Analysis of macrophage migration inhibitory factor (MIF) in patients with idiopathic pulmonary fibrosis

Respiratory Physiology & Neurobiology 167 (2009) 261–267

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol

Analysis of macrophage migration inhibitory factor (MIF) in patients with idiopathic pulmonary fibrosis E. Bargagli a,∗ , C. Olivieri a , N. Nikiforakis a , M. Cintorino b , B. Magi c , M.G. Perari a , C. Vagaggini a , D. Spina b , A. Prasse d , P. Rottoli a a

Dept of Clinical Medicine and Immunology, Respiratory Diseases Section, Siena University, Viale Bracci, 1, 53100 Siena, Italy Dept of Pathology, Siena University, Siena, Italy Dept of Molecular Biology, Siena University, Italy d Dept of Pneumology, Freiburg University, Freiburg, Germany b c

a r t i c l e

i n f o

Article history: Accepted 13 May 2009 Keywords: Macrophage migration inhibitory factor Bronchoalveolar lavage Lung tissue Interstitial lung diseases Idiopathic pulmonary fibrosis

a b s t r a c t By proteomic approach we previously characterised bronchoalveolar lavage (BAL) protein profiles of patients with idiopathic pulmonary fibrosis (IPF), sarcoidosis and systemic sclerosis. Among differently expressed proteins we identified macrophage migration inhibitory factor (MIF), a multi-function pleiotropic cytokine. This study was performed to validate our findings by a further proteomic approach and ELISA in a larger population of patients and controls. MIF expression in lung tissue was also evaluated by immunohistochemistry. MIF was identified in all 2-DE gels of IPF patients and it was significantly increased compared to controls (p < 0.05). This result was confirmed by ELISA: MIF concentrations were significantly higher in IPF patients than controls (p < 0.001) and were directly correlated with neutrophil percentages (p = 0.0095). Immunohistochemical analysis revealed enhanced expression in bronchiolar epithelium, alveolar epithelium, and fibroblastic foci. In conclusion, MIF is a pleiotropic cytokine that could be involved in the pathogenesis of IPF, being particularly abundant in BAL of these patients and mainly expressed in the areas of active fibrosis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Idiopathic pulmonary fibrosis (IPF) is a severe, rapidly progressive diffuse lung disease without any effective therapy. Specific biomarkers useful for diagnosis or prognosis have not yet been found (ATS, 2000; Strieter, 2005). In the last 10 years, we have used a proteomic approach to analyse the protein composition of bronchoalveolar lavage (BAL) of patients with different interstitial lung diseases (ILD) in order to identify potential biomarkers (Magi et al., 2002, 2006; Rottoli et al., 2005a,b). In IPF patients, we found a statistically significant increase in a group of low molecular weight proteins with respect to sarcoidosis and pulmonary fibrosis associated with systemic sclerosis patients. One of these proteins was macrophage migration inhibitory factor (MIF). MIF is a pleiotropic proinflammatory cytokine of 12.5 kDa, produced by many cells (e.g. macrophages, lymphocytes, eosinophils,

Abbreviations: MIF, macrophage migration inhibitory factor; BAL, bronchoalveolar lavage; ILD, interstitial lung diseases; IPF, idiopathic pulmonary fibrosis; UIP, usual interstitial pneumonia. ∗ Corresponding author. Tel.: +39 0577 586710; fax: +39 0577 586710. E-mail address: [email protected] (E. Bargagli). 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.05.004

neutrophils, endothelial and epithelial cells). It has multiple activities (endocrine and enzymic properties, involvement in innate and acquired immunity) (Lenzini et al., 1973; Calandra et al., 1995; Satoskar et al., 2001; Leng et al., 2003; Rodriguez-Sosa et al., 2003; Thiele and Bernhagen, 2005; Morand, 2005; Amin et al., 2006; Yu et al., 2007; Amano et al., 2007; Magalhaes et al., 2007). It has been studied in lung disorders such as ARDS (Donnelly et al., 1997; Makita et al., 1998; Beishuizen et al., 2001; Lue et al., 2002; Nunez et al., 2007), bronchial asthma (Rossi et al., 1998; Mizue et al., 2005; Kobayashi et al., 2006; Amano et al., 2007; Magalhaes et al., 2007), pulmonary tuberculosis (Bernhagen et al., 1996; Yamada et al., 2002; Kibiki et al., 2007) and in an animal model of bleomycininduced lung injury (Tanino et al., 2002). Polymorphism of the MIF gene was recently studied in sarcoidosis (Pakozdi et al., 2006; Plant et al., 2007) and asthma (Hizawa et al., 2004). Little information is available in the literature on a role of MIF in pulmonary fibrosis, however its different functions suggest that it could be involved in the pathogenesis of IPF. Regulation of cell redox status (Thiele and Bernhagen, 2005), inhibition of apoptosis (Morand, 2005), induction of matrix metalloproteinases, promotion of fibroblast proliferation (Yu et al., 2007) and modulation of Th1/Th2 immune responses (Satoskar et al., 2001; Rodriguez-Sosa

262

E. Bargagli et al. / Respiratory Physiology & Neurobiology 167 (2009) 261–267

Table 1 Demographic data, smoking history and lung function parameters of the study population (M ± SD).

