMN transmesothelial
migration both in vitro and in vivo. The ability of IFN- to
differentially modulate chemokine production and ultimately
transmesothelial leukocyte migration may be a further mechanism by
which IFN- promotes mononuclear phagocyte function and the
development of Th1-type responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
TNF-, and IFN- were obtained from R&D Systems (Abingdon, U.K.).
The specific activity of IFN- was 107 U/mg
protein. Recombinant murine IL-1 and IFN- were obtained from
PeproTech EC, (London, U.K.). Endotoxin levels in recombinant cytokines
were less than 0.1 ng/µg (1 EU/µg) protein. Culture inserts and
24-well companion plates, tissue culture flasks, and all multiwell
plates were obtained from Falcon (BD Biosciences, Oxford, U.K.).
Digoxigenin (DIG)-labeled mixtures of oligonucleotide probes to MCP-1,
RANTES, IL-8, and -actin were obtained from R&D Systems. All
reagents used for the detection of DIG-labeled probes were from
Boehringer Mannheim (Lewes, U.K.). Isolation and culture of HPMC
HPMC obtained from the omental tissue of consenting patients undergoing abdominal surgery, were isolated and characterized as described previously (19). Cells were maintained in Earle’s buffered M199 culture medium (Sigma) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM; all obtained from Life Technologies, Paisley, U.K.), transferrin (5 µg/ml), insulin (5 µg/ml), hydrocortisone (0.4 µg/ml; Sigma) and 10% (v/v) FCS (Perbioscience, Tattenhall, U.K.). Cultures were passaged with trypsin-EDTA-glucose (0.125% (w/v), 0.01% (w/v), 0.1% (w/v)) and maintained at 37°C in a humidified 5% CO2 atmosphere.
All data presented are from experiments performed with confluent HPMC from the second passage. All cells were washed and growth arrested for 48 h in serum-free, hydrocortisone-free culture medium before cytokine stimulation.
Establishment of a HPMC monolayer on the underside of porous culture inserts
Tissue culture inserts containing a 3-µm porous polycarbonate membrane (diameter, 6.25 mm; pore density, 1.6 x 106/cm2; BD Biosciences) were coated with human type IV collagen (Sigma) by the addition of 100 µl of collagen solution (100 µg/ml collagen in 0.1% (v/v) acetic acid) to the upper surface of the inverted insert for 1 h at room temperature. Excess collagen was washed off with PBS before the addition of 150 µl of neutralized rat tail collagen I solution (6 mg/ml in 0.1% (v/v) acetic acid) to the inner chamber of the insert placed upright in a 24-well insert culture plate (BD Biosciences). Inserts were incubated at 37°C for 30 min to allow the collagen I plugs to set. HPMC (3 x 105 cells/insert) in serum-free culture medium then were added to the upper (collagen IV-coated) surface of the inverted insert and incubated for 4 h at 37°C to allow cell attachment. Inserts were placed upright in 24-well plates containing 1 ml of medium supplemented with 5% (v/v) FCS, and 0.4 ml of serum-free medium was added to the inner chamber of the insert. HPMC were cultured on the underside of the inserts to allow leukocyte migration across the mesothelial monolayer in a physiological direction. The absence of FCS in the medium added to the upper chamber and the presence of a collagen I plug was necessary to maintain a monolayer of mesothelial cells on the underside of the membrane (7). Cells were grown for 5–7 days and the medium was replaced every day. The confluence and integrity of the mesothelial monolayer was routinely monitored by light microscopy and by measuring transmesothelial electrical resistance (Millipore ERS resistance meter; Millipore, Bedford, U.K.), which was corrected for the control resistance across an identical collagen I and IV-coated, cell-free insert. Transmesothelial electrical resistance increased from 10 ± 5 ohms/insert (mean ± SEM, n = 3) on days 1–45 ± 5 ohms/insert (mean ± SEM, n = 3) by days 5–7 of culture, and remained at 43 ± 3 ohms/insert (mean ± SEM, n = 3) after a 48-h growth-arrest period in serum-free medium. Monolayer integrity also was assessed by fixing the inserts in 2.5% glutaraldehyde for 1 h at room temperature before sectioning for light microscopy.
