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,1
* Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Messina, Italy;
Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Centro Neurolesi "Bonino-Pulejo," Messina, Italy;
Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, Tossicology and Chemioterapy, University of Perugia, and Polo Scientifico e Didattico di Terni, Terni, Italy; and
Department of Experimental Pharmacology, University of Naples "Federico II," Naples, Italy
2Correspondence: Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria, Gazzi, 98100 Messina Italy. E-mail: salvator{at}unime.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interleukin (IL)-1ß production; v) nitrotyrosine formation; and vi) NF-
B activation. The cotreatment of GITR+/+ mice with Fc-GITR fusion protein (6.25 µg/mouse) also significantly attenuated all of the above indicators of lung damage and inflammation. Our results clearly demonstrate that GITR-GITRL interaction plays an important role in the chronic lung injury induced by bleomycin in the mice.—Cuzzocrea, S., Ronchetti, S., Genovese, T., Mazzon, E., Agostini, M., Di Paola, R., Esposito, E., Muià, C., Nocentini, G., Riccardi, C. Genetic and pharmacological inhibition of GITR-GITRL interaction reduces chronic lung injury induced by bleomycin instillation.
Key Words: TNFR superfamily • mice • inflammation • fusion protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. While the pathogenesis is incompletely understood, a growing body of evidence suggests two different pathogenic routes for developing pulmonary fibrosis. The inflammatory pathway, where a shift to the so-called T-helper (Th) 2 type cytokine network is critical, and the epithelial pathway represented by idiopathic pulmonary fibrosis, by far the most aggressive interstitial lung diseases. Both routes may trigger a number of cytokines/growth factors inducing fibroblast migration/proliferation and phenotype change to myofibroblasts, with a consequent accumulation of extracellular matrix (ECM) (2)
.
The common pathological features in interstitial lung diseases include the fibrosis of the interstitium involving collagen, elastic, and smooth muscle elements, architectural remodeling of the interstitium, and chronic inflammation of the interstitium (i.e., variable increases in lymphocytes, plasma cells, macrophages, eosinophils, and mast cells), hyperplasia of type II cells and hyperplasia of endothelial cells (1)
.
Bleomycin is a drug widely used as an antineoplastic agent in the treatment of the germ-line tumors and Hodgkin lymphoma. An estimated 3–5% of patients develop a dose-dependent interstitial pulmonary fibrosis (3)
. For this reason, intratracheal instillation of the antitumor agent bleomycin is the most commonly used animal model for pulmonary fibrosis (4)
. This model is characterized by an early inflammatory response (predominantly neutrophilic), increased fibroblast proliferation, and enhanced collagen deposition due to increased collagen synthesis and decreased collagen degradation (5)
. In addition, it has been shown that fibroproliferative activity coexists with inflammation and that the major proliferative phase occurs during the first week after bleomycin-induced injury (6)
. Inflammation is a major component in the pathogenesis of interstitial lung disease that is orchestrated in part by endogenous and migrating leukocytes. These leukocytes, together with lung epithelial and endothelial cells, produce a feedback circle where stimuli from injury responses can activate alveolar and interstitial macrophages. Activated leukocytes release additional reactive oxygen and nitrogen species and proteases that sustain the injury/repair processes that are considered to contribute to the fibrotic processes (7)
.
The glucocorticoid-induced TNFR-related protein (GITR) is a receptor belonging to the TNFR superfamily (TNFRSF) (8)
. GITR is expressed in normal T lymphocytes, up-regulated on T cell activation and constitutively expressed at high levels in CD4+CD25+ regulatory T (Treg) cells (8
9
10
11
12)
. When activated, GITR costimulates effector T lymphocytes and negatively modulates Treg cell suppressor activity (9
10
11
, 13)
. Consequently, in GITR-deficient mice (GITR–/–), the response to TCR triggering of T lymphocytes is abnormal (14)
. GITR expression has also been shown on nonlymphoid cells, such as macrophages and neutrophils [polymorphonuclear leukocytes (PMNs); (10
, 15)
]. GITR is activated by its ligand (GITRL), which is expressed in antigen presenting cells (APC), including macrophages, and in endothelial cells (12
, 16
17
18
19)
. Following GITR-GITRL interaction, GITRL also delivers signals to APC (20
21
22)
. In vivo studies suggest that GITR triggering exacerbates autoimmune/inflammatory responses and potentates antiviral and antitumoral immunity (12
, 23
24
25)
. Recently, we have demonstrated that GITR modulates the acute inflammatory response (26
, 27)
.
