Role of IL-18 in Acute Lung Inflammation Jacqueline A

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Role of IL-18 in Acute Lung Inflammation Jacqueline A. Jordan, Ren-Feng Guo, Edward C. Yun, Vidya Sarma, Roscoe L. Warner, Larry D. Crouch, Giorgio Senaldi, Thomas R. Ulich and Peter A. Ward This information is current as of September 28, 2021. J Immunol 2001; 167:7060-7068; ; doi: 10.4049/jimmunol.167.12.7060 http://www.jimmunol.org/content/167/12/7060 Downloaded from References This article cites 52 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/167/12/7060.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Role of IL-18 in Acute Lung Inﬂammation1

Jacqueline A. Jordan,2* Ren-Feng Guo,2* Edward C. Yun,* Vidya Sarma,* Roscoe L. Warner,* Larry D. Crouch,† Giorgio Senaldi,‡ Thomas R. Ulich,‡ and Peter A. Ward3*

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2Ð4 h in inflamed lungs and remained elevated at 24 h, and the “mature” protein form of IL-18 (18 kDa) appeared in BAL fluids 1Ð8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-␣, IL-1␤, and cytokine-induced neutrophil che- Downloaded from moattractant, 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. The Journal of Immunology, 2001, 167: 7060Ð7068. http://www.jimmunol.org/

nterferon-␥ production by CD4ϩ helper T cells, CD8ϩ cy- converting enzyme (ICE4; also called caspase 1) is responsible for totoxic T cells, and NK cells plays an important role in the cleaving IL-1␤ and IL-18 to generate active, mature forms of these I immune response, especially in the Th1 pathway. IFN-␥ has proteins. Studies have shown that ICE-deficient mice have reduced the ability to activate macrophages, enhance NK cell activity, in- levels of IFN-␥ and mature IL-18 (13, 14). crease cytokine production, and protect cells from viral replication In the present study, we investigated the in vivo role of IL-18 in (1, 2). Only a few cytokines have been shown to induce the pro- acute lung inflammation in rats caused by deposition of IgG im- duction of IFN-␥, including TNF-␣, IL-2, and IL-12 (1–4). In mune complexes. This model is associated with production of ␣ 1989, a new cytokine termed IL-18 (or IGIF) was found to be a TNF- and IL-1, up-regulation of vascular adhesion molecules, by guest on September 28, 2021 potent inducer of IFN-␥ (5). IL-18 was first isolated from liver and CXC/CC chemokine expression, all of which facilitate the recruitment of blood neutrophils, activation of lung macrophages, extracts of mice sensitized with heat-killed Propionibacterium ac- and the release of oxidants and proteases (15, 16). The role of nes and subsequently challenged with LPS and was identified as an IL-18 after acute lung inflammation caused by IgG immune com- 18-kDa nonglycosylated protein secreted primarily by activated plex deposition has not been explored previously. The current macrophages/monocytes (5–7). Other cell types also have been studies also explore the effects of IL-18 binding protein (IL-18bp), shown to secrete IL-18, including epidermal keratinocytes, intes- a natural IL-18 antagonist (17). tinal epithelial cells, adrenal cortical cells, and osteoblasts (8–11). The three-dimensional structure of IL-18 indicates a relationship to Materials and Methods ␣ ␤ ␤ IL-1 (12%) and IL-1 (19%). Like IL-1 , IL-18 is synthesized in Reagents precursor form with an unusual signal peptide that allows secretion from the cell. The precursor form of IL-1␤ has a very low biolog- Except where noted, all reagents were purchased from Sigma (St. Louis, MO). The anti-IL-18 was affinity-purified rabbit IgG directed against mu- ical activity (defined by the induction of IFN-␥), whereas the rine IL-18. This Ab neutralizes the biological activity of IL-18 (R&D Sys- precursor form of IL-18 is totally devoid of activity (12). IL-1␤- tems, 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 *Department of Pathology, University of Michigan Medical School, Ann Arbor, MI Care of Animals. Ketamine was administered i.p. for sedation and anes- 48109; †Department of Physiology, School of Dentistry, University of Nebraska, Lincoln, NE 68198; and ‡Amgen, Thousand Oaks, CA 91320 thesia. Rabbit polyclonal IgG (1.5 or 2.5 mg) rich in Ab to BSA (anti-BSA) was intratracheally instilled in a volume of 300 ␮l of PBS (pH 7.4) during Received for publication June 1, 2001. Accepted for publication September 28, 2001. inspiration. This was followed by the i.v. injection of BSA (10 mg) with The costs of publication of this article were defrayed in part by the payment of page trace amounts of 125I-labeled BSA as a quantitative marker of permeability. charges. This article must therefore be hereby marked advertisement in accordance Rats were sacrificed 4 h later, the pulmonary circulation flushed, and lung with 18 U.S.C. Section 1734 solely to indicate this fact. injury quantified by increased vascular permeability. It has been shown 1 This work was supported by National Institutes of Health Grant HL-31963. elsewhere that inflammation and mediator presence in lung peak at this 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 4 Abbreviations used in this paper: ICE, IL-1␤-converting enzyme; IL-18bp, IL-18 Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Ar- binding protein; (rr)IL-18, rat recombinant IL-18; MIP, macrophage-inflammatory bor, MI 48109-0602. E-mail address: [email protected] protein; CINC, cytokine-induced neutrophil chemoattractant.

