Role of interferon-{gamma} in the evolution of murine bleomycin lung fibrosis

Michael J. Segel,1 Gabriel Izbicki,1 Pazit Y. Cohen,1 Reuven Or,2 Thomas G. Christensen,3 Shulamit B. Wallach-Dayan,1 and Raphael Breuer1,3

1Lung Cellular & Molecular Biology Laboratory, Institute of Pulmonology, 2Bone Marrow Transplantation Department, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel; and 3Mallory Institute of Pathology, Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118

Submitted 14 September 2002 ; accepted in final form 27 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
IFN-{gamma} production is upregulated in lung cells (LC) of bleomycin-treated C57BL/6 mice. The present study characterizes the time course, cellular source, and regulation of IFN-{gamma} expression in bleomycin-induced lung injury. IFN-{gamma} mRNA in LC from bleomycin-treated mice peaked 3 days after intratracheal instillation. IFN-{gamma} protein levels were increased at 6 days, as was the percentage of LC expressing IFN-{gamma}. CD4+, CD8+, and natural killer cells each contributed significantly to IFN-{gamma} production. IL-12 mRNA levels were increased at 1 day in LC of bleomycin-treated mice. Anti-IL-12 and anti-IL-18 antibodies decreased IFN-{gamma} production by these cells. To define the role of endogenous IFN-{gamma} in the evolution of bleomycin lung injury, we compared the effect of bleomycin in mice with a targeted knockout mutation of the IFN-{gamma} gene (IFN-{gamma} knockout) and wild-type mice. At 14 days after intratracheal bleomycin, total bronchoalveolar lavage cell counts and lung hydroxyproline were decreased in IFN-{gamma} knockouts compared with wild-type animals. There was no difference in morphometric parameters of fibrosis. Our data show that enhanced IFN-{gamma} production in the lungs of bleomycin-treated mice is at least partly IL-12 and IL-18 dependent. Absence of IFN-{gamma} in IFN-{gamma} knockout mice does not increase pulmonary fibrosis. Endogenous IFN-{gamma} may play a proinflammatory or profibrotic role in bleomycin-induced lung fibrosis.

interstitial lung disease; transgenic/knockout; cytokines


PULMONARY FIBROSIS IS A COMMON FEATURE of a diverse group of parenchymal lung diseases. In all of these, pathological specimens of lung tissue show a variable combination of inflammatory alveolitis and collagen fibrosis. Interferon-{gamma} (IFN-{gamma}) is a proinflammatory cytokine, primarily in its role as a key activator of macrophages. On the other hand, IFN-{gamma} has inhibitory effects on fibroblast proliferation and collagen production (10, 30). Thus it is difficult to surmise, a priori, what role IFN-{gamma} plays in inflammatory-fibrotic disorders of the lung.

In previous experiments we studied ex vivo cytokine production in mononuclear cells extracted from the lungs of mice with bleomycin-induced lung injury, a well-established model of lung fibrosis. Conditioned media of cells stimulated with concanavalin A (conA) were assayed for IFN-{gamma}, IL-2, IL-4, and IL-5. Of all cytokines studied, the dominant finding was a 20-fold increase in IFN-{gamma} production peaking at 3 days following bleomycin instillation (15).

In this study, we characterize the time course, cellular source, and regulation of increased IFN-{gamma} expression in bleomycin-induced lung injury. To determine the role of endogenous IFN-{gamma} in the pathogenesis of bleomycin-induced lung injury, we examined the response to intratracheal (IT) bleomycin instillation in C57BL/6 mice with a targeted knockout mutation of the IFN-{gamma} gene (IFN-{gamma} KO).


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals

Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME), 11-12 wk old, weighing 25-30 g, were used.