To investigate whether the GITR-GITRL system plays a role also in the induction of chronic inflammation, we studied the rodent model of bleomycin-induced lung injury. In particular, we demonstrate that genetic or pharmacological inhibition of GITR-GITRL interaction decreased the bleomycin-induced lung injury as investigated through i) infiltration of the lung with polymorphonuclear neutrophils (MPO activity); ii) edema formation; iii) histological evidence of lung injury; iv) tumor necrosis factor (TNF-) and IL-1ß (interleukin-1ß) production; v) nitrotyrosine formation; and vi) NF-B activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Animal care was in compliance with regulations in Italy (D.M. 116192), Europe (O.J. of E.C. L 358/1 12/18/1986), and the United States (Animal Welfare Assurance No A5594–01, Department of Health and Human Services).
Induction of lung injury by bleomycin
Mice received a single intratracheal instillation of saline (0.9%) or saline containing bleomycin sulfate (1 mg/kg body wt) in a volume of 50 µl and were killed after 7 d by pentobarbitone overdose.
Miniosmotic pumps and implantation
Alzet pumps are precision drug administration tools that were used to deliver fusion proteins at a constant rate. In particular, we used Alzet Model 2004 miniosmotic pumps (Charles River, Milan, Italy), placed 3 h after the administration of bleomycin. The Alzet miniosmotic pump was implanted subcutaneously (s.c.) in a mouse. A small incision was made in the skin between the scapulae. Using a hemostat, a small pocket was formed by spreading the s.c. connective tissues apart. The pump was inserted into the pocket with the flow moderator pointing away from the incision. The skin incision was closed with sutures. The pumping rate was 1 µl/h (±0.15 µl/h), and the reservoir vol was 200 µl.
Experimental groups
Mice were randomly allocated into the following groups:
i) bleomycin-treated GITR+/+ group: mice were subjected to intratracheal bleomycin instillation and given saline by miniosmotic pump; ii) bleomycin-treated GITR–/– group: the treatment was identical to that of bleomycin-treated GITR+/+ group; iii) control GITR+/+ group (sham): GITR+/+ mice were subjected to intratracheal saline instillation and received saline by miniosmotic pump; iv) control GITR–/– group (sham): the treatment was identical to that of control GITR+/+ group; v) bleomycin-Fc-GITR-cotreated GITR+/+ group: GITR+/+mice were subjected to intratracheal bleomycin instillation and received Fc-GITR (6.25 µg/mouse) by miniosmotic pump. Fc-GITR was purchased from Alexis and is a dimer of a fusion protein formed by the extracellular domain of GITR and the Fc portion of human IgG1; vi) bleomycin-Fc-cotreated GITR+/+ group: GITR+/+ mice were subjected to intratracheal bleomycin instillation and received Fc fusion protein (6.25 µg/mouse) by miniosmotic pump. Fc was purchased from Alexis and is a dimer of a fusion protein formed by the Fc portion of human IgG1; vii) control Fc-GITR-treated GITR+/+ group (sham+Fc-GITR): identical to control GITR+/+ group, except for the administration of Fc-GITR (6.25 µg mouse) by miniosmotic pump; viii) control Fc-treated GITR+/+ group (sham+Fc): identical to control GITR+/+, except for the administration of Fc fusion protein (6.25 µg mouse) by miniosmotic pump.
In the present study, mice (n=10/group) were sacrificed after 7 d for analyses of injury, inflammation, changes in body wt, and survival rate over the experimental period.
Measurement of fluid content in lung
The wet lung wt was measured 7 d after injection of bleomycin by careful excision of the lung from other adjacent extraneous tissues. The lung was exposed for 48 h at 180°C, and the dry wt was measured. Water content was calculated by subtracting dry wt from wet wt.