time point (18). The permeability index was determined by measuring the Western blot analysis amount of radioactivity (125I-labeled BSA) remaining in the lungs compared with the radioactivity present in 1.0 ml of blood obtained from the BAL fluids from IgG immune complex-injured animals were analyzed for inferior vena cava. Negative control animals were intratracheally instilled rat IL-18 immunoreactive protein. Five milliliters of PBS was instilled into with PBS only. In all experiments in which this model was used, n ϭ Ն 6 the lungs and collected three times, and cellular contents were removed by ϳ ␮ animals unless otherwise indicated. centrifugation. BAL fluids then were concentrated ( 5-fold) to 500 lby using a centricon (Amicon, Beverly, MA). Concentrated BAL fluids were subjected to SDS-PAGE (15%) according to the method of Laemmli. Cloning of rat IL-18 Equivalent amounts of protein (100 ␮g) were added to each lane. The ␮ Previously, it has been shown that IL-18 is strongly induced in the adrenal acrylamide gels then were transblotted to nitrocellulose (0.1 m) for 2 h at gland of rats after reserpine administration (10). In this study, we cloned rat 12 V. The membrane was blocked with TweenTris-buffered saline con- IL-18 after inflammatory challenge. Rat lungs were injured by IgG immune taining 5% milk for1hatroom temperature. Polyclonal goat anti-mouse ␮ complex deposition as described above. Four hours later, whole adrenal IL-18 (100 g/ml) then was added at a dilution of 1/1000, and the mem- glands (cortex and medulla) were removed and frozen in liquid nitrogen. brane was incubated overnight at 4°C. After washing, peroxidase-conju- ␮ Total RNA was extracted by using the guanidinium-isothiocyanate method gated donkey anti-goat IgG (800 g/ml) was added (1:10,000), and the (TRIzol; Life Technologies, Grand Islands, NY). First-strand cDNA was membrane was incubated for1hatroom temperature. The membrane was constructed by reverse-transcribing 10 ␮g of total RNA with an oligo(dT) developed with an ECL technique (Amersham Life Science, Piscataway, primer. cDNA amplification of rat IL-18 was performed with the above RT NJ). (rr)IL-18 was used as a positive control, and a m.w. marker was used products and the following rat primers: 5Ј,5Ј-AACAATGGCTGCCAT to estimate the size of the immunoreactive bands. Intensity of the protein GTCAG-3Ј; and 3Ј,5Ј-AGTGAACATTACAGATTTATCCC-3Ј. These band was determined as above. For experiments where indicated, the anti- primers result in a long 566-bp and a shorter 510-bp PCR band. The PCR IL-18 Ab was preabsorbed by the addition of (rr)IL-18 at a final concen- ␮ products were ligated into a PCR 2.1 vector (Invitrogen, Carlsbad, CA), tration of 1 g/ml. Downloaded from sequenced, and compared with the GenBank data bank. After confirmation BAL fluid analysis and cytokine and chemokine content 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. BAL fluid was collected from IgG immune complex-injured rat lungs 4 h after injury, and when used for Western blot analysis, was concentrated Recombinant rat (rr)IL-18 , anti-mouse IL-18 polyclonal Ab, 5-fold by using Centricon filters with a cut-off of 3 kDa (Amicon). Briefly, and IL-18bp 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.

(rr)IL-18 was purchased from R&D Systems. The protein was expressed in TNF-␣ activity was determined by using a standard WEHI cell cytotoxicity http://www.jimmunol.org/ Escherichia coli and was determined by SDS-PAGE to be Ͼ97% pure. The assay. BAL concentrations of macrophage-inflammatory protein (MIP)-2 endotoxin level was Ͻ0.1 ng per 1 ␮g of IL-18, and activity was confirmed and cytokine-induced neutrophil chemoattractant (CINC) were determined by its ability to induce mouse IFN-␥ production by activated mouse T cells. by ELISA as reported previously (19). The ELISA kits for detection of rat Where indicated, 1.0 or 2.0 ␮g of rat IL-18 was coinstilled intratracheally IL-1␤ and IFN-␥ were purchased from R&D Systems. (constant volume of 10 ␮l) alone or with anti-BSA (in a final volume of 300 ␮l) at the commencement of injury. Polyclonal goat anti-mouse IL-18 Immunostaining of alveolar macrophages Ab was derived from immunized goats. The endotoxin level in the IgG was Ͻ BAL fluids from control and injured animals were collected and centri- 0.1 ng per 1.0 mg of the Ab. Cross-reactivity with rat IL-18 was deter- fuged at 450 ϫ g for 10 min and were layered onto Ficoll-Paque to remove mined by Western blotting. The Ab was reconstituted from the lyophilized RBCs and neutrophils (450 ϫ g for 30 min). The upper layer containing form with sterile PBS to a concentration of 100 ␮g/ml. Our studies, shown