Leukocyte isolation
PMN preparations were freshly isolated from venous blood taken from healthy human volunteers. Citrated blood was dextran-sedimented and leukocytes separated by Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation. PMN preparations were routinely >99% pure, as determined by morphological assessment of Neat-stained (Guest Medical, Sevenoaks, U.K.) cytospin preparations.
Transmigration experiments
Collagen type I plugs were removed from growth-arrested HPMC insert cultures before apical stimulation with 1 ml of fresh serum-free medium, in the presence or absence of cytokines. Fresh serum-free medium (0.4 ml) was added to the upper chamber. After a 24-hour stimulation period, 240 µl of supernatant in the upper chamber was replaced with 240 µl of fresh serum-free medium. This adjustment was performed to correct for the different medium volumes present on either side of the HPMC monolayer, thus allowing any secreted (in apical vs basal direction) chemokine gradient to form. PMN (2 x 106) in 30 µl of M199 were added to the upper chamber and the inserts incubated at 37°C. After a 1-h incubation period, the inserts were removed and the number of transmigrated PMN in the lower well quantitated with a myeloperoxidase assay as described previously (3).
Chemokine synthesis measurements
Growth-arrested HPMC in 48-well culture plates were stimulated
at 37°C with 0.5 ml of fresh serum-free medium in the presence or
absence of IL-1, TNF-, or IFN-, alone or in combination, for
the times and doses specified in Results. HPMC supernatants
were harvested, centrifuged to remove cellular debris, and stored at
-80°C until assayed by ELISA. Cell monolayers were carefully washed
twice with PBS and solubilized with 0.1 M NaOH. Total cellular protein
was analyzed by the modified Bradford method with BSA as the standard
(20). Repeated cell counts revealed that 1 µg of
cellular protein was equivalent to 3400 ± 900 cells
(n = 5). All data for chemokine production are
expressed as pg/µg of cell protein. Chemokine levels in cell
supernatants were quantitated with specific sandwich ELISA. MCP-1
assays were performed as previously described (21), and
IL-8 and RANTES ELISA were established with Ab pairs from R&D systems,
according to the manufacturers instructions. Intraperitoneal murine KC
levels were analyzed in lavage fluid with the Quantakine M mouse KC
ELISA (MKC00; R&D Systems) as per the manufacturer’s instructions.
RNA extraction and isolation
Total cellular RNA was extracted from control or cytokine-stimulated HPMC cultured in six-well plates by lysis with 1 ml of RNA isolator (Genosys Biotechnologies, Cambridge, U.K.). RNA was separated from DNA and proteins by the addition of chloroform and precipitated with isopropanol, essentially as described in the manufacturer’s instructions (Genosys Biotechnologies). RNA was quantitated by measuring OD260 before reverse transcription and PCR amplification or Northern blot analysis.
Reverse transcription and PCR amplification
Total cellular RNA was reversed-transcribed into cDNA with M-MLV
Superscript reverse transcriptase (Life Technologies) by the random
hexamers method, as described previously (22). To ensure
exponential phase amplification, PCR was performed with 25 cycles for
-actin, 25 cycles for MCP-1, 33 cycles for RANTES, and 30 cycles for
IL-8. One-tenth of the PCR product was separated by flat-bed
electrophoresis in 3% (w/v) NuSieve GTG agarose gels (Flowgen
Instruments, Sittingbourne, U.K.), stained with ethidium bromide (1
µg/ml), and photographed. The negatives were analyzed by densitometry
(Model GS670 video densitometer; Bio-Rad Laboratories, Hemel Hempstead,
U.K.) and the density of the chemokine bands compared with those of the
housekeeping gene.
Oligonucleotide synthesis
The -actin-, MCP-1-, RANTES-, and IL-8-specific
oligonucleotide amplification primers were synthesized by Genosys
Biotechnologies. The primer sequences were as follow: -actin:
forward, 5'-ATCCCCCAAAGTTCACAA-3' and reverse,
5'-CTGGGCCATTCTCCTTAG-3' (147 bp; Ref. 23);
MCP-1: forward, 5'-TCGCCTCCAGCATGAAAGTCT-3' and reverse,
5'-TGGGGAAAGCTAGGGGAAAAT-3' (369 bp; Ref. 24); RANTES:
forward, 5'-ACGCCTCGCTGTCATCCTCAT-3' and reverse,
5'-TTCTTCTCTGGGTTGGCACAC-3' (219 bp; Ref. 25); IL-8:
forward, 5' –TGACTTCCAAGCTGGCCGTG-3' and reverse,
5'-CCACGTCTCCCAACACCTCT-3'(270 bp; Ref. 26).