Bronchoalveolar lavage (BAL)
Seven days after bleomycin or saline solution instillation, mice were euthanized and the trachea was immediately cannulated with an I.V. polyethylene catheter (Neo Delta Ven 2, delta Med, Viadana, Italy) equipped with a 24-gauge needle on a 1 ml syringe. Lungs were lavaged once with 0.5 ml D-phosphate buffer saline (D-PBS, GIBCO, Paisley, U.K.). In >95% of the mice, the recovery vol was over 0.4 ml. The BAL fluid was spun at 800 rpm, the supernatant was removed, and the pelleted cells were collected. Total BAL cells were enumerated by counting on a hemocytometer in the presence of trypan blue.
Histological examination
Lung biopsies were taken 7 d after instillation of bleomycin. They were fixed for 1 wk in 10% (w/v) PBS-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ, USA). After embedding in paraffin, the sections were prepared and stained by trichrome stain. All sections were studied using light microscopy (Dialux 22 Leitz, Ziess, Milan, Italy). The severity of fibrosis was semiquantitatively assessed according to the method proposed by Ashcroft and coworkers (28)
. Briefly, the grade of lung fibrosis was scored on a scale from 0 to 8 by examining randomly chosen sections, with fields per sample at a magnification of x100. Criteria for grading lung fibrosis were as follows: grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening of walls without obvious damage to lung architecture; grade 5, increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses; grade 7, severe distortion of structure and large fibrous areas; grade 8, total fibrous obliteration of fields.
Immunohistochemical localization of TNF-, IL-1ß, MPO, and nitrotyrosine
Lungs were taken 7 d after instillation of bleomycin and treated as previously reported (29)
. Briefly, they were fixed in 10% (w/v) PBS-buffered formaldehyde and 8 µm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were incubated overnight with anti-TNF- polyclonal antibody (pAb) (1:500 in PBS, v/v), or with anti-interleukin-1ß pAb (1:500 in PBS, v/v), or with antinitrotyrosine pAb (1:500 in PBS, v/v), or with anti-MPO pAb (1:500 in PBS, v/v). Sections were washed with PBS and incubated with secondary antibody (Ab). Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA, Milan, Italy).
Preparation of protein extracts from whole lung
All the extraction procedures were performed on ice using ice-cold reagents as previously reported (26)
. Briefly, lung tissues from each mouse were suspended in extraction buffer (0.32 M sucrose; 10 mM TRIS-HCl, pH 7.4; 1 mM ethylenebis(oxyethylenenitrilo) tetra-; ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA); 2 mM ethylenediaminetetraacetic acid (EDTA); 5 mM NaN3; 10 mM 2-mercaptoethanol; 50 mM NaF; 0.2 mM phenylmethylsulphonylfluoride (PMSF); 0,15 µg/ml pepstatin A; 20 µM leupeptin; 1 mM sodium orthovanadate) and homogenized at the highest setting for 2 min in a Polytron PT 3000 tissue homogenizer. The homogenates were chilled on ice for 15 min and then centrifuged (10 min, 1000 g f 4°C). The pellets were suspended in the supplied complete lysis buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM TRIS-HCl pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM PMSF, 20 µM, and 0.2 mM sodium orthovanadate and then centrifuged (30 min, 15,000 g, 4°C) to yield the nuclear fraction. Protein concentration in the supernatant was determined by the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), and stored at –80°C.
Western blot analysis for I B-, phospho-NF-B p65 (serine 536), NF-B p65
The levels of IB- and phospho-NF-B p65 (serine 536) were quantified in citosolic fraction from lungs collected at 7 d after bleomycin instillation by Western blot analysis. Proteins were transferred onto nitrocellulose membranes, according to the manufacturer’s instructions. Briefly, the membranes were saturated by incubation at room temperature for 1 h with 10% (w/v) nonfat dry milk in PBS and then incubated overnight at 4°C with anti-IB- (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti phospho-NF-B p65 (serine 536) (1:1000, Cell Signaling Technology, Boston, MA, USA). Nuclear fractions were incubated with anti-NF-B p65 (1:500; Santa Cruz Biotechnology). Membranes were washed three times with 1% (w/v) Tween 20 in PBS and then incubated with peroxidase-conjugated bovine anti-mouse IgG secondary Ab or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. The immune complexes were visualized using the SuperSignal West Pico chemiluminescence substrate (Pierce, Milan, Italy).