alveolar macrophages was removed and resuspended in PBS containing by guest on September 28, 2021 below, indicate that the anti-IL-18 Ab, when absorbed with (rr)IL-18, loses 1% BSA to a concentration of 250,000 cells/ml. Slides were prepared by its ability to detect IL-18 in BAL fluids from inflamed lungs. Human re- ␮ ␮ adding 100 l of this cell suspension to a cytospin and centrifuging at combinant IL-18bp (1.5, 6, or 18 g) was coinstilled intratracheally with 450 ϫ g for 7 min. Cytospin slides were fixed in 100% methanol and stored anti-BSA at the commencement of inflammation. Human IL-18bp was ex- at Ϫ20°C. For immunostaining, slides were washed in PBS and incubated pressed in mammalian cells and purified by HPLC, and shown to inhibit ␮ ␥ with anti-IL-18 (1 g/ml) for1hinahumidified chamber. Slides then were LPS-induced IFN- production in rat lungs (G. Senaldio, unpublished washed two times and incubated for 1 h with biotinylated anti-goat IgG (1 observations). ␮g/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 dia- Northern Blot Analysis minobenzidine reagent for 10 min and later counterstained with hematoxylin. Total RNA was extracted (TRIzol Reagent; Life Technologies) from whole lungs of rats injured with IgG immune complexes. The pulmonary circu- Isolation and culture of rat lung cells lation was flushed with 10 ml of PBS, and the lungs were surgically removed and immediately frozen in liquid nitrogen. Twenty micrograms of Alveolar macrophages were isolated from BAL fluids from normal rat RNA was fractionated in 1% agarose formaldehyde gel and transferred to lungs as described (20). Cells were pelleted and then resuspended in a nylon membrane (Magna Charge; MSSI, Westboro, MA). The cDNA for DMEM supplemented with 10% FBS and penicillin-streptomycin rat IL-18 was [32P]d-CTP radiolabeled by random priming with Klenow (DMEM-FBS). After allowing cells to adhere to the plate (5 ϫ 105 cells per enzyme to generate a highly specific activity probe (1.5 ϫ 107 cpm; Am- well in 24-well tissue culture plates; Corning, Corning, NY), nonadherent ersham Life Science, Piscataway, NJ). Hybridization was performed at cells were removed with two washes. Cell monolayers then were stimu- 68°C for 4 h (QuikHyb; Stratagene, La Jolla, CA), and the autoradiograph lated with the appropriate agonists suspended in DMEM-FBS in a 5% CO2 was developed on Kodak Biomax-MR film (Kodak, Rochester, NY). In- humidified incubator at 37°C for 24 h. Rat alveolar fibroblasts were iso- tensity of RNA bands was analyzed with image analysis software (Adobe lated from normal lungs. Lungs were removed, minced into 2-mm pieces, Systems, San Jose, CA). ␤-Actin radiolabeled probe was used as an inter- and the cells allowed to adhere to an inverted 25-mm tissue culture flask nal standard to ensure equal RNA loading. containing no medium for 48 h. The flasks then were righted, DMEM containing 10% FBS was added, and cultures were allowed to grow out ELISA for rat IL-18 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 Anti-IL-18 Ab, biotinylated Ab, and (rr)IL-18 protein were purchased from (Corning). All cultures were used between passage 1 and passage 3 and R&D Systems. A-sandwich ELISA was performed with 100 ␮lof1␮g/ml were plated at 5 ϫ 105 cells per well in 24-well tissue culture plates (Corn- of anti-IL-18 Ab (primary Ab) in borate buffer to coat ELISA plates. A 1% ing) containing DMEM-FBS. Cells were stimulated with appropriate ago-

BSA solution in Dulbecco’s PBS was used to block nonspeciﬁc binding. nists suspended in DMEM-FBS in 5% CO2 humidiﬁed incubator at 37°C After washing, samples and standards were added to individual wells and and allowed to grow for 24 h. Type II alveolar epithelial cells were isolated incubated for2hat37°C. This was followed by washing and incubation from normal rat lung using elastase cell dispersion and IgG panning by the with 100 ␮l of biotinylated Ab (1 ␮g/ml) for1hat37°C. Subsequently, method of Warner and associates (21). Cells were plated in DMEM-FBS at after a 1-h incubation with a streptavidin-HRP conjugate, the assay was 5 ϫ 105 cells per well in 24-well tissue culture plates and incubated at 37°C

developed by addition of o-phenylenediamine substrate. The developing in 5% CO2. Cells were stimulated with appropriate agonists suspended in reaction was stopped by3MH2SO4. The plate was read on an ELISA plate DMEM-FBS and allowed to grow for 24 h. Microvascular endothelial cells reader at 492 nm. were isolated from peripheral lungs of normal 21-day-old rats (22). Strips 7062 IL-18 AND ACUTE LUNG INFLAMMATION

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 ϫ 105 cells per well in 24-well tissue culture plates and incubated at

37°Cin5%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 ␮g/ml) in borate-coating buffer was used to coat each well in a 96-well ELISA plate overnight at 4°C.A1% BSA solution in Dulbecco’s PBS was used to block nonspeciﬁc binding (1 hat37°C). Various concentrations of rat IL-18 and BSA (100 ␮l) were added per well and incubated for1hat37°C. After the washing step, goat anti-rat IL-18 (R&D Systems) was added and incubated for1hat37°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 lof3MH2SO4 and read at 492 nm.