For in vivo studies of lung injury, IFN-{gamma} KO (C57BL/6JIFN{gamma}tm1Ts) were used in addition to the wild-type animals (Wild). The IFN-{gamma} KO mutation was confirmed at the mRNA level. Total RNA was extracted from conA-stimulated splenocytes extracted from mice of the Wild and IFN-{gamma} KO groups. Equal quantities of RNA were reverse transcribed to cDNA, and aliquots were amplified by PCR using primers for IFN-{gamma} over 34 cycles and for GAPDH over 30 cycles. The primers used were the same as those used for semiquantitative RTPCR as described below. A signal for GAPDH cDNA (903 bp) was obtained in all samples. A signal for IFN-{gamma} cDNA (365 bp) was obtained in samples from Wild but not IFN-{gamma} KO animals.

All procedures involving animals were approved by the Hebrew University-Hadassah Medical School committee of animal care. Mice were housed in a specific pathogen-free environment in plastic cages on hardwood shavings. A 12:12-h light/dark cycle was maintained, and mice had access to water and rodent laboratory chow ad libitum. Mice were acclimated to these conditions at least 1 wk before experiments commenced.

IT Instillation

Mice were anesthetized by injection of 0.05-0.07 ml of 40 mg/ml Ketalar ip (Parke-Davis, Pontypool, Gwent, UK) and 0.5 mg/ml Droperidol (Janssen Pharmaceutica, Beerse, Belgium). The skin and subcutaneous tissues overlying the proximal portion of the trachea were exposed by a 5-mm transverse incision to allow for direct external visualization of the trachea. A metal cannula fitted to a tuberculin syringe was carefully passed into the trachea. A dose of 0.06-0.08 units of bleomycin (H. Lundbeck, Copenhagen, Denmark) dissolved in 0.1 ml of saline solution, or 0.1 ml of saline alone was slowly injected.

Lung Cell Isolation and Culture

Mice were killed with a lethal dose of pentobarbital 1, 3, 6, 10, and 14 days after IT instillation. The abdominal aorta was transected, and the animal was exsanguinated. To eliminate airway and alveolar space cells, we perfused the lungs with normal saline through the right ventricle and performed bronchoalveolar lavage (BAL). A polyethylene cannula (PE 205; Clay Adams, Parsippany, NJ) was placed in the trachea, and eight aliquots of 0.5 ml of normal saline were slowly injected and withdrawn.

Lung cells (LC) were extracted as follows: lungs were removed, minced, and incubated (37°C, 5% CO2 air) for 45 min in PBS containing 1 mg/ml collagenase (Sigma, C0130). After enzyme treatment, lung tissue was gently passed through a cell dissociation sieve (Sigma), then washed twice in PBS. For cell culture experiments, cells were resuspended in culture medium (RPMI 1640 supplemented with 30% FCS, 100 µg/ml penicillin, 100 mg/ml streptomycin, 25 mM HEPES buffer, and 2 mM L-glutamine).

RNA Isolation

Total cellular RNA was isolated from cell pellets using Tri Reagent (Sigma, T9424) supplemented by 1 µg/ml of glycogen, according to the protocol supplied by the manufacturer. To assess quality of the RNA, an aliquot of each sample was analyzed by 1% agarose gel electrophoresis for integrity of the RNA, and to verify the absence of a high-molecular-weight band representing contamination with genomic DNA. RNA was also analyzed by spectrophotometer for assessment of purity (A260:A280 >1.8) and quantification.

RT-PCR

RNA was reverse transcribed to cDNA using an avian myeloblastosis virus-RT-based protocol and random primers as well as poly(dT) (Reverse Transcription System; Promega, Madison, WI). One microgram of each sample was uniformly used for reverse transcription.

Semiquantitative RT-PCR

Primers for PCR are detailed in Table 1. Taq DNA polymerase (Promega) was added to each tube after a hot start of 10 min at 95°C. This was followed by 28-34 cycles of denaturation (45 s at 94°C), annealing (45 s at 60°C), and extension (2 min at 72°C), followed by a final extension step of 7 min. The number of cycles used (32 for IFN-{gamma}, 34 for IL-12 and IL-18, and 28 for GAPDH) was predetermined to be the greatest number of cycles in which amplification was within the linear range. PCR products were analyzed by electrophoresis on a 1% agarose gel stained with ethidium-bromide, and the intensity of the fluorescent signal emitted by the PCR products was determined by densitometry (Fluor-S-Multiimager, Bio-Rad). We determined relative cDNA concentration by comparing the results for samples with those of external standards (1:2 serial dilutions of a reference cDNA). Each cDNA was amplified in triplicate, and the median of the three values was used. Amplification was repeated with a smaller quantity of substrate if the densitometric signal was beyond the predetermined linear range.