To ascertain that blots were loaded with equal amounts of protein lysates, we incubated the blots in the presence of the Ab against ß-tubulin protein (1:10,000 Sigma-Aldrich Corp.). Subsequently, the relative expression of the proteins was quantified by densitometry scanning of the X-ray films with GS-700 Imaging Densitometer (GS-700, Bio-Rad Laboratories, Milan, Italy) and a computer program (Molecular Analyst, IBM).
Myeloperoxidase activity
Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as described previously (30)
. MPO activity was defined as the quantity of enzyme degrading 1 µmol of peroxide/min at 37°C and was expressed in units per gram of wet tissue.
Measurement of cytokines
Portions of lung, collected at 7 d after bleomycin administration, were homogenized in PBS containing 2 mmol/L of phenyl-methyl sulfonyl fluoride (Sigma Chemical Co., Milan, Italy), and tissue levels of TNF- and IL-1ß were evaluated. The assay was performed by using a colorimetric, commercial kit (Calbiochem-Novabiochem Corporation, San Diego, CA, USA) according to the manufacturer instructions. All cytokines determinations were performed in duplicate serial dilutions.
Materials
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd. All other chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; J.T. Baker, Deventer, Holland).
Analysis
All values in the figures and text are expressed as mean ± SEM (SE) of N observations. For the in vivo studies N represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. Data sets were examined by one- or two-way ANOVA, and individual group means were then compared with Student’s unpaired t test. A P value of less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). GITR–/– mice that underwent to bleomycin tracheal instillation showed a lower increase of BAL cellularity as compared to bleomycin GITR+/+ mice (Fig. 1)
. The above presented results demonstrate that GITR participates in the lung inflammatory response to bleomycin.
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Histological examination of lung sections from bleomycin-treated GITR+/+ mice demonstrated an important lung injury characterized by extensive inflammatory cell infiltration (Fig. 2
B, 3a, Bb, F). Furthermore, bleomycin instillation elicited an inflammatory response characterized by the accumulation of water in lung tissue, indicating formation of edema (Fig. 3
). The absence of GITR gene significantly reduced the extent and severity of the histological signs of lung injury (Fig. 2C, Ca, F
), as well as the accumulation of water in the lung (Fig. 3)
. To further confirm that the inhibition of GITR-GITRL interaction negatively modulates the development of the bleomycin-induced lung injury, we cotreated GITR+/+ mice with bleomycin and an Fc-GITR fusion protein. This protein binds GITRL, inhibiting GITR activation. The treatment of GITR+/+ mice with Fc-GITR fusion protein gave results similar to those obtained with GITR–/– mice (Fig. 2D, Da, and F
and Fig. 3
). On the contrary, the treatment of GITR+/+ mice with Fc fusion protein did not reduce the histological alteration (Fig. 2E, Ea, and F
), as well as the accumulation of water in lung induced by bleomycin instillation (Fig. 3)
, suggesting that the effect of Fc-GITR is not due to the interaction with the Fc-receptor. No histological alteration was observed in sham-treated mice (Fig. 2A, Aa, F
).
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Genetic and pharmacological inhibition of GITR-GITRL interaction reduced lung neutrophil infiltration
The above histological pattern of lung injury appeared to be correlated with the influx of leukocytes into the lung tissue. Therefore, we investigated the role of GITR-GITRL interaction on the neutrophil infiltration by measurement of the myeloperoxidase (MPO) activity. MPO activity was significantly elevated (P<0.001) after bleomycin administration in GITR+/+ mice (Fig. 4
). In GITR–/– mice subjected to bleomycin instillation, lung MPO activity was significantly reduced (P<0.01) in comparison to those of GITR+/+ mice (Fig. 4)
. Similarly, the treatment of GITR+/+ mice with Fc-GITR fusion protein significantly reduced the bleomycin-induced MPO activity (Fig. 4)
. The presence of MPO positive staining correlated with MPO activity. In fact, positive staining for MPO (Fig. 5
B, F) increased along the bronchial epithelium and inflammatory cells in tissue section obtained from bleomycin-treated GITR+/+ mice. In bleomycin-treated GITR–/– mice (Fig. 5C, F
) as well as in GITR+/+ cotreated with Fc-GITR (Fig. 5D, F
), the staining for MPO was visibly and significantly reduced in comparison to bleomycin-alone treated GITR+/+ mice. As expected, no positive staining for MPO was observed in lungs from saline-treated mice (Fig. 5A, F
). Of note, the treatment of GITR+/+ mice with Fc fusion protein did not reduce the lung MPO activity (Fig. 4)
as well as MPO expression induced by bleomycin instillation (Fig. 5E, F
).