Statistical analysis Downloaded from All data are presented as means Ϯ 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 Ͻ 0.05.

Results http://www.jimmunol.org/ IL-18 mRNA and protein expression in IgG immune complex- induced inflamed rat lungs IL-18 mRNA content varied in normal rat tissues based on North- ern blot analysis. In the adult rat, IL-18 mRNA (ϳ1.35 kb) was FIGURE 1. Northern blot for IL-18 mRNA in various rat tissues. A, most abundant in liver, with moderate expression in spleen and Two micrograms of poly(A)ϩ RNA from eight different rat tissues were low but measurable constitutive expression in the lungs, kidneys, loaded into 1.2% formaldehyde agarose gel. The Northern blot was probed and heart (Fig. 1A). Little if any mRNA for IL-18 was found in with a 32P-labeled full-length cDNA for rat IL-18. Equal loading was con- skeletal muscle and testis. Equal loading of RNA was confirmed firmed by ␤-actin probing (data not displayed). B, mRNA for IL-18 in lung by stripping the blot and reprobing with radiolabeled ␤-actin (data 0–4 h after initiation of the inflammatory response. GAPDH content for by guest on September 28, 2021 not shown). To assess changes in endogenous IL-18 mRNA and equal loading is shown. The lowest frame is a densitometry of the upper protein during the pulmonary inflammatory response, lungs from portion of B. 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) re- lung inflammation. As shown in Fig. 2B, IL-18 levels in BAL vealed increases (ϳ50%, as determined by densitometry) in fluids from normal lungs were Ͻ1 ng/ml, whereas there was a mRNA for IL-18, 2 and 4 h after initiation of the inflammatory progressive increase in BAL IL-18, peaking at4h(atϳ9 ng/ml), response (Fig. 1B, bottom). Equal loading was confirmed by prob- followed by a decline. When lung homogenates were evaluated ing with GADPH (Fig. 1B, bottom). over the same period of time, normal lungs contained 1025 Ϯ 128 As determined by Western blot analysis with anti-rat IL-18 rab- ng/g lung weight (Fig. 2B). In the inflamed lungs, there was very bit polyclonal IgG, changes in IL-18 content in BAL fluids (5-fold little measurable increase in IL-18 during the first2hofinflam- concentrated) are shown in Fig. 2A as a function of time. In lane mation, but by 4 and 6 h, the IL-18 content was 2585 Ϯ 172 and 1, (rr)IL-18 gave a single band in the 18-kDa position, consistent 2441 Ϯ 301 ng/g lung, respectively. These data indicate that IL-18 with the mature form of IL-18. At time 0 (lane 2), a single band is induced in lungs of rats during deposition of IgG immune reactive with anti IL-18 Ab was found in BAL fluids in the 26-kDa complexes. position, which is consistent with the “pro” form of IL-18. There- after, between 1 and 8 h, IL-18 bands were found in both the 26- Effects of (rr)IL-18 on vascular permeability and BAL content and 18-kDa positions (lanes 3Ð6), whereas at 16 and 24 h (lanes The effects of the exogenously administered rat IL-18 during IgG- 7 and 8) bands in the former position were prominent, but bands in induced inflammation were determined in the lung model. For the 18-kDa position were barely visible. When the anti-IL-18 prep- these studies, the dose of anti-BSA used was 1.25 mg, to detect in aration (1.0 ␮g/ml) was preabsorbed by addition of (rr)IL-18 (1.0 a more sensitive manner increases in the parameters of lung in- ␮g/ml final concentration), both the 18-kDa and the 26-kDa bands flammation and vascular leak. The vascular permeability (leakage disappeared (lane 9, using the 4-h BAL sample), indicating that of 125I-labeled albumin) was measured at 4 h, the time at which the both bands are related to IL-18. As is also shown in Fig. 2A, the permeability index peaks (18). The mean permeability value in the double banding pattern found in BAL fluids obtained 4 h after negative control lungs was 0.17 Ϯ 0.01 (Fig. 3A). Instillation of initiation of this inflammatory response (lane 10) disappeared 2.0 ␮g of IL-18 alone did not significantly augment vascular per- from similarly treated rats that also received 400 ␮g of anti-IL-18 meability in otherwise normal lungs (0.19 Ϯ 0.03). Positive con- intratracheally at time 0 (lane 11). trol rats not otherwise treated demonstrated a 40% increase in vas- By using a custom-prepared ELISA for rat IL-18, BAL fluids cular permeability (to a value of 0.29 Ϯ 0.02). In a companion (which were concentrated 5-fold) were evaluated after induction of group of rats that also received 2.0 ␮g of IL-18 in the presence of The Journal of Immunology 7063