Northern blot analysis
Total cellular RNA (10 µg) was fractionated on a formaldehyde,
1% agarose gel. RNA was transferred overnight to a positively charged
nylon membrane (Boehringer Mannheim) by capillary action and fixed by
UV irradiation. Membranes were hybridized and DIG-labeled probes
detected, as described previously (27). Visualization was
achieved by using the chemiluminescent substrate CSPD (Boehringer
Mannheim). Blots were exposed to x-ray film for 
1–2 h at room
temperature. Membranes were stripped by incubating twice for 30 min
each in boiling 0.1% (w/v) SDS in RNase-free water, rehybridized with
fresh DIG-labeled oligonucleotide probe, and developed as described
above. Autoradiographs were quantitated by densitometry and expressed
as a ratio of the house-keeping gene, -actin.
In vivo experiments
Recombinant murine IL-1, recombinant murine IFN-, or
combinations of both (PeproTech) were administered i.p. (250 µl) to
groups of 5–9 wild-type BALB/c mice (25–27 g; Harlan U.K., Bicester,
U.K.) as described previously described (28). Control
animal received 250 µl of PBS. At periods between 30 min and 24
h animals, were sacrificed and the peritoneum lavaged with 2 ml of PBS
containing 5 IU/ml heparin (CP Pharmaceuticals, Wrexham, U.K.). Total
leukocyte counts were assessed by Coulter counting (Coulter ZM, High
Wycombe, U.K.) and corrected for specific cell phenotypes by
differential leukocyte counts on Neat-stained (Guest Medical) cytospin
preparations. Lavage fluid was rendered cell-free by centrifugation
(150 x g, 10 min) and stored at -70°C before
chemokine assay.
Statistical analysis
Data are presented as mean ± SEM. The values stated in the results section for fold increases or percentage inhibitions of chemokine production are the mean of the fold increases or percentage inhibitions observed in each individual experiment. Numbers (n) for all data refer to the number of different donors tested. Statistical analyses were performed by the Wilcoxon matched-pairs signed ranks test or unpaired Students-t test as appropriate (Statview SE; Abacus Concepts, Berkeley, CA) with a p value of less than 0.05 considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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regulation of MCP-1, RANTES, and IL-8 protein production
IFN- stimulation of HPMC resulted in a dose-dependent increase
in MCP-1 production, and 100 U/ml IFN- had similar efficacy for
MCP-1 production as 1000 pg/ml TNF- (Fig. 1
A). Stimulation with a combination of IFN- and IL-1
(100 pg/ml) or TNF- (1000 pg/ml) resulted in significant
(p < 0.05) synergistic up-regulation of MCP-1
production at a 100 U/ml dose of IFN-, where production was above
the levels produced by any of the stimuli alone and significantly above
the predicted additive value. Combination of 100 U/ml IFN- with
either IL-1 or TNF- resulted in a mean fold increase in MCP-1
levels above the expected additive values of 1.8 ± 0.3 (range of
1.1- to 3.0-fold; n = 5) and 1.8 ± 0.2 (range of
1.1- to 2.1-fold; n = 5), respectively.
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alone resulted in a minor, nonsignificant
(p < 0.05) induction of RANTES peptide
production at 100 U/ml dose of IFN- (Fig. 1
B). However,
the combination of IFN- with either IL-1 or TNF- resulted in
marked synergistic, dose-dependent up-regulation of RANTES production
compared with the expected additive value. The potentiating effects of
IFN- were greater for the TNF- response, where a mean fold
increase of 84 ± 35 (range of 21- to 202-fold; n
= 5) was observed above the expected additive values at a 100 U/ml dose
of IFN-, compared with a fold increase of 8.9 ± 1.8 (range of
5- to 16-fold, n = 5) in the IL-1 response.
In marked contrast, IFN- caused a significant
(p < 0.05) down-regulation of basal and
IL-1- and TNF--induced IL-8 production (Fig. 1C). The
inhibitory activity of 10 U/ml IFN- on basal IL-8 production was
86 ± 8.5% (range of 66–100%; n = 5), on
IL-1-induced IL-8 was 31 ± 8.0% (range of 15–54%;
n = 5), and on the TNF- response was 35 ± 10%
(range of 8.1–64%; n = 5). The inhibitory activity of
IFN- on basal and TNF- induced-IL-8 was maximal at a 10 U/ml
dose, whereas IFN- at 10 or 100 U/ml caused similar down-regulation
of IL-1-induced IL-8 production.
