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*
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109;
Department of Physiology, School of Dentistry, University of Nebraska, Lincoln, NE 68198; and
Amgen, Thousand Oaks, CA 91320
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
, and
cytokine-induced neutrophil chemoattractant, whereas intratracheal
instillation of anti-IL-18 greatly reduced these changes and
prevented increases in BAL content of IFN-
. Intratracheal
administration of the natural antagonist of IL-18, IL-18 binding
protein, resulted in suppressed lung vascular permeability and
decreased BAL content of neutrophils, cytokines, and chemokines. These
findings suggest that endogenous IL-18 functions as a proinflammatory
cytokine in this model of acute lung inflammation, serving as an
autocrine activator to bring about expression of other inflammatory
mediators. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by CD4+ helper T cells,
CD8+ cytotoxic T cells, and NK cells plays an
important role in the immune response, especially in the Th1 pathway.
IFN- has the ability to activate macrophages, enhance NK cell
activity, increase cytokine production, and protect cells from viral
replication (1, 2). Only a few cytokines have been shown
to induce the production of IFN-, including TNF-, IL-2, and IL-12
(1, 2, 3, 4). In 1989, a new cytokine termed IL-18 (or IGIF) was
found to be a potent inducer of IFN- (5). IL-18 was
first isolated from liver extracts of mice sensitized with heat-killed
Propionibacterium acnes and subsequently challenged with LPS
and was identified as an 18-kDa nonglycosylated protein secreted
primarily by activated macrophages/monocytes (5, 6, 7). Other
cell types also have been shown to secrete IL-18, including epidermal
keratinocytes, intestinal epithelial cells, adrenal cortical cells, and
osteoblasts (8, 9, 10, 11). The three-dimensional structure of
IL-18 indicates a relationship to IL-1 (12%) and IL-1 (19%).
Like IL-1, IL-18 is synthesized in precursor form with an unusual
signal peptide that allows secretion from the cell. The precursor form
of IL-1 has a very low biological activity (defined by the induction
of IFN-), whereas the precursor form of IL-18 is totally devoid
of activity (12). IL-1-converting enzyme
(ICE4; also called
caspase 1) is responsible for cleaving IL-1 and IL-18 to generate
active, mature forms of these proteins. Studies have shown that
ICE-deficient mice have reduced levels of IFN- and mature IL-18
(13, 14).
In the present study, we investigated the in vivo role of IL-18 in
acute lung inflammation in rats caused by deposition of IgG immune
complexes. This model is associated with production of TNF- and
IL-1, up-regulation of vascular adhesion molecules, and CXC/CC
chemokine expression, all of which facilitate the recruitment of blood
neutrophils, activation of lung macrophages, and the release of
oxidants and proteases (15, 16). The role of IL-18 after
acute lung inflammation caused by IgG immune complex deposition has not
been explored previously. The current studies also explore the effects
of IL-18 binding protein (IL-18bp), a natural IL-18 antagonist
(17).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Except where noted, all reagents were purchased from Sigma (St. Louis, MO). The anti-IL-18 was affinity-purified rabbit IgG directed against murine IL-18. This Ab neutralizes the biological activity of IL-18 (R&D Systems, Minneapolis, MN).
IgG immune complex-mediated alveolitis
Adult male Long-Evans rats weighing 275–300 g were used in all
studies (specific pathogen-free). All experimental protocols have been
approved by the University of Michigan Institutional Animal Committee
for the Use and Care of Animals. Ketamine was administered i.p. for
sedation and anesthesia. Rabbit polyclonal IgG (1.5 or 2.5 mg) rich in
Ab to BSA (anti-BSA) was intratracheally instilled in a volume of
300 µl of PBS (pH 7.4) during inspiration. This was followed by the
i.v. injection of BSA (10 mg) with trace amounts of
125I-labeled BSA as a quantitative marker of
permeability. Rats were sacrificed 4 h later, the pulmonary
circulation flushed, and lung injury quantified by increased vascular
permeability. It has been shown elsewhere that inflammation and
mediator presence in lung peak at this time point (18).
The permeability index was determined by measuring the amount of
radioactivity (125I-labeled BSA) remaining in the
lungs compared with the radioactivity present in 1.0 ml of blood
obtained from the inferior vena cava. Negative control animals were
intratracheally instilled with PBS only. In all experiments in which
this model was used, n = 
6 animals unless otherwise
indicated.