absence of exogenously administered IL-18 (108 Ϯ 14.8 pg/ml). The coadministration intratracheally of anti-BSA and 1.0 or 2.0 ␮g IL-18 further increased IL-1␤ production, to 132 Ϯ 5.4 and 177 Ϯ 16.5 pg/ml, respectively, ( p Ͻ 0.05 for the latter). Noninflamed lungs contained low levels of CINC (146 Ϯ 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 Ϯ 23.5 ng/ml; Fig. 3E). Positive control rats demonstrated a 3-fold increase in BAL levels of CINC (1837 Ϯ 97.5). In the positive control group with coinstillation of 2.0 ␮g of IL-18, CINC levels were increased further, by 20% (to 2211 Ϯ 104 ng/ml; p Ͻ 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 Downloaded from could alter events in positive control animals. As indicated above, FIGURE 2. Rat IL-18 protein expression in BAL fluids (concentrated this Ab is reactive with rat IL-18. As shown in Fig. 4A, the ex- 5-fold) after IgG immune complex deposition. As determined by Western travascular leakage of 125I-labeled albumin was measured 4 h after blot analysis as a function of time (A), lane 1 contains (rr)IL-18, whereas the initiation of injury in lungs receiving 2.5 mg of anti-BSA to- lane 9 contains the same, but the anti-IL-18 was first preabsorbed with gether with 400 ␮g of preimmune goat IgG. As compared with the (rr)IL-18. Lane 10 is BAL fluid 4 h after initiation of the reaction, whereas ␮ negative control group, the positive control group receiving 400 http://www.jimmunol.org/ lane 11 is similar, but in rats receiving 400 g of anti-IL-18 intratracheally ␮ at time 0. B, Rat IL-18 was measured in BAL fluids as a function of time g of normal rabbit IgG intratracheally had a more than 4-fold Ϯ after initiation of lung inflammation. C, Whole lung homogenates were increase in vascular permeability (to a value of 0.60 0.04). The evaluated for IL-18 by ELISA, as a function of time after initiation of the addition of 400 ␮g of anti-IL-18 IgG resulted in a 30% reduction lung inflammatory response. in vascular permeability (to a value of 0.42 Ϯ 0.04; p Ͻ 0.05). BAL fluids collected at 4 h were evaluated for neutrophil content (Fig. 4B). Intrapulmonary deposition of IgG immune complexes anti-BSA, the permeability index was further increased, to 0.44 Ϯ caused a 10-fold increase in BAL neutrophil numbers (to 6.8 ϫ 0.04 (a 35% increase) as compared with the positive control group 106). The coinstillation of anti-BSA and 400 ␮g of anti-IL-18 de- ( p Ͻ 0.05). Next, we determined whether these changes were as- creased neutrophil counts by 50% (to 3.5 ϫ 106; p Ͻ 0.05). by guest on September 28, 2021 sociated with enhanced neutrophil recruitment. BAL fluids were In parallel, we determined whether BAL content of inflamma- collected at 4 h and evaluated for neutrophil content (Fig. 3B). In tory mediators would be altered by airway instillation of 400 ␮gof the negative control animals, low levels (Ͻ5 ϫ 105) neutrophils neutralizing Ab to IL-18. BAL fluids were obtained 4 h after ini- were found; the intratracheal administration of 2.0 ␮g of IL-18 tiation of lung inflammatory reactions and cytokine levels deter- caused no significant increase in BAL content of neutrophils. In- mined. In rats receiving 400 ␮g of normal rabbit IgG intratrache- trapulmonary instillation of 1.25 mg of anti-BSA together with 10 ally, BAL levels of all three mediators that are known to be mg of BSA (which was intravenously administered) resulted in a involved in the pathogenesis of these inflammatory responses were 4-fold increase in neutrophil counts (to 1.6 ϫ 106). The coinstil- significantly increased (Fig. 4, C–E) to 2880 Ϯ 229 pg/ml for lation of IL-18 (1.0 ␮gor2.0␮g) together with anti-BSA further TNF-␣,to250Ϯ 25 pg/ml for TNF-␣, and to 3984 Ϯ 210 ng/ml increased neutrophils counts (to 3.1 ϫ 106 and 5.0 ϫ 106, respec- for CINC. The coadministration of 400 ␮g of anti-IL-18 IgG with tively, p Ͻ 0.05). anti-BSA decreased levels of TNF-␣ by 33% (to 1908 Ϯ 330 pg/ Because TNF-␣, IL-1, and CINC are important cytokines and ml), IL-1␤ levels by 60% (98 Ϯ 15 pg/ml), and CINC levels by chemokines involved in the recruitment of inflammatory cells in 27% (to 2892 Ϯ 368 ng/ml). In companion experiments, we also this lung model (19, 23, 24), we examined the effects of exog- assessed the effects of administration of anti-IL-18 on BAL levels enously administered IL-18 on BAL levels of these mediators after of IFN-␥, because, as described above, IL-18 is well known to IgG immune complex-induced lung inflammation. TNF-␣ levels in induce expression of IFN-␥. In noninflamed (negative control) the BAL fluids (obtained at 4 h) were determined. Negative control lungs, BAL levels of IFN-␥ were 50 Ϯ 4.0 pg/ml, whereas in the animals had low BAL levels of TNF-␣ (5.6 Ϯ 0.8 pg/ml; Fig. 3C). inflamed lungs of animals treated with preimmune IgG, the levels Intrapulmonary instillation of 2.0 ␮g of IL-18 alone caused a small rose to 120 Ϯ 8.2 pg/ml (Fig. 4F). In the presence of anti-IL-18, increase in TNF-␣ content (to 9.0 Ϯ 1.1 pg/ml). After immune BAL levels of IFN-␥ fell to negative control levels, 50 Ϯ 3.9 complex deposition, BAL levels of TNF-␣ substantially increased pg/ml. These data suggest that intrapulmonary blockade of IL-18 (to 1279 Ϯ 118 pg/ml). The coadministration of 1.25 mg of anti- prevents the expression of IFN-␥, consistent with the known bio- BSA and 1.0 or 2.0 ␮g of IL-18 further increased TNF-␣ levels, to logical activity of IL-18. Collectively, these data suggest that en- 1590 Ϯ 109 and 2776 Ϯ 130 pg/ml, respectively ( p Ͻ 0.05). dogenous IL-18 enhances the lung inflammatory response in this IL-1␤ levels also were determined in BAL fluids by ELISA, model by enhancing production of mediators. using the same experimental conditions (Fig. 3D). Negative control animals had low levels of IL-1␤ (13.4 Ϯ 0.50 pg/ml). The Immunostaining of alveolar macrophages for IL-18 instillation of IL-18 into otherwise normal lungs caused no signif- To assess the possible source(s) of endogenous IL-18, immuno- icant changes in BAL levels of IL-1␤. A nearly 8-fold increase in staining for rat IL-18 was done by using BAL alveolar macro- BAL levels of IL-1␤ was found in the positive control group in the phages retrieved from normal and lungs with immune complex 7064 IL-18 AND ACUTE LUNG INFLAMMATION