The regulatory activity of IFN- on IL-1- or TNF--stimulated
chemokine production was dependent on the dose of IL-1 (or TNF-)
used (Fig. 2). The synergistic effect of combined stimulation with IFN- (100
U/ml) and IL-1 (or TNF-) on MCP-1 production became significant
(p < 0.05) above the additive values, at doses
of 100 pg/ml IL-1 (Fig. 2A) or 1000 pg/ml TNF- and
above (data not shown). Maximal synergy was observed at 100 pg/ml
IL-1 and 1000 pg/ml TNF-. For RANTES production by HPMC, the
synergistic effects of IFN- became significant
(p < 0.05) above the additive values at and
above a 10 pg/ml dose of IL-1 (Fig. 2B) and 1000 pg/ml of
TNF-. Maximal synergy was observed with 100 pg/ml IL-1 and 10,000
pg/ml TNF- (data not shown).
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on IL-8 production first became
significant (p < 0.05) at a 10 pg/ml dose of
IL-1 and 100 pg/ml dose of TNF-. At these cytokine doses, maximum
down-regulation of 96 ± 2.9% (range of 85–100%;
n = 5) and 78 ± 14% (range of 33–100%;
n = 5) respectively, were observed (Fig. 2, C and D). The regulatory effects of IFN- on
IL-8 production therefore were maximal at 
10-fold lower doses
of IL-1 or TNF- than MCP-1 or RANTES. The percentage of
inhibition of cytokine-induced IL-8 production by IFN- then
decreased with increasing dose of IL-1 or TNF-. Interestingly,
the regulatory effect of IFN- on TNF--induced IL-8 production was
biphasic across the TNF- dose range used, as IFN- in combination
with 10,000 pg/ml TNF-, the highest dose used, resulted in increased
IL-8 generation compared with stimulation with a corresponding dose of
TNF- alone.
Time-dependent regulation of IL-1-induced chemokine expression
and production by IFN-
PCR analysis was performed to determine whether the regulatory
effects of IFN- on IL-1-induced chemokine generation were
observed at the level of gene expression. Stimulation with a
combination of IFN- (100 U/ml) and IL-1 (100 pg/ml) resulted in
increased expression of IL-1-induced MCP-1 and RANTES mRNA levels,
compared with either stimuli alone (Fig. 3). The potentiating effect of IFN- on IL-1-induced MCP-1
expression was observed by 1 h, was maximal by 6 h, and
remained similarly up-regulated over the whole time course used (24 h).
The combination of IFN- and IL-1 induced a prolonged time course
of RANTES mRNA expression compared with IL-1 alone, with
up-regulation first observed at 12 h and continuing to increase up
to 24 h. In comparison, down-regulation of IL-1-induced IL-8
mRNA expression was observed in the presence of IFN- (Fig. 3C). The inhibitory effect of IFN- was greatest after
cytokine stimulation for 1 to 6 h; after 6 h, the inhibitory
effect of IFN- on IL-1 induced IL-8 mRNA levels decreased.
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-induced MCP-1 or RANTES protein production by
IFN- was maximal after longer cytokine stimulation times (of 36
h or more for protein production). In contrast, the inhibition of
IL-1-induced IL-8 production by IFN- was maximal after a 24-hour
stimulation, and decreased thereafter (Fig. 4A–C).
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regulation of chemokine mRNA expression
To quantitate the changes in chemokine mRNA abundance, we examined
IFN- (100 U/ml)-induced changes in basal and IL-1 (100 pg/ml)-
and TNF- (1000 pg/ml)-induced chemokine mRNA expression by Northern
analysis with appropriate cytokine stimulation times for each
chemokine. After a 4-h cytokine stimulation of HPMC, IFN- increased
basal and IL-1- and TNF--induced MCP-1 mRNA expression by a fold
increase of 7.4 ± 2.2, 1.7 ± 0.5, and 4.0 ± 1.0
(n = 3), respectively (Fig. 5A). Probing of the same blots for IL-8 showed basal IL-8
expression was inhibited by 72 ± 28%, IL-1 induced IL-8 by
47 ± 14%, and TNF- induced IL-8 by 69 ± 10%
(n = 3; Fig. 5A). Basal expression of RANTES
mRNA transcripts examined after a 24-h stimulation period were
undetectable, whereas stimulation with IFN- alone caused a minor
induction of RANTES mRNA expression (Fig. 5B). The
combination of IFN- with IL-1 or TNF- fold-increased RANTES
expression by 12 ± 2.4 and 28 ± 9.5 (n =
2), respectively, compared with IL-1 or TNF- stimulation
alone.