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Genetic and pharmacological inhibition of GITR-GITRL interaction reduced production and expression of TNF- and IL-1ß after bleomycin administration
To test whether GITR-GITRL interaction modulates the inflammatory process through the regulation of the secretion of cytokines, we analyzed the lung levels of proinflammatory cytokines TNF- and IL-1ß. A substantial increase of TNF- and IL-1ß formation was found in lung samples collected from GITR+/+ mice after bleomycin instillation (Fig. 6
). Positive staining for TNF- (Fig. 7
B, F) and for IL-1ß (Fig. 8
B, F) was mainly localized in the infiltrated inflammatory cells in damaged tissues. Lung levels of TNF- and IL-1ß were significantly reduced in GITR–/– mice as well as in GITR+/+ mice treated with Fc-GITR in comparison to those of GITR+/+ animals (Fig. 6)
. In bleomycin-treated GITR–/– mice (Figs. 7C, F
and 8C, F
, respectively) as well as in GITR+/+ mice cotreated with Fc-GITR fusion protein (Figs. 7D, F
and 8D, F
, respectively), the staining for TNF- and for IL-1ß was visibly and significantly reduced in comparison to the GITR+/+ mice. There was no staining for either TNF- or IL-1ß in lungs from sham groups (Figs. 7A, F
and 8A, F
, respectively). Treatment of GITR+/+ mice with Fc fusion protein did not reduce the lung production (Fig. 6)
and expression (Figs. 7E, F
and 8E, F
, respectively) of TNF- and IL-1ß induced by bleomycin instillation.
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Genetic and pharmacological inhibition of GITR-GITRL interaction reduced the bleomycin-induced nitrotyrosine formation
Peroxynitrite is a powerful oxidant produced in bleomycin-induced lung fibrosis deriving from NO and stress oxidative products. Nitrotyrosine, a specific marker of peroxynitrite, was evident in lung sections from bleomycin-treated GITR+/+ mice (Fig. 9
B, F) mainly localized in nuclei of inflammatory cells. In bleomycin-treated GITR–/– mice (Fig. 9C, F
), as well as in GITR+/+ mice cotreated with Fc-GITR fusion protein (Fig. 9D, F
), the staining for nitrotyrosine was visibly and significantly reduced in comparison to the GITR+/+ mice. There was no staining for nitrotyrosine in lungs obtained from the sham groups (Fig. 9A, F
). Of note, the treatment of GITR+/+ mice with Fc fusion protein did not reduce the nitrotyrosine formation induced by bleomycin instillation (Fig. 9E, F
).
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Lower activation levels of NF-B in lungs from bleomycin-treated GITR–/– mice
Most inflammatory mediators, including inducible NOS (iNOS), TNF-, and IL-1ß are controlled by NF-B transcription factor, which is kept inactive by IB. NF-B transactivation potential is increased by phosphorylation of the p65 subunit (31)
. Since the levels of proinflammatory markers evaluated are lower in lungs from bleomycin-treated GITR–/– compared with GITR+/+ mice and it is known that GITR triggering activates TRAFs and NF-B (12
, 32)
, we evaluated IB, phospho-p65, and NF-B p65 expression by Western blot analysis. A basal level of IB- was detected in the lung tissues from sham-treated GITR+/+ and GITR–/– mice, whereas in bleomycin-treated GITR+/+ mice IB- levels were substantially reduced (Fig. 10
Aa). Slight but significant reduced levels of IB- degradation were observed in the lung tissues collected from GITR–/– mice (Fig. 10A
).