Cloning of rat IL-18
Previously, it has been shown that IL-18 is strongly induced in the adrenal gland of rats after reserpine administration (10). In this study, we cloned rat IL-18 after inflammatory challenge. Rat lungs were injured by IgG immune complex deposition as described above. Four hours later, whole adrenal glands (cortex and medulla) were removed and frozen in liquid nitrogen. Total RNA was extracted by using the guanidinium-isothiocyanate method (TRIzol; Life Technologies, Grand Islands, NY). First-strand cDNA was constructed by reverse-transcribing 10 µg of total RNA with an oligo(dT) primer. cDNA amplification of rat IL-18 was performed with the above RT products and the following rat primers: 5', 5'-AACAATGGCTGCCATGTCAG-3'; and 3', 5'-AGTGAACATTACAGATTTATCCC-3'. These primers result in a long 566-bp and a shorter 510-bp PCR band. The PCR products were ligated into a PCR 2.1 vector (Invitrogen, Carlsbad, CA), sequenced, and compared with the GenBank data bank. After confirmation of the sequence of both transcripts with the published rat IL-18 sequence, the longer sequence was used as a cDNA probe for Northern blot analysis.
Recombinant rat (rr)IL-18 , anti-mouse IL-18 polyclonal Ab, and IL-18bp
(rr)IL-18 was purchased from R&D Systems. The protein was
expressed in Escherichia coli and was determined
by SDS-PAGE to be >97% pure. The endotoxin level was <0.1 ng per 1
µg of IL-18, and activity was confirmed by its ability to induce
mouse IFN- production by activated mouse T cells. Where indicated,
1.0 or 2.0 µg of rat IL-18 was coinstilled intratracheally (constant
volume of 10 µl) alone or with anti-BSA (in a final volume of 300
µl) at the commencement of injury. Polyclonal goat anti-mouse
IL-18 Ab was derived from immunized goats. The endotoxin level in the
IgG was <0.1 ng per 1.0 mg of the Ab. Cross-reactivity with rat IL-18
was determined by Western blotting. The Ab was reconstituted from the
lyophilized form with sterile PBS to a concentration of 100 µg/ml.
Our studies, shown below, indicate that the anti-IL-18 Ab, when
absorbed with (rr)IL-18, loses its ability to detect IL-18 in BAL
fluids from inflamed lungs. Human recombinant IL-18bp (1.5, 6, or 18
µg) was coinstilled intratracheally with anti-BSA at the
commencement of inflammation. Human IL-18bp was expressed in mammalian
cells and purified by HPLC, and shown to inhibit LPS-induced IFN-
production in rat lungs (G. Senaldio, unpublished observations).
Northern Blot Analysis
Total RNA was extracted (TRIzol Reagent; Life Technologies) from
whole lungs of rats injured with IgG immune complexes. The pulmonary
circulation was flushed with 10 ml of PBS, and the lungs were
surgically removed and immediately frozen in liquid nitrogen. Twenty
micrograms of RNA was fractionated in 1% agarose formaldehyde gel and
transferred to a nylon membrane (Magna Charge; MSSI, Westboro, MA). The
cDNA for rat IL-18 was [32P]d-CTP radiolabeled
by random priming with Klenow enzyme to generate a highly specific
activity probe (1.5 x 107 cpm; Amersham
Life Science, Piscataway, NJ). Hybridization was performed at 68°C
for 4 h (QuikHyb; Stratagene, La Jolla, CA), and the
autoradiograph was developed on Kodak Biomax-MR film (Kodak, Rochester,
NY). Intensity of RNA bands was analyzed with image analysis software
(Adobe Systems, San Jose, CA). -Actin radiolabeled probe was used as
an internal standard to ensure equal RNA loading.
ELISA for rat IL-18
Anti-IL-18 Ab, biotinylated Ab, and (rr)IL-18 protein were purchased from R&D Systems. A-sandwich ELISA was performed with 100 µl of 1 µg/ml of anti-IL-18 Ab (primary Ab) in borate buffer to coat ELISA plates. A 1% BSA solution in Dulbecco’s PBS was used to block nonspecific binding. After washing, samples and standards were added to individual wells and incubated for 2 h at 37°C. This was followed by washing and incubation with 100 µl of biotinylated Ab (1 µg/ml) for 1 h at 37°C. Subsequently, after a 1-h incubation with a streptavidin-HRP conjugate, the assay was developed by addition of o-phenylenediamine substrate. The developing reaction was stopped by 3 M H2SO4. The plate was read on an ELISA plate reader at 492 nm.