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Table 1. Primers for PCR

 

Flow Cytometry

LC IFN-{gamma} production was assessed by indirect immunofluorescence of intracellular cytokine and analyzed by flow cytometry. Cytokine secretion was blocked by incubating cells (37°C, 4 h) in culture medium (2 x 106 cells/2 ml) containing 2 µM monensin. After two washes in cold PBS, cells were fixed in 4% paraformaldehyde preheated to 37°C for 10 min at room temperature. Cells were rendered permeable by suspension in saponin buffer (0.5 x 106 cells/0.1 ml 1% saponin in PBS containing 1% BSA and 1 mM HEPES), pelleted, resuspended in saponin buffer, and incubated overnight (4°C) with 0.5 µg/0.1 ml of FITC-conjugated anti-mouse IFN-{gamma} MAb (18114A; Pharmingen, San Diego, CA) and 0.2 µg of R-phycoerythrin-conjugated MAbs for cell surface markers: anti-mouse natural killer (NK) 1.1 (cat. no. 01295A0; Pharmingen), anti-CD45/B220 (cat. no. 01125A; Pharmingen), anti-CD4 (Pharmingen, 09425A), or anti-CD8 (Serotec, MCA609PE). Before staining with anti-CD4 or anti-B220, nonspecific binding was blocked by treating cells with anti-CD16/CD32 MAb (cat. no. 01241D; Pharmingen). After double-staining, cells were washed twice with FACS buffer (3% FCS in PBS) and analyzed by flow cytometry (FACStar; Becton-Dickinson, Mountain View, CA). Lymphocytes were gated to analyze cell surface markers.

ELISA

We assayed conditioned media for IFN-{gamma} by ELISA using rat anti-mouse IFN-{gamma} MAbs. Purified R4-6A2 was used as the capture antibody and biotinylated XMG1.2 as the detection antibody, using the cytokine ELISA protocol provided by the manufacturer (70-CK-ELISA protocol, rev. 040897; Pharmingen).

Lung extracts were also assayed for IFN-{gamma} by ELISA (2). Mice were killed for study 3, 6, 14, and 21 days after IT instillation. The left lung was removed, weighed, and immersed in 0.1 ml ELISA buffer/10 mg tissue. Tissue was homogenized with a Polytron for 20 s. The homogenate was centrifuged (10 min, 14,000 rpm, 4°C), and supernatant was removed and assayed by ELISA as above.

Regulation of IFN-{gamma} Production

To study the regulation of IFN-{gamma} production, we extracted LC 3 days after IT instillation of bleomycin. This time point was chosen based on previous studies of similar design (15). Cells were cultured with neutralizing antibodies as follows: rat IgG2a anti-murine IL-12 (p40/p70) or isotype-matched control antibody (Pharmingen), and rabbit anti-murine IL-18 or control rabbit immunoglobulin (Cytolab). IFN-{gamma} levels in conditioned media were studied by ELISA.

In Vivo Studies of Bleomycin Lung Injury in IFN-{gamma} KO Mice

All animals received IT instillations of either bleomycin or saline on day 0. There were four experimental groups: 1) Wild IT bleomycin, n = 7; 2) Wild IT saline, n = 12; 3) IFN-{gamma} KO IT bleomycin, n = 10; and 4) IFN-{gamma} KO IT saline, n = 12. Animals were killed for study 14 days following IT instillation. Lung injury was evaluated by analysis of BAL cells, lung hydroxyproline measurement, and quantitative morphological studies as previously described (4, 19, 23).

BAL. BAL was carried out by injection and withdrawal of eight aliquots of 0.5 cc of Ca2+/Mg2+-free PBS. Total cell count was estimated by a hemocytometer and cell viability by trypan blue exclusion. We performed differential cell count on 200 cells using cytospin slides stained with Diff-Quik (Baxter).