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Furthermore, bleomycin instillation caused a significant increase in the phosphorylation of p65 on Ser-536 in the lung tissues from GITR+/+ mice and a significant reduction of p65 phosphorylation in the lung tissues from GITR–/– mice (Fig. 10Ba
). Moreover, NF-B p65 subunit presence in the nuclear fractions from lung tissue was also significantly increased after bleomycin instillation compared to the sham-treated mice (Fig. 10C, Cc
). A significant reduction of the NF-B p65 nuclear levels was observed in the tissues from GITR–/– mice (Fig. 10C
). These results confirm previous observation suggesting that GITR can induce NF-B activation and are consistent with the role of NF-B in regulation of inflammatory mediators such as iNOS, TNF-, and IL-1ß (33
, 34)
.
Effects of genetic and pharmacological inhibition of GITR-GITRL interaction on changes in body wt and survival rate
In BLM-treated GITR+/+ mice, the severe lung injury caused by BLM administration was associated with a significant loss in body wt (Fig. 11
A). The survival of animals was monitored for 7 d. BLM-treated mice developed severe lung injury, and 30% of GITR+/+ mice died within 7 d of BLM administration (Fig. 11B
). In bleomycin-treated GITR–/– mice as well as in GITR+/+ mice cotreated with Fc-GITR fusion protein, a significant reduction of the decrease in body wt loss (Fig. 11A
) as well as mortality rate (Fig. 11B
) was observed. Of note, the treatment of GITR+/+ mice with Fc fusion protein did not reduce the loss in body wt and the mortality rate induced by bleomycin instillation (Fig. 11)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 36)
. To date no satisfactory treatment exists for idiopathic pulmonary fibrosis. Various antiinflammatory agents such as corticosteroids (37)
, colchicines (38)
, and cytotoxic agents such as azathioprine (39)
and cyclophospamide (40)
have been used alone or in combination to treat the disease. However, fewer than one-third of patients respond to treatment with corticosteroids and/or cytotoxic therapy.
This study provides evidence that mice lacking GITR (GITR–/–) have (i) a decreased development of bleomycin-induced lung injury; (ii) a decreased PMN infiltration of the lung; (iii) lower levels of TNF-, IL-1ß, and stress oxidative products; and (iv) lower activation levels of NF-B. To be certain that these results were due to the lack of GITR/GITRL interaction and not to dysfunction (such as deficit of maturation/differentiation) of GITR–/– mice, we cotreated GITR+/+ mice with bleomycin and Fc-GITR, a fusion protein that can bind GITRL, thus inhibiting GITR engagement by its ligand. Results were similar to those obtained with GITR–/– mice, suggesting that GITR plays a role in chronic inflammation of the lungs. What is, then, the mechanism by which the genetic and pharmacological inhibition of GITR-GITRL interaction reduces lung fibrosis induced by bleomycin?
It is well known that GITR plays a co-accessory function in effector T cell activation, further potentate by the inhibition of Treg cell function on its triggering (12)
. Moreover, we have demonstrated that in response to Candida Albicans infection, GITR+/+ mice demonstrate a prevalent Th2 polarization and GITR–/– mice a prevalent Th1 polarization (22)
. Among the different pathogenesis hypothesis explaining pulmonary fibrosis development, it has been proposed that macrophages stimulated with Th2 cytokines play a pivotal role in the development of pulmonary fibrosis (41)
. Thus, GITR triggering in effector CD4+ T lymphocytes may play a role in the development of chronic inflammation.
We have recently demonstrated that GITR–/– mice are less prone to develop acute inflammation reaction. In particular, we used the carrageenan-induced pleurisy and the shock following the occlusion and reperfusion of the splanchnic artery (SAO) models (26
, 27)
. In these models, we observed a decreased recruitment of cells in the area of inflammation in GITR–/– mice as compared to GITR+/+ mice and hypothesized that it was due the decreased levels of proinflammatory molecules and to the decreased expression of adhesion molecules (i.e., P-selectin and ICAM-1), which play a crucial role in this process (42
, 43)
. Thus, it is possible that a GITR/GITRL system participates to the extravasation process, increasing its efficiency. Possibly, this phenomenon contributes to explain also the results here presented. In fact, genetic and pharmacological inhibition of GITR-GITRL interaction significantly reduces leukocyte infiltration on bleomycin treatment as assessed by the specific granulocyte enzyme MPO as well as the lung tissue damage. However, it is unlikely that the decreased chronic inflammation of GITR–/– mice and of GITR+/+ mice cotreated with Fc-GITR can be explained only on the basis of a less-efficient extravasation process.