Western blot analysis
BAL fluids from IgG immune complex-injured animals were analyzed
for rat IL-18 immunoreactive protein. Five milliliters of PBS was
instilled into the lungs and collected three times, and cellular
contents were removed by centrifugation. BAL fluids then were
concentrated (
5-fold) to 500 µl by using a centricon (Amicon,
Beverly, MA). Concentrated BAL fluids were subjected to SDS-PAGE (15%)
according to the method of Laemmli. Equivalent amounts of protein (100
µg) were added to each lane. The acrylamide gels then were
transblotted to nitrocellulose (0.1 µm) for 2 h at 12 V. The
membrane was blocked with TweenTris-buffered saline containing 5% milk
for 1 h at room temperature. Polyclonal goat anti-mouse IL-18
(100 µg/ml) then was added at a dilution of 1/1000, and the membrane
was incubated overnight at 4°C. After washing, peroxidase-conjugated
donkey anti-goat IgG (800 µg/ml) was added (1:10,000), and the
membrane was incubated for 1 h at room temperature. The membrane
was developed with an ECL technique (Amersham Life Science, Piscataway,
NJ). (rr)IL-18 was used as a positive control, and a m.w. marker was
used to estimate the size of the immunoreactive bands. Intensity of the
protein band was determined as above. For experiments where indicated,
the anti-IL-18 Ab was preabsorbed by the addition of (rr)IL-18 at a
final concentration of 1 µg/ml.
BAL fluid analysis and cytokine and chemokine content
BAL fluid was collected from IgG immune complex-injured rat
lungs 4 h after injury, and when used for Western blot analysis,
was concentrated 5-fold by using Centricon filters with a cut-off of 3
kDa (Amicon). Briefly, 5 ml of PBS was intratracheally instilled
three times into the lungs. Cell counts were normalized to the volume
of BAL fluid recovered per rat. TNF- activity was determined by
using a standard WEHI cell cytotoxicity assay. BAL concentrations of
macrophage-inflammatory protein (MIP)-2 and cytokine-induced neutrophil
chemoattractant (CINC) were determined by ELISA as reported previously
(19). The ELISA kits for detection of rat IL-1 and
IFN- were purchased from R&D Systems.
Immunostaining of alveolar macrophages
BAL fluids from control and injured animals were collected and centrifuged at 450 x g for 10 min and were layered onto Ficoll-Paque to remove RBCs and neutrophils (450 x g for 30 min). The upper layer containing alveolar macrophages was removed and resuspended in PBS containing 1% BSA to a concentration of 250,000 cells/ml. Slides were prepared by adding 100 µl of this cell suspension to a cytospin and centrifuging at 450 x g for 7 min. Cytospin slides were fixed in 100% methanol and stored at -20°C. For immunostaining, slides were washed in PBS and incubated with anti-IL-18 (1 µg/ml) for 1 h in a humidified chamber. Slides then were washed two times and incubated for 1 h with biotinylated anti-goat IgG (1 µg/ml). After washing in PBS, the slides were incubated for 30 min with streptavidin-HP (1:3000). After a final wash, slides were reacted with diaminobenzidine reagent for 10 min and later counterstained with hematoxylin.
Isolation and culture of rat lung cells
Alveolar macrophages were isolated from BAL fluids from normal rat lungs as described (20). Cells were pelleted and then resuspended in DMEM supplemented with 10% FBS and penicillin-streptomycin (DMEM-FBS). After allowing cells to adhere to the plate (5 x 105 cells per well in 24-well tissue culture plates; Corning, Corning, NY), nonadherent cells were removed with two washes. Cell monolayers then were stimulated with the appropriate agonists suspended in DMEM-FBS in a 5% CO2 humidified incubator at 37°C for 24 h. Rat alveolar fibroblasts were isolated from normal lungs. Lungs were removed, minced into 2-mm pieces, and the cells allowed to adhere to an inverted 25-mm tissue culture flask containing no medium for 48 h. The flasks then were righted, DMEM containing 10% FBS was added, and cultures were allowed to grow out from the edges of tissue pieces for 2 wk. Cells were removed from the flask by using 0.5% trypsin and then recultured into 25-mm tissue culture flasks (Corning). All cultures were used between passage 1 and passage 3 and were plated at 5 x 105 cells per well in 24-well tissue culture plates (Corning) containing DMEM-FBS. Cells were stimulated with appropriate agonists suspended in DMEM-FBS in 5% CO2 humidified incubator at 37°C and allowed to grow for 24 h. Type II alveolar epithelial cells were isolated from normal rat lung using elastase cell dispersion and IgG panning by the method of Warner and associates (21). Cells were plated in DMEM-FBS at 5 x 105 cells per well in 24-well tissue culture plates and incubated at 37°C in 5% CO2. Cells were stimulated with appropriate agonists suspended in DMEM-FBS and allowed to grow for 24 h. Microvascular endothelial cells were isolated from peripheral lungs of normal 21-day-old rats (22). Strips of peripheral lungs were removed, minced, and incubated in gelatin-coated tissue culture flasks in DMEM containing 10% FBS and endothelial cell growth factor. After 65 h, tissues were removed. Cultures consisted primarily of endothelial cell. Cells were maintained in culture and grown to 80–90% confluence at passage 2 by 10 days. For all experiments, cells were used at 80–90% confluence at passage 2 and plated in DMEM-FBS at 5 x 105 cells per well in 24-well tissue culture plates and incubated at 37°C in 5% CO2. Cells were stimulated with appropriate agonists suspended in DMEM-FBS and allowed to grow for 24 h.