Lung hydroxyproline content. After BAL and thoracotomy, the right main bronchus was ligated and cut distally to the suture, and the lung was dissected free of extraneous tissues and hydrolyzed in 6 N HCl for 24 h at 106°C. An aliquot was analyzed on an amino acid analyzer (Beckman 6300). Hydroxyproline results are expressed as nanomoles per lung.

Morphological examination. After removal of the right lung, the left lung was fixed by IT infusion with 4% formalin-1% glutaraldehyde-0.1 M cacodylate buffer (pH 7.4) maintained at 25 cm of hydrostatic pressure for 15 min. The trachea was ligated, and both heart and lung were removed en bloc and immersed in fixative for an additional 24 h. Three 0.2-cm-thick transverse sections of the left lung were embedded in paraffin. Paraffin tissue blocks were cut to provide 4- to 6-µm sections. The sections were stained with hematoxylin-eosin and modified Masson's trichrome stains to be used for morphological assessment of the inflammatory-fibrotic injury.

Quantitative image analysis. Pathological assessment of the degree of fibrotic lung injury was performed by computer-assisted morphometry, using the Optimas image analysis computer program (Optimas, Bothell, WA) as previously described (4, 19, 23). We quantified the degree of fibrosis by analyzing slides that were stained with a modified trichrome stain, to enhance the blue-stained collagen. By adjusting image contrast, brightness, and color threshold settings, we configured the image analysis program to detect areas of blue-stained collagen within each of 20 randomly selected fields per slide via a x40 objective lens. The percentage area of blue-stained collagen (fibrosis fraction) for each field, a constant 135 x 95 µm, was averaged for each animal.

Statistical Analysis

Comparison of IFN-{gamma} mRNA and protein levels between treatment and control groups was done by the nonparametric Mann-Whitney test. Paired values were compared by the Wilcoxon rank-sum test.

To compare the effect of bleomycin between the different strains of mice, z-scores [= (Xi - mean saline)/SD saline] (18) were calculated for the measured parameters in the Wild IT bleomycin and IFN-{gamma} KO IT bleomycin groups, so as to normalize the data of these groups to the corresponding saline-treated group. Z-scores were compared by the Mann-Whitney test.

Significance was attributed to probability values <0.05.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Increased IFN-{gamma} Production

We first sought to establish that the increased IFN-{gamma} expression in response to IT bleomycin that we described in previous work (15) is a bona fide phenomenon, rather than an artifact of ex vivo manipulation. IFN-{gamma} mRNA levels in LC extracted at different times after IT instillation of bleomycin and control saline are shown in Fig. 1A. In LC from bleomycin-treated animals, the IFN-{gamma} mRNA was increased 1 and 3 days post-IT (P < 0.05) by as much as threefold. By day 6 post-IT, IFN-{gamma} mRNA had returned to control levels.



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Fig. 1. IFN-{gamma} expression at different times after intratracheal (IT) instillation of bleomycin ({bullet}) or saline ({circ}). Each point represents a single animal. Median values are denoted by a horizontal bar. A: IFN-{gamma} mRNA levels, normalized to GAPDH mRNA, in lung cells (LC). Values are the ratio of IFN-{gamma} to GAPDH mRNA. B: IFN-{gamma} levels in lung tissue extract as measured by ELISA. C: scatter plots from flow cytometry of LC after staining intracellular IFN-{gamma} with FITC-labeled anti-IFN-{gamma} MAb. The percentage of IFN-{gamma}-positive cells (right lower quadrant) is noted in the upper right corner of each plot. Data shown are from 1 of 2 duplicate experiments. *P < 0.05 for bleomycin vs. saline.