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addition
The mRNA expression studies demonstrated that the regulatory
effects of IFN- on IL-1- or TNF--induced chemokine production
were observed at the level of gene expression, indicating that IFN-
modulates the IL-1 and TNF- signal transduction pathways at a
pretranslational level. To begin to investigate the mechanisms involved
in the IFN- regulation of CC and CXC chemokine synthesis, we
examined the effect of timing of IFN- addition on its ability to
regulate IL-1-induced RANTES and IL-8 production. Fig. 6A shows the potentiating effect of IFN- on IL-1-induced
RANTES production was significantly (p < 0.05)
greater when cells were pretreated (for 5 h) with IFN-,
compared with the simultaneous addition of both cytokines for 24
h. Both of these conditions resulted in significantly
(p < 0.05) increased RANTES production
compared with pretreatment of HPMC with IL-1 for 5 h before
IFN- addition. These findings indicated that IFN- might act to
prime the cells for subsequent induction of RANTES by IL-1.
Therefore, we examined the effect of sequential addition of IL-1 and
IFN- on RANTES production (Table I). HPMC were incubated in the presence or absence of IL-1 or IFN-
for 24 h before washing and culturing the cells for a further
24 h in the absence or presence of the cytokines. Incubation with
IFN- for the first 24 h and IL-1 for the second 24 h
resulted in a potentiation of RANTES production. In contrast, IFN-
had no effect when added after IL-1. Similar results were obtained
when studying the combination of IFN- and TNF- (data not shown).
These results indicate IFN- can prime HPMC for the stimulatory
activity of IL-1 (or TNF-) on RANTES generation. No significant
difference in the down-regulation of IL-1-induced IL-8 production by
IFN- was observed in any of the three initial stimulation conditions
used (Fig. 6B), indicating the timing of IFN- addition
did not effect its potential to inhibit IL-8 generation.
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To determine whether the regulatory effect of IFN- on HPMC IL-8
production translated into a functional effect of IFN- on
transmesothelial PMN migration, we used an in vitro transmigration
system. HPMC were stimulated apically with doses of IL-1 (10 pg/ml)
or TNF- (100 pg/ml) which, in combination with IFN- (10 U/ml)
were known to result in maximal inhibition of IL-8 production. Fig. 7
shows that in the presence of IFN--, IL-1 (188, 772 ± 165,
600 PMN; n = 3)-, or TNF- (52, 705 ± 26, 834
PMN; n = 3)-stimulated PMN transmigration were reduced
by 56 ± 11% and 52 ± 14% compared with control
(unstimulated) levels (n = 3; p <
0.05). No significant inhibition of PMN transmigration by IFN- was
observed when higher doses of IL-1 (100 pg/ml) or TNF- (1000
pg/ml) were used (data not shown).
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To determine whether our in vitro observations on the control of
PMN recruitment were operative in the peritoneal cavity in vivo,
recombinant murine IL-1 and IFN- were injected, either singly or
in combinations, i.p. in wild-type BALB/c mice. Initial time course
experiments identified that PMN recruitment peaked at 3 h after
injection in response to IL-1 or staphylococcal supernatant-treated
mice (Ref. 28 ; Fig. 8A). This increase in PMN numbers was preceded by a rapid and
time-dependent increase in KC (the murine counterpart of human GRO),
peaking 1 h after injection (Fig. 8B). In subsequent
experiments at 1 and 3 h, injection of IL-1 induced a
dose-dependent and significant increase in both KC and PMN levels
compared with PBS-injected control animals (Fig. 8, C and
D). In subsequent experiments at these time points,
combinations of IL-1 (a dose of 100 ng/mouse was selected based on
the dose response experiments) and increasing IFN- concentrations
were injected simultaneously into wild type mice. In these experiments,
i.p. PMN numbers at 3 h increased from 1.66 ± 1.04
(x105) in PBS injected animals to 7.61 ±
2.12 (x105) in animals injected with 100
ng/mouse of IL-1 (mean ± SEM; n = 5;
p < 0.05; Fig. 8E). When IFN- (0.05–50
ng) was combined with IL-1 (100 ng/ml) there was a dose-dependent
decrease in PMN numbers in the peritoneal lavage fluid. At doses
between 0.5 and 50 ng/mouse, PMN numbers were significantly reduced
compared with IL-1 treatment alone. At these higher IFN- doses,
PMN numbers were reduced by 60.58, 68.47, and 80.29% for IFN- doses
of 0.5, 5.0, and 50 ng/mouse, respectively (n = 5–7;
p < 0.05 for all; Fig. 8E). Injection of
IL-1 (100 ng/mouse) into wild-type mice resulted in a significant
increase in i.p. KC concentrations at 1 h (Fig. 8F).