Binding of rat IL-18 to solid phase human IL-18bp
Fifty microliters of human IL-18bp (10 µg/ml) in borate-coating buffer was used to coat each well in a 96-well ELISA plate overnight at 4°C. A 1% BSA solution in Dulbecco’s PBS was used to block nonspecific binding (1 h at 37°C). Various concentrations of rat IL-18 and BSA (100 µl) were added per well and incubated for 1 h at 37°C. After the washing step, goat anti-rat IL-18 (R&D Systems) was added and incubated for 1 h at 37°C. After a 1-h incubation with donkey anti-goat IgG-HRP conjugate (Santa Cruz Biotechnology, Santa Cruz, CA), the assay was developed by addition of o-phenylenediamine substrate. The developing reaction was stopped by adding 50 µl of 3 M H2SO4 and read at 492 nm.
Statistical analysis
All data are presented as means ± SEM. Sample size was 4–6 animals per group unless otherwise noted. One-way analysis of variance was used to compare treatment groups. Significant differences between groups were determined using Tukey’s test. Statistical significance equal to p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-18 mRNA content varied in normal rat tissues based on Northern
blot analysis. In the adult rat, IL-18 mRNA (1.35 kb) was most
abundant in liver, with moderate expression in spleen and low but
measurable constitutive expression in the lungs, kidneys, and heart
(Fig. 1
A). Little if any mRNA
for IL-18 was found in skeletal muscle and testis. Equal loading of RNA
was confirmed by stripping the blot and reprobing with radiolabeled
-actin (data not shown). To assess changes in endogenous IL-18 mRNA
and protein during the pulmonary inflammatory response, lungs from
injured animals were evaluated (0–4 h) for changes in mRNA and protein
expression. Northern blot analysis of IgG immune complex-inflamed lungs
(using 1.25 mg anti-BSA intratracheally) revealed increases
(50%, as determined by densitometry) in mRNA for IL-18, 2 and
4 h after initiation of the inflammatory response (Fig. 1B, bottom). Equal loading was confirmed by
probing with GADPH (Fig. 1B, bottom).
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A as a function of time. In
lane 1, (rr)IL-18 gave a single band in the 18-kDa position,
consistent with the mature form of IL-18. At time 0 (lane
2), a single band reactive with anti IL-18 Ab was found in BAL
fluids in the 26-kDa position, which is consistent with the "pro"
form of IL-18. Thereafter, between 1 and 8 h, IL-18 bands were
found in both the 26- and 18-kDa positions (lanes
3–6), whereas at 16 and 24 h (lanes 7 and
8) bands in the former position were prominent, but bands in
the 18-kDa position were barely visible. When the anti-IL-18
preparation (1.0 µg/ml) was preabsorbed by addition of (rr)IL-18 (1.0
µg/ml final concentration), both the 18-kDa and the 26-kDa bands
disappeared (lane 9, using the 4-h BAL sample),
indicating that both bands are related to IL-18. As is also shown in
Fig. 2A, the double banding pattern found in BAL fluids
obtained 4 h after initiation of this inflammatory response
(lane 10) disappeared from similarly treated rats
that also received 400 µg of anti-IL-18 intratracheally at time 0
(lane 11).