FIGURE 3. Enhancing effects of (rr)IL-18 on acute lung inﬂammation. Permeability index of rats receiving PBS intratracheally 2.0 ␮g of (rr)IL-18 alone Downloaded from or together with anti-BSA (A) and BAL content of neutrophils in companion groups (B); and BAL content of TNF-␣ (C), IL-1␤ (D) or CINC (E). http://www.jimmunol.org/ by guest on September 28, 2021

deposits (at 4 h). Alveolar macrophages isolated from the nonin- human IL-18bp (10 ␮g/ml) were used to coat each well of the flamed lungs demonstrated low constitutive expression of rat IL-18 microtiter plate. Rat IL-18 bound to human IL-18bp in a dose- (Fig. 5A). After intrapulmonary deposition of IgG immune com- dependent manner but not to solid phase BSA (Fig. 6A). The bind- plex-induced injury, alveolar macrophages showed strong staining ing was detected at a dose of rat IL-18 as low as 15.6 ng/ml. These for rat IL-18 (Fig. 5B). Thus, alveolar macrophages represent a data demonstrate that rat IL-18 can bind to human IL-18bp. To source of IL-18 in the inflamed lung. investigate the effects of exogenously administered IL-18bp in the IgG immune complex model, recombinant human IL-18bp (0–18 Lung cell sources of IL-18 ␮g) was added to 2.5 mg of anti-BSA IgG before their intratra- As indicated above, several lung cells types were used in vitro: cheal instillation. The permeability index and BAL content of neu- fibroblasts, alveolar macrophages, type II alveolar epithelial cells, trophils and cytokines/chemokines were determined at the 4-h time and lung microvascular endothelial cells. The cells were cultured point. The results are shown in Fig. 6. At a dose of 1.5 or 6 ␮gof in the absence or presence of LPS (10 ␮g/ml) for 24 h at 37°C, and IL-18bp, there was no statistically significantly effect on the per- the supernatant fluids then were evaluated for IL-18 by ELISA. As meability index. However, at the 18-␮g dose of IL-18bp, there was shown in Table I, stimulated fibroblasts and alveolar macrophages a 52% decrease in the lung permeability index (Fig. 6B). Under the produced 0.38 Ϯ 0.37 and 1.14 Ϯ 0.61 ng/ml, respectively, whereas fluids from the other cell cultures were negative (Ͻ200 same conditions, neutrophil counts in BAL fluids dropped (by ϫ 6 ϫ 6 Ͻ ϭ pg/ml). Thus, the source of IL-18 in the inflamed lung appears to 43%), from 6.43 10 to 3.65 10 ( p 0.05; n 4; Fig. 6C). ␣ ␤ be restricted. Reduction in BAL content of TNF- , IL-1 , and CINC were 38% ( p Ͻ 0.05; n ϭ 4), 55% ( p Ͻ 0.01; n ϭ 4), and 34% ( p Ͻ 0.01; Effects of exogenously administered IL-18bp n ϭ 4), respectively (Fig. 6, D–F). These data suggest that IL-18bp As a further strategy to assess functional effects of IL-18 in the attenuates lung inflammation induced by IgG immune complexes lung inflammatory model, we used recombinant human IL-18bp and support the concept that endogenous IL-18 functions as a that can intercept IL-18. Human IL-18bp binding to rat IL-18 was proinflammatory factor in this model of lung injury by enhancing determined by the methods described above. Fifty microliters of production of proinflammatory mediators. The Journal of Immunology 7065