Coinjection of IL-1 (100 ng/mouse) with increasing concentrations of
IFN- (0.05–50 ng/mouse), resulted in a dose-dependent decrease in
i.p. KC levels (Fig. 8F). At the highest doses of IFN-
used, these represented 65.9 and 92.5% reductions of the levels
induced by IL-1 alone (n = 6–8 animals/group;
p < 0.05 for both) (Fig. 8F). IFN- (50
ng/mouse) alone did not induce significant PMN influx or elevation of
KC levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a Th1-type cytokine, in mononuclear-specific chemokine
induction (33, 36). In contrast, IFN- has been reported
to possess inhibitory activity for IL-8 generation in some, but not in
all cell types examined (16, 37).
In the present study, we examined the potential of IFN- to regulate
mononuclear and neutrophil-specific chemokine production and control
the transcellular migration of leukocytes across HPMC monolayers in
vitro and into the peritoneal cavity in vivo. We have examined the
regulatory effects of IFN- on constitutive as well as
proinflammatory cytokine (IL-1 and TNF-)-induced HPMC-chemokine
production with doses of cytokines known to be present in the
peritoneal cavity during bacterial peritonitis (14, 15).
Our in vitro culture studies show that IFN- alone was an efficient
stimulus for MCP-1 production, and the combination of IFN- with
IL-1 (or TNF-) resulted in significant synergistic up-regulation
of MCP-1 synthesis. In contrast, stimulation of HPMC with combinations
of IFN- and IL-1 (or TNF-) was required for significant
induction of RANTES gene expression and protein production. This
requirement for IFN-, in combination with IL-1 or TNF-, for
significant RANTES generation, has been documented previously
(17, 35), and synergistic induction of IL-1-stimulated
MCP-1 production by IFN- (or IFN-) has been observed in human
Hep-2 epithelial cells. However, in diploid fibroblasts, these two
cytokines had an additive effect on MCP-1 production
(38).
In comparison to the two CC chemokines, IFN- treatment resulted in
both constitutive and IL-1- or TNF--induced IL-8 gene expression
and synthesis being significantly down-regulated. Previous reports on
the ability of IFN- to regulate IL-8 synthesis contrast in their
findings. Oliveira et al. (16) described an inhibitory
effect of IFN- (and IFN-) on IL-1- or TNF--induced IL-8
expression and production in FS-4 fibroblasts. Similarly, IFN-- and
IFN--inhibited neutrophil-specific chemokine production (IL-8,
ENA-78) in LPS or IL-1 stimulated monocytes (37). In
contrast, Yasumoto et al. (39) reported synergistic
up-regulation of IL-8 production with the combination of IFN- and
TNF- in a human gastric cancer cell line, and in HPMC, Visser et al.
(8) described no effect of IFN- on basal or IL-1- or
TNF--induced IL-8 production. Our findings that the potential of
IFN- to regulate IL-8 production was critically dependent on the
dose of IL-1 or TNF- used, with inhibition evident at lower doses
but stimulation present at higher ones, may in part explain the
observed differences in the published literature.