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B, IL-18 levels in BAL
fluids from normal lungs were <1 ng/ml, whereas there was a
progressive increase in BAL IL-18, peaking at 4 h (at 9 ng/ml),
followed by a decline. When lung homogenates were evaluated over the
same period of time, normal lungs contained 1025 ± 128 ng/g lung
weight (Fig. 2B). In the inflamed lungs, there was very
little measurable increase in IL-18 during the first 2 h of
inflammation, but by 4 and 6 h, the IL-18 content was 2585 ±
172 and 2441 ± 301 ng/g lung, respectively. These data indicate
that IL-18 is induced in lungs of rats during deposition of IgG immune
complexes. Effects of (rr)IL-18 on vascular permeability and BAL content
The effects of the exogenously administered rat IL-18 during
IgG-induced inflammation were determined in the lung model. For these
studies, the dose of anti-BSA used was 1.25 mg, to detect in a more
sensitive manner increases in the parameters of lung inflammation and
vascular leak. The vascular permeability (leakage of
125I-labeled albumin) was measured at 4 h,
the time at which the permeability index peaks (18). The
mean permeability value in the negative control lungs was 0.17 ±
0.01 (Fig. 3A). Instillation
of 2.0 µg of IL-18 alone did not significantly augment vascular
permeability in otherwise normal lungs (0.19 ± 0.03). Positive
control rats not otherwise treated demonstrated a 40% increase in
vascular permeability (to a value of 0.29 ± 0.02). In a companion
group of rats that also received 2.0 µg of IL-18 in the presence of
anti-BSA, the permeability index was further increased, to
0.44 ± 0.04 (a 35% increase) as compared with the positive
control group (p < 0.05). Next, we determined
whether these changes were associated with enhanced neutrophil
recruitment. BAL fluids were collected at 4 h and evaluated for
neutrophil content (Fig. 3B). In the negative control
animals, low levels (<5 x 105) neutrophils were found;
the intratracheal administration of 2.0 µg of IL-18 caused no
significant increase in BAL content of neutrophils. Intrapulmonary
instillation of 1.25 mg of anti-BSA together with 10 mg of BSA
(which was intravenously administered) resulted in a 4-fold increase in
neutrophil counts (to 1.6 x 106). The
coinstillation of IL-18 (1.0 µg or 2.0 µg) together with
anti-BSA further increased neutrophils counts (to 3.1 x
106 and 5.0 x 106,
respectively, p < 0.05).
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, IL-1, and CINC are important cytokines and chemokines
involved in the recruitment of inflammatory cells in this lung model
(19, 23, 24), we examined the effects of exogenously
administered IL-18 on BAL levels of these mediators after IgG immune
complex-induced lung inflammation. TNF- levels in the BAL fluids
(obtained at 4 h) were determined. Negative control animals had
low BAL levels of TNF- (5.6 ± 0.8 pg/ml; Fig. 3C).
Intrapulmonary instillation of 2.0 µg of IL-18 alone caused a small
increase in TNF- content (to 9.0 ± 1.1 pg/ml). After immune
complex deposition, BAL levels of TNF- substantially increased (to
1279 ± 118 pg/ml). The coadministration of 1.25 mg of
anti-BSA and 1.0 or 2.0 µg of IL-18 further increased TNF-
levels, to 1590 ± 109 and 2776 ± 130 pg/ml, respectively
(p < 0.05).
IL-1 levels also were determined in BAL fluids by ELISA, using the
same experimental conditions (Fig. 3D). Negative control
animals had low levels of IL-1 (13.4 ± 0.50 pg/ml). The
instillation of IL-18 into otherwise normal lungs caused no significant
changes in BAL levels of IL-1. A nearly 8-fold increase in BAL
levels of IL-1 was found in the positive control group in the
absence of exogenously administered IL-18 (108 ± 14.8 pg/ml). The
coadministration intratracheally of anti-BSA and 1.0 or 2.0 µg
IL-18 further increased IL-1 production, to 132 ± 5.4 and
177 ± 16.5 pg/ml, respectively, (p <
0.05 for the latter).
Noninflamed lungs contained low levels of CINC (146 ± 36.2
ng/ml). The instillation of IL-18 alone (in the absence of immune
complexes) caused a modest (but significant) increase in CINC (to
543 ± 23.5 ng/ml; Fig. 3E). Positive control rats
demonstrated a 3-fold increase in BAL levels of CINC (1837 ±
97.5). In the positive control group with coinstillation of 2.0 µg of
IL-18, CINC levels were increased further, by 20% (to 2211 ± 104
ng/ml; p < 0.05). Thus the copresence of IL-18 with
IgG immune complexes in the lung enhanced lung vascular permeability
and augmented levels of neutrophils, IL-1, TNF-, and
CINC.