FIGURE 4. Protectiveeffectsofanti- IL-18 on acute lung inﬂammation. Pul- monary vascular permeability index

(A) and BAL content of neutrophils Downloaded from (B), TNF-␣ (C), IL-1␤ (D), CINC (E), and IFN-␥ (F). http://www.jimmunol.org/ by guest on September 28, 2021

Discussion results in damage of capillary endothelial cells and alveolar epi- In the current study, we used a model of acute lung inﬂammation thelial cells, neutrophil inﬂux, and intraalveolar hemorrhage. This induced by the intrapulmonary deposition of IgG immune com- attendant activation of lung macrophages leads to induction of the plexes. Alveolar deposition of IgG immune complexes was early response cytokines TNF-␣ and IL-1␤. These cytokines not achieved by the intratracheal instillation of rabbit polyclonal IgG only activate macrophages but lead to the expression of vascular against BSA followed by the intravenous injection of BSA. This adhesion molecules and chemokines involved in the recruitment of

FIGURE 5. Immunohistochemical staining of alveolar macrophages for rat IL-18 after IgG immune complex lung inﬂammation. A, BAL macrophages from PBS-instilled lung; B, BAL macrophages obtained 4 h after deposition of IgG immune complexes. Counter- stained with hematoxylin, ϫ100. 7066 IL-18 AND ACUTE LUNG INFLAMMATION

Table I. IL-18 production by cultured lung cells (10, 31, 32). In numerous studies, it has been concluded that the 1.35-kb mRNA encodes the precursor (pro) form, which, once IL-18 Content in Supernatant Fluids (ng/ml) translated, is cleaved by ICE (caspase-1) to a functional, mature protein of ϳ18.3 kDa. By Western blot analysis of BAL fluids, the Type II Endothelial ϳ Stimulusa Fibroblasts Macrophages cells cells IL-18 precursor protein ( 26 kDa), but not the mature form (ϳ18.3 kDa), was found in noninflamed lungs (Fig. 2). The mature b None ND ND ND ND form of IL-18 was detected in BAL fluids 1–6 h after initiation of LPS (10 ␮g/ml 0.38 Ϯ 0.37 1.14 Ϯ 0.61 ND ND for 24 h) lung injury. The absence of the mature form of IL-18 in noninflamed lungs is consistent with the finding of others who have a See text for details of culture conditions and cell isolation. b ND, not detectable (Ͻ200 pg/ml). 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 neutrophils (16). Complement, especially C5a, plays a key role in BAL fluids from inflamed lungs were both related to IL-18, be- these events (25). Oxidants and proteases released from neutro- cause both bands essentially disappeared after absorption by IL-18 phils and activated lung macrophages damage lung cells and tissue (Fig. 2, lane 9). It should be noted that IL-18 mRNA was evaluated matrix. Previously, we have shown that ILs have various regula- by using whole-lung RNA. When concentrated BAL fluids were tory roles during the immune response. For instance, IL-4, IL-6, evaluated, mature IL-18 protein was found by 1 h (Fig. 2A), IL-10, and IL-13 have strong anti-inflammatory effects in this lung whereas the mRNA increase wasn’t apparent until 2 h (Fig. 1). injury model (26–29). The role of IL-18 in this model of lung This discrepancy probably is attributable to the two different Downloaded from injury has not been explored. The current study provides evidence sources of mRNA (whole lung) and protein (BAL fluids). The data that endogenous IL-18 acts as a proinflammatory cytokine and in Fig. 2, B and C, suggest that BAL fluids are a more sensitive contributes to IgG immune complex-induced lung inflammation. indicator of the presence of IL-18 than are lung homogenates be- IL-18 mRNA is constitutively expressed in various rat tissues, in- cause of earlier detection of IL-18 (1 h as seen in Fig. 2, A and B, cluding lung. Constitutive expression of IL-18 also has been ob- vs4hasseen in Fig. 2C). Alternatively, it is likely that alveolar served in specific cell types, including Kupffer cells, macrophages, macrophages are the chief source of IL-18, resulting in earliest http://www.jimmunol.org/ keratinocytes, and articular chondrocytes (8, 30). Up-regulation of presence in the BAL compartment. Tight regulation in expression IL-18 mRNA was found after onset of pulmonary inflammation of the mature form of IL-18 also has been found in stimulated initiated by intrapulmonary deposition of IgG immune complex macrophages and chondrocytes (11, 33). In the current study, it is (Fig. 1). The early up-regulation of IL-18 has been observed after possible that ICE plays a key role in expression of the mature form insults induced by mycobacteria, contact allergens, and cold stress of IL-18. by guest on September 28, 2021