Initial investigations into the mechanisms involved in the IFN-
regulation of chemokine synthesis by IL-1- or TNF--stimulated
HPMC by PCR and Northern analysis demonstrated the regulatory effects
were observed at the level of gene expression. This indicates the
cross-talk between the signal transduction pathways used by these
cytokines for MCP-1, RANTES, and IL-8 regulation occurred at the
pretranslational level. Studies on the effect of timing of IFN-
addition in relation to IL-1 demonstrated that IFN- could
"prime" HPMC to the subsequent actions of IL-1 (and TNF-,
data not shown) for RANTES induction. The ability of IFN- to prime
cells for the subsequent actions of TNF- for RANTES production has
been reported previously in human endothelial and airway smooth muscle
cells (17, 35). Priming effects may be mediated at the
level of receptor expression, and the ability of IFN- to up-regulate
cell surface TNF- receptors has been demonstrated in several human
tumor cell lines (40, 41, 42). However, this was only observed
in cells with low constitutive TNF receptor expression
(40). Although the potential of IFN- to modulate
IL-1 or TNF- receptor expression in HPMC was not determined in
the present study, HPMC have previously been shown to express
relatively high levels (7 x 103/cell) of
the ubiquitous type 1 receptor (43). Therefore, the
mechanism by which IFN- potentiates TNF--induced RANTES
expression in HPMC may be independent of TNF receptor modulation. In
contrast, IFN- has been reported to down-regulate basal and
cytokine-induced IL-1 receptor (type I and type II) mRNA expression in
human monocytes (44). Therefore, priming of HPMC by
IFN- for RANTES (and possibly MCP-1) production would appear to
occur independently of IL-1 or TNF receptor modulation, although
clearly this requires further investigation.
The synergy between IFN- and TNF- for RANTES production
demonstrated in endothelial cells did not occur at the level of surface
TNF- receptor expression, but occurred further downstream at the
level of transcriptional control (17). Cooperation between
the transcription factors NF-
B and STAT-1 have been shown to
mediate, in part, the synergy between TNF- and IFN- for Mig and
RANTES gene expression (45). IFN- also has been shown
to mediate negative regulatory effects on gene expression at the level
of transcription and mRNA stability. The role of these mechanisms in
controlling HPMC chemokine synthesis remains to be defined. The
potential of IFN- to prime HPMC for subsequent RANTES synthesis, but
not IL-8 induction, together with the significant differences observed
in the dose responses and time courses of these events, suggests that
several distinct signaling mechanisms may be involved in the observed
differential regulation.
Previous in vitro data has suggested that the amount of IL-8 and the
creation of a basolateral to apical concentration gradient is the
primary determinant in directing PMN transmigration across HPMC
monolayers (46). Investigation of the functionality of the
IFN- regulation of HPMC chemokine production in relation to PMN
recruitment was achieved both in vitro and in vivo in the present
study. With an in vitro transwell system we demonstrated that although
both IL-1 and TNF- induced increases in PMN transmigration across
HPMC monolayers, this increase could be attenuated by the simultaneous
addition of IFN-. IFN- itself had no effect on PMN migration in
this system. This ability to down-regulate PMN migration correlated
directly with the ability of IFN- to inhibit proinflammatory
cytokine-induced IL-8 synthesis (80% inhibition of IL-8 observed
when parallel cytokine doses were used). As observed for the IFN-
regulation of IL-8 production, the doses of IL-1 or TNF- were
critical in determining the degree of PMN transmigration. Little or no
inhibition of stimulated PMN migration was observed when higher doses
of IL-1 (100 pg/ml) or TNF- (1000 pg/ml) were used (data not
shown), conditions which resulted in only partial (31–35%) inhibition
of IL-8 secretion. Therefore, down-regulation of HPMC IL-8 production
appears to be a major mechanism involved in mediating this inhibitory
action of IFN- on cytokine induced PMN transmigration.
IFN- also can regulate PMN migration by exerting effects at the
level of HPMC adhesion molecule expression. Previous reports indicate
IFN- up-regulates ICAM-1 expression on many cell types, including
mesothelium (4, 47, 48). We have shown previously that
ICAM-1 was required for both basal and IL-1-induced transmesothelial
PMN migration (7). Furthermore, Issekutz and Lopes
(49) have shown IFN- superinduced LPS-stimulated ICAM-1
expression on human endothelial cells, prolonged E-selectin expression,
and sustained LPS-induced PMN transmigration across freshly washed
endothelial cell monolayers. Interestingly, the reported potentiating
effects of IFN- on ICAM-1 expression do not appear to fit with our
current model of a role for IFN- in inhibiting PMN recruitment.