Effects of anti-IL-18 on vascular permeability and BAL content
Because we observed an increase in vascular permeability after the
coinstillation of IL-18 with anti-BSA, we evaluated whether an
affinity-purified, neutralizing goat polyclonal Ab to mouse IL-18 could
alter events in positive control animals. As indicated above, this Ab
is reactive with rat IL-18. As shown in Fig. 4A, the extravascular leakage
of 125I-labeled albumin was measured 4 h
after the initiation of injury in lungs receiving 2.5 mg of
anti-BSA together with 400 µg of preimmune goat IgG. As compared
with the negative control group, the positive control group receiving
400 µg of normal rabbit IgG intratracheally had a more than 4-fold
increase in vascular permeability (to a value of 0.60 ± 0.04).
The addition of 400 µg of anti-IL-18 IgG resulted in a 30%
reduction in vascular permeability (to a value of 0.42 ± 0.04;
p < 0.05). BAL fluids collected at 4 h were
evaluated for neutrophil content (Fig. 4B). Intrapulmonary
deposition of IgG immune complexes caused a 10-fold increase in BAL
neutrophil numbers (to 6.8 x 106). The
coinstillation of anti-BSA and 400 µg of anti-IL-18 decreased
neutrophil counts by 50% (to 3.5 x 106;
p < 0.05).
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, C–E) to
2880 ± 229 pg/ml for TNF-, to 250 ± 25 pg/ml for
TNF-, and to 3984 ± 210 ng/ml for CINC. The coadministration
of 400 µg of anti-IL-18 IgG with anti-BSA decreased levels of
TNF- by 33% (to 1908 ± 330 pg/ml), IL-1 levels by 60%
(98 ± 15 pg/ml), and CINC levels by 27% (to 2892 ± 368
ng/ml). In companion experiments, we also assessed the effects of
administration of anti-IL-18 on BAL levels of IFN-, because, as
described above, IL-18 is well known to induce expression of IFN-.
In noninflamed (negative control) lungs, BAL levels of IFN- were
50 ± 4.0 pg/ml, whereas in the inflamed lungs of animals treated
with preimmune IgG, the levels rose to 120 ± 8.2 pg/ml (Fig. 4F). In the presence of anti-IL-18, BAL levels of
IFN- fell to negative control levels, 50 ± 3.9 pg/ml. These
data suggest that intrapulmonary blockade of IL-18 prevents the
expression of IFN-, consistent with the known biological activity of
IL-18. Collectively, these data suggest that endogenous IL-18 enhances
the lung inflammatory response in this model by enhancing production of
mediators. Immunostaining of alveolar macrophages for IL-18
To assess the possible source(s) of endogenous IL-18,
immunostaining for rat IL-18 was done by using BAL alveolar macrophages
retrieved from normal and lungs with immune complex deposits (at 4
h). Alveolar macrophages isolated from the noninflamed lungs
demonstrated low constitutive expression of rat IL-18 (Fig. 5A). After intrapulmonary
deposition of IgG immune complex-induced injury, alveolar macrophages
showed strong staining for rat IL-18 (Fig. 5B). Thus,
alveolar macrophages represent a source of IL-18 in the inflamed
lung.
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As indicated above, several lung cells types were used in vitro:
fibroblasts, alveolar macrophages, type II alveolar epithelial cells,
and lung microvascular endothelial cells. The cells were cultured in
the absence or presence of LPS (10 µg/ml) for 24 h at 37°C,
and the supernatant fluids then were evaluated for IL-18 by ELISA. As
shown in Table I, stimulated fibroblasts
and alveolar macrophages produced 0.38 ± 0.37 and 1.14 ±
0.61 ng/ml, respectively, whereas fluids from the other cell cultures
were negative (<200 pg/ml). Thus, the source of IL-18 in the inflamed
lung appears to be restricted.
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As a further strategy to assess functional effects of IL-18 in the
lung inflammatory model, we used recombinant human IL-18bp that can
intercept IL-18. Human IL-18bp binding to rat IL-18 was determined by
the methods described above. Fifty microliters of human IL-18bp (10
µg/ml) were used to coat each well of the microtiter plate. Rat IL-18
bound to human IL-18bp in a dose-dependent manner but not to solid
phase BSA (Fig. 6A). The
binding was detected at a dose of rat IL-18 as low as 15.6 ng/ml. These
data demonstrate that rat IL-18 can bind to human IL-18bp. To
investigate the effects of exogenously administered IL-18bp in the
IgG immune complex model, recombinant human IL-18bp (0–18
µg) was added to 2.5 mg of anti-BSA IgG before their
intratracheal instillation. The permeability index and BAL content of
neutrophils and cytokines/chemokines were determined at the 4-h time
point. The results are shown in Fig. 6. At a dose of 1.5 or 6 µg of
IL-18bp, there was no statistically significantly effect on the
permeability index. However, at the 18-µg dose of IL-18bp, there was
a 52% decrease in the lung permeability index (Fig. 6B).