 

Increased IFN-{gamma} mRNA levels have been described without concomitant increase in protein (33). We therefore proceeded to measure tissue IFN-{gamma} protein levels (Fig. 1B). Here the increased IFN-{gamma} level in bleomycin-treated animals was significant only 6 days post-IT. The percentage of LC that stained for intracellular IFN-{gamma}, as assessed by flow cytometry (Fig. 1C), was also increased 6 days post-IT bleomycin to almost 9% of the total LC population vs. only 1.5% of LC extracted from control mice (P < 0.05). On day 3 post-IT there was a nonsignificant trend towards increased tissue levels and percentage of IFN-{gamma}-producing LC.

Cellular Source of IFN-{gamma}

The cellular source of IFN-{gamma} was studied on day 6 post-IT bleomycin, when the number of IFN-{gamma}-producing cells peaked (Table 2). The majority (54%) of the IFN-{gamma}-producing cells were CD4+. In addition, 27% were NK1.1+, 11% CD8+, and 5% B220+ (Fig. 2). The CD4/CD8 ratio among the IFN-{gamma}-producing LC was 4.9.


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Table 2. Cellular source of IFN-{gamma}

 


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Fig. 2. Cellular source of IFN-{gamma}. LC extracted 6 days after IT bleomycin were double-stained for intracellular IFN-{gamma} and cell surface markers and analyzed by flow cytometry (Table 2). Median values of n = 5 (1-3 animals each) are represented.

 

Regulation of IFN-{gamma}

To determine the intercellular signals responsible for the observed increase in IFN-{gamma}, we studied LC mRNA levels of IFN-{gamma}-inducing cytokines IL-12 and IL-18. IL-12 levels were increased almost twofold on day 1, but not at later times studied (Fig. 3A). When cells from bleomycin-treated mice were cultured ex vivo in the presence of neutralizing anti-IL-12 antibody (Fig. 3B), IFN-{gamma} levels in conditioned media were significantly decreased compared with control cells (P < 0.05).



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Fig. 3. A: IL-12 mRNA levels, normalized to GAPDH mRNA, in LC from mice killed at different times after IT instillation of bleomycin ({bullet}) or saline ({circ}). Values are the ratio of IL-12/GAPDH mRNA. Each point represents a single animal. Median values are denoted by a horizontal bar. *P < 0.05 for bleomycin vs. saline. B: IFN-{gamma} levels as measured by ELISA, in conditioned media of LC extracted 3 days after IT bleomycin. Paired values are shown for wells cultured for 24 h with neutralizing anti-IL-12 or isotype-matched control MAbs (40 µg/ml). P < 0.05.

 

IL-18 mRNA levels in cells from the lungs of bleomycin-treated mice were no different from controls (data not shown). Nevertheless, IFN-{gamma} production by cultured LC was decreased when the cells were treated with neutralizing anti-IL-18 antibody (Fig. 4).



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Fig. 4. IFN-{gamma} levels as measured by ELISA in conditioned media of LC extracted 3 days after IT bleomycin. Paired values are shown for wells cultured for 24 h with neutralizing anti-IL-18 or control antibodies (5 µg/ml).

 

Bleomycin Lung Injury in IFN-{gamma} KO Mice

To determine the role of endogenously expressed IFN-{gamma}, we compared the response to IT instillation of bleomycin in IFN-{gamma} KO mice on a C57BL/6 background with Wild C57BL/6 mice. The results obtained in the bleomycin-treated groups were normalized to the corresponding saline-treated control groups. Mean z-score values for BAL total cell count and lung hydroxyproline levels were significantly lower (P < 0.05) in bleomycin-treated IFN-{gamma} KO compared with Wild mice (Fig. 5). There was no significance difference between the two groups in the morphometric fibrosis fraction. There were also no significant differences in the differential counts of the BAL cells (not shown).



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Fig. 5. Z-score values of bronchoalveolar lavage (BAL)-total cell count, hydroxyproline, and fibrosis fraction in bleomycin-treated wild-type (solid bars) and IFN-{gamma} knockout mice (open bars) normalized to respective saline-treated groups. Data are presented as means ± SD. *P < 0.05, IFN-{gamma} knockout compared with wild type.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
IFN-{gamma} plays a complex role within networks of cytokines regulating fibroblast activity. IFN-{gamma} inhibits proliferation (10) and collagen production (30, 32) by cultured fibroblasts but enhances fibronectin production (32). IFN-{gamma} eliminates collagen synthesis induced by TNF-{alpha} and IL-1 (9), IL-4 (26), and transforming growth factor-{beta} (32).