GITR and GITRL are expressed in several cells participating to inflammatory response other than T lymphocytes. In particular, GITR is also expressed in PMN and macrophages (12
, 26)
. Moreover, GITRL is expressed in antigen presenting cells (such as macrophages and dendritic cells), PMN, and endothelial cells (12
, 26)
. GITR and GITRL expression is modulated during inflammation and is very likely that GITR interacts with its ligand in the inflamed tissues. The understanding of the effect of GITR/GITRL interaction is further complicated by the possibility that not only GITR but also GITRL can deliver signals inside the cells. For example, it has been demonstrated that GITRL triggering exerts proinflammatory effects in macrophages (20)
. From this point of view, GITR–/– mice may behave differently from Fc-GITR-treated GITR+/+ mice. In fact, in GITR–/– mice both GITR and GITRL signaling lack, but when the Fc-GITR fusion protein is used, GITR activation is inhibited while GITRL can be triggered by the fusion protein (21)
. Since Fc-GITR cotreated GITR+/+ mice had a lower bleomycin-induced inflammation, similar to those observed in GITR–/– mice, it is likely that, at least in this experimental system, GITR triggering inhibition is more important than the possible GITRL stimulation by Fc-GITR fusion protein. This would suggest that GITR activation (more than GITRL activation) plays an active role in the development of chronic inflammation. In fact, a role of GITR signaling expressed on macrophages has been proposed in a viral infection model (24)
.
It is well known that GITR has a coaccessory role in T cells, where it participates to NF-B activation via TRAFs adaptor molecules (12
, 32)
. Present data suggest that it may play a similar role in inflammatory cells different from T cells. In fact, we report here that bleomycin instillation caused a significant increase in the phosphorylation of Ser-536 on NF-B subunit p65, whereas the genetic or pharmacological inhibition of GITR-GITRL interaction significantly reduced this phosphorylation. Moreover, inhibition of GITR-GITRL interaction slightly decreased IB- degradation. As a consequence, NF-B translocation induced by bleomycin treatment was clearly impaired by inhibition of GITR signaling.
The decreased NF-B activation following GITR blocking correlates with the decreased expression of proinflammatory mediators. It is well known that NF- B plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in inflammation such as TNF-, IL-1ß, iNOS, and COX-2 (33
, 34
, 44)
. There is good evidence that TNF- and IL-1ß are clearly involved in chronic inflammation and the pathogenesis of lung fibrosis, since these cytokines are present in lung tissues and can be detected immunohistochemically in the inflamed tissues (45)
. Direct evidence that TNF- and IL-1ß play a role in the pathogenesis of experimental lung injury has been obtained in animal models in which blocking of the action of these cytokines has been shown to delay the onset of experimental lung injury, suppress inflammation, and ameliorate lung destruction that corresponds to the antiinflammatory response (46)
. We confirm that the model of lung injury used here leads to a substantial increase in the levels of TNF- and IL-1ß in the lung. Interestingly, we report in the present study that GITR blocking reduces the biosynthesis of the proinflammatory cytokines TNF- and IL-1ß. These findings, therefore, suggest that GITR triggering modulates the expression of proinflammatory genes.
We found that the tissue damage induced by bleomycin in GITR+/+ mice was associated with an intense immunostaining of nitrotyrosine formation also suggesting that a structural alteration of lung had occurred, most probably due to the formation of highly reactive nitrogen-derivatives. Recent evidence indicates that bleomycin is a well-known cause of intracellular oxidative stress that may also play a role in the pathogenesis of bleomycin-induced lung injury (47)
. In this study we clearly demonstrate that the genetic and pharmacological inhibition of GITR activation prevents the formation of nitrosative stress.
In conclusion, the results of the present study enhance our understanding of the role of GITR-GITRL interaction in the pathophysiology of chronic lung inflammation. Our results also imply that inhibition of GITR triggering may be useful in the therapy of chronic inflammation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication June 20, 2006. Accepted for publication July 24, 2006.
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