Under the same conditions, neutrophil counts in BAL fluids dropped (by
43%), from 6.43 x 106 to 3.65 x
106 (p < 0.05;
n = 4; Fig. 6C). Reduction in BAL content of
TNF-, IL-1, and CINC were 38% (p <
0.05; n = 4), 55% (p < 0.01;
n = 4), and 34% (p < 0.01;
n = 4), respectively (Fig. 6, D–F). These data suggest that IL-18bp attenuates
lung inflammation induced by IgG immune complexes and support the
concept that endogenous IL-18 functions as a proinflammatory factor in
this model of lung injury by enhancing production of proinflammatory
mediators.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1. These cytokines not only activate macrophages but lead to
the expression of vascular adhesion molecules and chemokines involved
in the recruitment of neutrophils (16). Complement,
especially C5a, plays a key role in these events (25).
Oxidants and proteases released from neutrophils and activated lung
macrophages damage lung cells and tissue matrix. Previously, we have
shown that ILs have various regulatory roles during the immune
response. For instance, IL-4, IL-6, IL-10, and IL-13 have strong
anti-inflammatory effects in this lung injury model
(26, 27, 28, 29). The role of IL-18 in this model of lung injury
has not been explored. The current study provides evidence that
endogenous IL-18 acts as a proinflammatory cytokine and contributes to
IgG immune complex-induced lung inflammation. IL-18 mRNA is
constitutively expressed in various rat tissues, including lung.
Constitutive expression of IL-18 also has been observed in specific
cell types, including Kupffer cells, macrophages, keratinocytes, and
articular chondrocytes (8, 30). Up-regulation of IL-18
mRNA was found after onset of pulmonary inflammation initiated by
intrapulmonary deposition of IgG immune complex (Fig. 1). The early
up-regulation of IL-18 has been observed after insults induced by
mycobacteria, contact allergens, and cold stress (10, 31, 32). In numerous studies, it has been concluded that the 1.35-kb
mRNA encodes the precursor (pro) form, which, once translated, is
cleaved by ICE (caspase-1) to a functional, mature protein of 18.3
kDa. By Western blot analysis of BAL fluids, the IL-18 precursor
protein (26 kDa), but not the mature form (18.3 kDa), was found
in noninflamed lungs (Fig. 2). The mature form of IL-18 was detected in
BAL fluids 1–6 h after initiation of lung injury. The absence of the
mature form of IL-18 in noninflamed lungs is consistent with the
finding of others who have observed the absence of the mature form of
IL-18 in unstimulated cells (reviewed in Ref. 12).
Preabsorption of anti-IL-18 by (rr)IL-18 revealed that the bands at
positions 26 and 18 kDa in BAL fluids from inflamed lungs were both
related to IL-18, because both bands essentially disappeared after
absorption by IL-18 (Fig. 2, lane 9). It should be noted
that IL-18 mRNA was evaluated by using whole-lung RNA. When
concentrated BAL fluids were evaluated, mature IL-18 protein was found
by 1 h (Fig. 2A), whereas the mRNA increase wasn’t
apparent until 2 h (Fig. 1). This discrepancy probably is
attributable to the two different sources of mRNA (whole lung) and
protein (BAL fluids). The data in Fig. 2, B and
C, suggest that BAL fluids are a more sensitive indicator of
the presence of IL-18 than are lung homogenates because of earlier
detection of IL-18 (1 h as seen in Fig. 2, A and
B, vs 4 h as seen in Fig. 2C).
Alternatively, it is likely that alveolar macrophages are the chief
source of IL-18, resulting in earliest presence in the BAL compartment.
Tight regulation in expression of the mature form of IL-18 also has
been found in stimulated macrophages and chondrocytes (11, 33). In the current study, it is possible that ICE plays a key
role in expression of the mature form of IL-18. Experimental studies have suggested that IL-18 protects against fungal, bacterial, and viral agents (34, 35, 36, 37). This protection is thought to be attributable in part to the infiltration of inflammatory cells after treatment with IL-18. In the current studies, we show that the in vivo exogenous administration of IL-18 after IgG immune complex deposition increases neutrophil recruitment as well as vascular permeability, a marker of lung injury. Conversely, IL-18 blockade by neutralizing Ab or by IL-18bp reduced evidence of lung inflammation. These results suggest that the protection afforded by IL-18 against infectious agents may be related to its ability to facilitate recruitment of inflammatory cells to sites of infectious agents. Reduction in inflammatory injury by treatment with anti-IL-18 has been observed in endotoxin-induced injury and in experimental autoimmune encephalomyelitis (5, 38, 39). IL-18bp appears as a soluble decoy receptor for IL-18 and has been described as a natural inhibitor for biological activities of IL-18 (40, 41). Our studies suggest that endogenous IL-18 has a role in enhancing the inflammatory response by causing enhanced cytokine and chemokine generation, which is consistent with our findings.