IFN-{gamma} also has an important role in regulating inflammation. IFN-{gamma} induces major histocompatibility complex class I and II antigens (28) and Fc-{gamma} receptor on macrophages (16) and ICAM-1 and VCAM-1 on fibroblasts (29). IFN-{gamma} also regulates production of key inflammatory mediators such as nitric oxide (21), TNF-{alpha}, IL-1, and IL-6 (20).

IFN-{gamma} also has several effects that might play a role at the interface between inflammation and fibrosis in bleomycin-induced lung injury. For example, alveolar macrophages stimulated with IFN-{gamma} express PDGF, a potent mediator of fibroblast proliferation and chemotaxis (3). Furthermore, IFN-{gamma} greatly enhances Fas-mediated apoptosis of lung epithelial cells (8). Alveolar epithelial cell apoptosis appears to play an important role in the pathogenesis of pulmonary fibrosis (7).

Thus on the basis of evidence at the cellular level, it appears that, whereas the direct effect of IFN-{gamma} on fibrosis is suppressive, it may have a more complex overall influence through effects on inflammation and tissue responses to injury.

In this study we confirm and expand upon previous work showing that IFN-{gamma} production is increased in bleomycin lung injury. We (15) and others (27) found that conditioned media of mononuclear cells extracted from the lungs of bleomycin-treated mice and stimulated with conA contain more IFN-{gamma} than media of cells from control mice. It has also been shown that IFN-{gamma} mRNA levels are increased in cells from lungs of bleomycin-treated mice (17). The data presented here confirm, in vivo, that the number of IFN-{gamma}-producing cells, as well as the concentration of the IFN-{gamma} in lung tissue, is increased in the early inflammatory stage of the injury cascade triggered by IT instillation of bleomycin. This increase in IFN-{gamma} production can be ascribed, at least in part, to increased IFN-{gamma} gene expression, as shown by the increase in mRNA levels.

IFN-{gamma} is a pleiotropic cytokine expressed mainly by T lymphocytes and NK cells. An earlier study found that IFN-{gamma} expression is increased in bleomycin-treated SCID mice (17). The authors postulated that the source of the IFN-{gamma} in this system must therefore be NK cells. Our results show that both CD4 lymphocytes and NK cells contribute to the increase in IFN-{gamma} expression. These two cell types account for >80% of IFN-{gamma}-producing cells. Interestingly, we found that B lymphocytes also have a small contribution to the IFN-{gamma}-producing cell population. It has been shown that stimulation with IL-12 and IL-18 can induce IFN-{gamma} production by B cells in vivo and in vitro (34). IFN-{gamma} production by B lymphocytes has also been demonstrated in human tonsils (1) and in mice infected with Borrelia burgdorferi (13). IFN-{gamma} expression appears to be limited to immature B cells (1, 12) and has autocrine effects on cell migration (12).

Studies (25) using cultured lymphocytes have implicated IL-12 as a key inducer of IFN-{gamma} expression. IL-18 synergizes with IL-12 to produce much greater IFN-{gamma} production than either of the two cytokines alone (25). We investigated what regulates the increased IFN-{gamma} production observed in vivo in bleomycin lung injury. Our results show increased IL-12, but not IL-18, mRNA in LC from bleomycin-treated mice, just before the peak increase in IFN-{gamma}. The time course of the increase in IL-12 and IFN-{gamma} mRNA is consistent with induction of IFN-{gamma} by IL-12. We confirmed this hypothesis by showing that LC cultured ex vivo with anti-IL-12 antibody produce significantly less IFN-{gamma} than untreated control cells.