However, previous data suggests that the direction of PMN (and
mononuclear cell) migration across HPMC monolayers is primarily
dependent on the creation of a chemotactic gradient and only
secondarily on the up-regulation of adhesion molecule expression
(7).
IFN- may also regulate PMN migration in vivo at the level of
chemokine receptor expression, and IFN- has been shown recently to
have a complex array of effects on chemokine receptors expressed on PMN
(50), monocytes (51), and T lymphocytes
(52). These may function to modulate leukocyte
infiltration to sites of inflammation, as well as their subsequent
emigration. In light of the varied effects of IFN- on factors known
to play a role in mediating PMN migration, it is important to emphasize
that in this study, under the experimental conditions used
(particularly the cytokine doses), the regulatory effects of IFN-
culminated in significant inhibition of IL-1-induced PMN migration
both in vitro and in vivo. Our in vivo data demonstrating the ability
of IFN- to abolish IL-1-induced acute PMN recruitment supports
the in vitro findings on the ability of IFN- to control PMN
migration across HPMC. These data provide evidence for this pathway
being important in switching off PMN recruitment during peritoneal
inflammation. The mechanism controlling this effect in vivo appears to
be the specific down-regulation of murine KC synthesis by IFN-.
These data parallel our observations in IL-6-deficient mice that
suggests that modulation of PMN recruitment during murine peritonitis
is dependent on the control of macrophage-inflammatory protein-2 and KC
synthesis (the murine equivalents of IL-8 and GRO; Ref.
28). However, there was a large difference in magnitude of
the observed effects on PMN migration between the in vitro and in vivo
migration assays and the regulation of HPMC IL-8 synthesis and mRNA
expression by IFN-. These data suggest that in the in vitro
transmigration assay where only PMN and HPMC are present and in the in
vivo situation (where many cell types reside) that other soluble
inhibitory/antagonistic factors serve to facilitate the down-regulation
of IL-8 expression by IFN-. In this respect, ongoing studies suggest
that soluble IL-6R, present in the peritoneal cavity during
inflammation, plays a central role in this process (28).
However, to date, it is not known whether IFN- plays a role in the
generation of soluble IL-6R production (53).
Although mesothelial cell-derived chemokine secretion may be the
primary source of i.p. chemotactic activity at the onset of infection,
inflammatory leukocytes subsequently migrate into the peritoneum and
become major cell populations (54). These leukocytes will
become a major source of chemokine secretion. The reported potential of
IFN- to modulate chemokine secretion by mononuclear cells and PMN,
(37, 55, 56) in an analogous manner to HPMC suggests that
IFN- plays an important role in controlling leukocyte recruitment
throughout the inflammatory response.
In conclusion, we have shown that IFN- differentially regulates
IL-1- or TNF--induced mononuclear and neutrophil-specific
chemokine production by HPMC, an effect that correlated with an
inhibitory action of IFN- on IL-1-induced transmesothelial PMN
migration. Therefore, IFN- may play a role in controlling PMN
recruitment during the evolution of an immune response. The role of
IFN- in controlling the temporal influx of neutrophils is supported
by recent observations in IFN-- and / T cell-deficient mice
that suggest that its absence results in their delayed removal,
resulting in inadequate clearance of infecting organisms and subsequent
tissue damage (57). This observation is supported by data
generated after targeted disruption of the murine STAT-1- and Jak-1
genes that have cast further light on the importance of this signaling
pathway in IFN--mediated responses (58, 59, 60). STAT-1
deficiency (resulting in complete nonresponsiveness to IFN- or
IFN-) is associated with increased susceptibility to bacterial and
viral infection (59, 60). In the context of the present
study, these data confirm the central role of IFN- in the hosts
response to infection and suggest that similar experiments in
STAT-1-deficient animals might identify the cellular mechanisms
controlling immunity against invading pathogens.
Taken together, these data suggest that IFN- plays a central role in
controlling neutrophil influx and may be one mechanism that polarizes
the immune response toward a Th1 phenotype and adequate resolution of
infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nicholas Topley, Institute of Nephrology, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, U.K. E-mail address: topley{at}cf.ac.uk
3 Abbreviations used in this paper: HPMC, human peritoneal mesothelial cells; GRO, growth-related oncogene; MCP, monocyte chemoattractant protein; PMN, polymorphonuclear leukocyte; DIG, digoxigenin.
Received for publication November 6, 2000. Accepted for publication May 14, 2001.
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