In vitro studies suggest that IL-18 acts as a proinflammatory cytokine
during injury. Mature IL-18 is induced in human peripheral blood
mononuclear cells after exposure to IL-8, MIP-1, MCP-1, or TNF-.
Inhibition of TNF- resulted in an 80% reduction in IL-18
production, suggesting that the primary action of IL-18 is via a
TNF--dependent pathway (42). Previously, we have shown
that lung inflammation and related events require the production of
TNF-, MIP-2, and CINC (19). In the current studies, we
have shown the airway instillation of IL-18 together with deposition of
IgG immune complexes enhanced TNF- and IL-1 levels in BAL fluids.
Conversely, the addition of IL-18bp or neutralizing Ab to IL-18
suppressed the increase in BAL levels of cytokines after injury. This
suggests that IL-18 has an endogenous role in enhancing production of
early response cytokines during acute lung inflammation. It is unknown
whether the increase in these inflammatory cytokines is attributable in
part to the induction of IFN- (or another cytokine) and the
subsequent activation of macrophages by IL-18. The effectiveness of
IL-18 blockade in vivo by Ab is suggested by the reduction to baseline
levels of IFN- in BAL fluids (Fig. 4F). It seems that in
the model used increases in TNF- and IL-1 in the presence of
IL-18 (endogenous or exogenous) would be linked to enhanced
up-regulation of lung vascular ICAM-1 and E selection. It also is
likely that endogenous IL-18 causes enhanced production of MIP-2 and
CINC, causing increased recruitment of neutrophils. The protective
effects of IL-18bp are consistent with the outcomes when anti-IL-18
was used in vivo.
Enhanced levels of TNF- and the subsequent up-regulation of
chemokines are associated with the activation of NF-
B
(43). We have shown recently that IL-10 and IL-13 regulate
proinflammatory cytokine production by the suppression of the
transcription factor NF-B (28, 44). Many cytokines
signal through different cell surface receptors to activate the
transcription of NF-B. TNFR-associated factor-6 protein appears to
be linked to IL-1/IL-1R complex that causes NF-B activation
(45). The importance of activation of NF-B after the
administration of IL-18 during inflammatory injury induced by IgG
immune complex is unknown and currently is being evaluated.
The current study suggests that IL-18 has a role in lung inflammation
induced by the deposition of IgG immune complexes. In many respects,
the role of IL-18 in this model is similar to MIP-1, which is a
product of activated macrophages and has the ability to cause autocrine
stimulation of macrophages, resulting in enhanced generation of
cytokines and chemokines (46). It is clear that IL-18 is a
pleiotropic cytokine with numerous functions. Recent studies have shown
IL-18 directly induces IFN- promotor activity, up-regulates ICAM-1
expression in human myelomonocytic cells, and has synergistic effects
when combined with IL-12 (47). Our recent data indicate
that some, but not all polyclonal rabbit Abs to rat chemokines, are
protective in the IgG immune complex model of acute lung injury. For
example, in this model Abs to MIP-2, CINC, MIP-1, and MIP-1 were
protective (46, 48, 49) but not Abs to MCP-1 or RANTES
(49). In contrast, the same anti-RANTES Ab
significantly delayed rejection of allografted rat hearts
(50) and the same anti-MCP-1 enhanced the lethal
effects of infused LPS in mice (51). Conversely, infusion
of MCP-1 in the same mouse model was protective (52).
Thus, there is a certain specificity of these blocking Abs, depending
on the inflammatory model under study. The current studies identify an
in vivo proinflammatory role for IL-18 in the lung inflammatory
model used.
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Acknowledgments |
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Footnotes |
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2 J.A.J. and R.-F.G. contributed equally to the body of work.
3 Address correspondence and reprint requests to Dr. Peter A. Ward, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail address: pward{at}umich.edu
4 Abbreviations used in this paper: ICE, IL-1-converting enzyme; IL-18bp, IL-18 binding protein; (rr)IL-18, rat recombinant IL-18; MIP, macrophage-inflammatory protein; CINC, cytokine-induced neutrophil chemoattractant.
Received for publication June 1, 2001. Accepted for publication September 28, 2001.
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