FIGURE 6. Protective effects of IL-18bp on immune complex-induced lung inﬂammation. A, binding of (rr)IL-18 to solid-phase human IL-18bp. A total of 50 ␮l of human IL-18bp (10 ␮g/ml) was used to coat each well. Bound rrIL-18 was detected with polyclonal goat anti-mouse IL-18. B, Effects of airway instillation of IL-18bp on lung vascular permeability index at 4 h. When used, 0–18 ␮g were coinstilled with 2.5 mg of anti-BSA. BAL content of PMN (C), TNF-␣ (D), IL-1␤ (E), and CINC (F). The Journal of Immunology 7067

Experimental studies have suggested that IL-18 protects against is a pleiotropic cytokine with numerous functions. Recent studies fungal, bacterial, and viral agents (34–37). This protection is have shown IL-18 directly induces IFN-␥ promotor activity, up- thought to be attributable in part to the infiltration of inflammatory regulates ICAM-1 expression in human myelomonocytic cells, and cells after treatment with IL-18. In the current studies, we show has synergistic effects when combined with IL-12 (47). Our recent that the in vivo exogenous administration of IL-18 after IgG im- data indicate that some, but not all polyclonal rabbit Abs to rat mune complex deposition increases neutrophil recruitment as well chemokines, are protective in the IgG immune complex model of as vascular permeability, a marker of lung injury. Conversely, acute lung injury. For example, in this model Abs to MIP-2, CINC, IL-18 blockade by neutralizing Ab or by IL-18bp reduced evi- MIP-1␣, and MIP-1␤ were protective (46, 48, 49) but not Abs to dence of lung inflammation. These results suggest that the protec- MCP-1 or RANTES (49). In contrast, the same anti-RANTES Ab tion afforded by IL-18 against infectious agents may be related to significantly delayed rejection of allografted rat hearts (50) and the its ability to facilitate recruitment of inflammatory cells to sites of same anti-MCP-1 enhanced the lethal effects of infused LPS in infectious agents. Reduction in inflammatory injury by treatment mice (51). Conversely, infusion of MCP-1 in the same mouse with anti-IL-18 has been observed in endotoxin-induced injury and model was protective (52). Thus, there is a certain specificity of in experimental autoimmune encephalomyelitis (5, 38, 39). IL- these blocking Abs, depending on the inflammatory model under 18bp appears as a soluble decoy receptor for IL-18 and has been study. The current studies identify an in vivo proinflammatory role described as a natural inhibitor for biological activities of IL-18 for IL-18 in the lung inflammatory model used. (40, 41). Our studies suggest that endogenous IL-18 has a role in enhancing the inflammatory response by causing enhanced cyto- Acknowledgments kine and chemokine generation, which is consistent with our We thank Robin Kunkel, Lisa Riggs, and Beverly Schumann for their Downloaded from findings. assistance in the preparation of this manuscript. In vitro studies suggest that IL-18 acts as a proinflammatory cytokine during injury. Mature IL-18 is induced in human periph- References ␣ 1. Tomura, M., X. Y. Zhou, S. Maruo, H. J. Ahn, T. Hamaoka, H. Okamura, eral blood mononuclear cells after exposure to IL-8, MIP-1 , K. Nakanishi, T. Tanimoto, M. Kurimoto, and H. Fujiwara. 1998. A critical role MCP-1, or TNF-␣. Inhibition of TNF-␣ resulted in an 80% reduc- for IL-18 in the proliferation and activation of NK1.1ϩ CD3Ϫ cells. J. Immunol. tion in IL-18 production, suggesting that the primary action of 160:4738. http://www.jimmunol.org/ ␣ 2. Tomura, M., S. Maruo, J. Mu, X. Y. Zhou, H. J. Ahn, T. Hamaoka, H. Okamura, IL-18 is via a TNF- -dependent pathway (42). Previously, we K. Nakanishi, S. Clark, M. Kurimoto, and H. Fujiwara. 1998. Differential capac- have shown that lung inflammation and related events require the ities of CD4ϩ, CD8ϩ, and CD4ϪCD8Ϫ T cell subsets to express IL-18 receptor ␥ production of TNF-␣, MIP-2, and CINC (19). In the current stud- and produce IFN- in response to IL-18. J. Immunol. 160:3759. 3. Arai, K. I., F. Lee, A. Miyajima, S. Miyatake, N. Arai, and T. Yokota. 1990. ies, we have shown the airway instillation of IL-18 together with Cytokines: coordinators of immune and inflammatory responses. Annu. Rev. Bio- deposition of IgG immune complexes enhanced TNF-␣ and IL-1␤ chem. 59:783. 4. Dinarello, C. A. 1999. IL-18: A Th1-inducing, proinflammatory cytokine and levels in BAL fluids. Conversely, the addition of IL-18bp or neu- new member of the IL-1 family. J. Allergy Clin. Immunol. 103:11. tralizing Ab to IL-18 suppressed the increase in BAL levels of 5. Nakamura, K., H. Okamura, M. Wada, K. Nagata, and T. Tamura. 1989. Endo- cytokines after injury. This suggests that IL-18 has an endogenous toxin-induced serum factor that stimulates ␥ interferon production. Infect. Immun.

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