Although we did not find increased IL-18 gene expression, our studies using anti-IL-18 antibodies suggest IL-18 may nevertheless play a role in inducing IFN-{gamma} in this system. Unlike IL-12, IL-18 is expressed constitutively by various types of cells, including dendritic cells (31) and lung epithelium (5). It is possible that constitutively expressed IL-18 synergizes with IL-12 in this system as well. An analogous situation was observed in cultured murine splenocytes stimulated only with IL-12, in which anti-IL-18 antibody strongly reduced their IFN-{gamma} production (11).

Among the myriad functions of IFN-{gamma} are many that, a priori, might affect the complex cascade of inflammation and fibrosis that occurs in the lung as a result of injury by bleomycin, making it difficult to predict its role in fibrotic lung disorders. We have previously demonstrated that IFN-{gamma} production by pulmonary mononuclear cells in bleomycin-treated mice is increased in the bleomycin-sensitive C57BL/6 strain, but not in "resistant" BALB/c mice (15). However, the administration of IFN-{gamma} to bleomycin-treated animals can ameliorate the fibrosis (14). Moreover, in a preliminary clinical trial, treatment with IFN-{gamma} was beneficial for patients suffering from idiopathic pulmonary fibrosis (35).

To clarify the role of endogenous IFN-{gamma} in the pathogenesis of bleomycin lung injury, we studied the effect of bleomycin in IFN-{gamma}-deficient mice. Surprisingly, lung hydroxyproline levels and BAL total cell count were significantly lower in bleomycin-treated IFN-{gamma} KO mice compared with Wild mice. There was no difference in the morphometric fibrosis fraction. These results imply that endogenous IFN-{gamma} is clearly not protective in the bleomycin-induced lung fibrosis model and even suggest the possibility that IFN-{gamma} plays a profibrotic role. Our findings confirm and expand upon work published recently (6) that demonstrated significantly less inflammation 10 days after IT instillation of bleomycin in IFN-{gamma} KO mice compared with Wild controls. Results from a separate small-scale experiment at 21 days postbleomycin suggests that lung collagen is reduced.

What is the reason for the apparent discrepancy between the experiments that showed a beneficial effect of IFN-{gamma} in bleomycin lung injury and our findings and those of others (6) that IFN-{gamma} produced within the lung seems to be part and parcel of the pathogenesis of the process? The issue is further exemplified by the paradoxical findings that administration of both IL-12 (22) and neutralizing antibodies against IL-12 (24) ameliorates bleomycin lung injury. Moreover, the beneficial effect of IL-12 was neutralized by the administration of anti-IFN-{gamma} antibodies, whereas mice treated with anti-IL-12 antibodies produced higher levels of IFN-{gamma} than control animals.

We see the explanation rooted in the minutiae of cellular responses to IFN-{gamma}. As discussed above, IFN-{gamma} has myriad effects on cells present in the lung that potentially have diverse effects on inflammation and fibrosis. The overall effect of IFN-{gamma} is therefore dependent on the location, kinetics, and magnitude of IFN-{gamma} activity, as well as the cytokine milieu that forms the context of the phenomenon.

The role of IFN-{gamma} in the pathogenesis of lung fibrosis may be more complex than once thought. A simplistic model would be that IFN-{gamma} enhances chronic lung inflammation and, in contrast, depresses components of fibrosis. The end result depends on the balance between these opposite effects in each specific pathological situation. Detailed characterization of the effects of IFN-{gamma} in this system may help refine cytokine therapy of fibrotic lung diseases.


    DISCLOSURES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported in part by the David Shainberg Fund, the Israel Lung Association-Tel-Aviv, the Chief Scientist's Office of the Ministry of Health (Israel), and by National Heart, Lung, and Blood Institute Grant P50-HL-56386. G. Izbicki is a recipient of grants from the Swiss National Science Foundation (Fellowship 81GE-050068) and the Swiss Foundation for Medical and Biological Grants.


    ACKNOWLEDGMENTS
 
We thank Dr. H. Lorberboum-Galski for technical advice and constructive criticism of the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Breuer, Inst. of Pulmonology, Hadassah Univ. Hospital, POB 12000, Jerusalem 91120, Israel (E-mail: raffi{at}hadassah.org.il).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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