Subjects, n Age (yr) Sex M:F (%) Smoker Current Past Never FEV1, %pred FVC, %pred TLC, %pred DLCO, %pred PaO2 (mmHg) PaCO2 (mmHg) pH

IPF

Controls

30 64.22 ± 9.50 23(77%):7(23%)

12 38.50 ± 11.68 6(50%):6(50%)

2 14 14 73.60 ± 21.51 67.75 ± 20.15 70.32 ± 16.79 42.11 ± 5.76 77.08 ± 9.39 40.70 ± 4.08 7.42 ± 0.03

2 2 8 94.88 ± 6.19 98.68 ± 5.79 90.00 ± 12.73 104.00 ± 4.90 – – –

p-Value (t-test)

NS NS NS 0.0002 <0.0001 NS <0.0001

et al., 2003; Amano et al., 2007; Magalhaes et al., 2007) are some of the main functions of MIF. Interestingly, MIF counteracts the immunosuppressive effects of glucocorticoids mainly by suppression of inflammatory cytokine secretion by activated macrophages (TNF␣, IL-1, IL-6, IL-8) (Calandra et al., 1995) and by up-regulating expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) (Amano et al., 2007). On this basis we set out to validate the previously found increase in MIF in BAL of patients with idiopathic pulmonary fibrosis, using a proteomic approach. Further BAL samples from IPF patients were analysed by a quantitative assay (ELISA) and MIF expression in lung tissue was studied by immunohistochemistry for insights into potential involvement of this cytokine in the pathogenesis of IPF. 2. Materials and methods 2.1. Study population We analysed the proteome of BAL from 10 patients with IPF and 5 healthy controls, then we performed ELISA on BAL of a larger population of 30 IPF patients and 12 healthy controls. We did immunohistochemistry in 8 of these 30 patients with histologically demonstrated IPF and five controls who were donors for lung transplant. Patients included in the study had never been treated with steroids or other immunosuppressants, had no history of concomitant pathology and had been regularly followed-up (since onset or for at least the last 12 months) at our Sarcoidosis and Interstitial Lung Diseases Regional Referral Centre in Siena. IPF was diagnosed according to international ATS/ERS criteria (ATS, 2000), with a lung biopsy in 8/30 patients and with a clinical–radiological diagnosis in 22/30 patients. Clinical–radiological features and respiratory function parameters (including single-breath diffusing capacity for carbon monoxide) were obtained. Chest X-ray was performed in

the posterior–anterior and lateral projections and classified by a single experienced radiologist. High resolution computed tomography of the chest (HRCT) was read independently by two experienced radiologists. The following parameters were recorded: age, sex, smoking habits, BAL differential cell count, BAL lymphocyte phenotype, lung function tests, oxygen saturation during 6 min walking test, PaO2 by blood gas analysis and radiological impairment (by chest X-ray and HRCT). Control subjects were healthy Caucasians with no history of asthma or allergy and were not taking any medicines. The 12 healthy controls underwent BAL procedure for minimal clinical problems (i.e. after a single episode of hemoptysis). In all cases the bronchoscopy did not reveal any alterations. These subjects did not develop any lung disease in a follow-up period of 1 year. Healthy subjects underwent bronchoscopy for unrelevant clinical reasons (i.e. single episode of hemoptysis) or after specific request of the subjects to exclude lung diseases. No alterations were observed and they did not develop lung diseases in a 12-month follow-up period. Patients and controls gave their written informed consent to the study which was approved by the local ethics committee. 2.2. Bronchoalveolar lavage Bronchoalveolar lavage was performed as previously described (Haslam and Baughman, 1999; Magi et al., 2002). Samples were obtained by instillation of four aliquots (about 60 ml each) of saline solution through a fiberoptic bronchoscope (Olympus IT-10). The first BAL sample was kept separate from the others and was not used for immunological tests. Sub-samples were cultured for microbes, fungi and viruses to exclude infections. Cells were separated by centrifuge and the supernatant was frozen for enzyme assays. Cell differential counts were performed. Lymphocyte phenotype was analysed by flow cytometry (Becton & Dickinson Facs-Calibur) using anti-CD3, CD4 and CD8 monoclonal antibodies (Becton & Dickinson), as previously described (Rottoli et al., 2005a). In healthy controls, BAL was performed in the mid-right lobe. In patients with IPF, areas with evident lung fibrosis, such as macroscopic honeycombing, were avoided. 2.3. Two-dimensional electrophoresis Two-dimensional electrophoresis (2-DE) was performed as previously described (Magi et al., 2002; Rottoli et al., 2005a,b). Briefly, BAL samples of patients and controls were dialyzed against water and dissolved in lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris base, 65 mM dithioerythritol (DTE) and trace amounts of bromophenol blue), so that 100 ␮L of sample (the volume loaded on each gel) contained Bjellqvist 45 mg of proteins. 2-DE was performed essentially as described by Bjellqvist et al. (1993). The first dimension was run on a nonlinear wide-range immobilized pH gradient IPG (pH 3.5–10, 18 cm long IPG strips; Amersham Biosciences, Uppsala, Sweden). The second dimension was run on 9–16% polyacrylamide

Table 2 Bronchoalveolar lavage (BAL)—total cells and differential counts.

Subjects, n Recovery rate (%) Cell number Cell/ml Macrophages (%) Lymphocytes (%) Neutrophils (%) Eosinophils (%) CD4/CD8 Data are expressed as median (range).

IPF

Controls

p-Value (Mann–Whitney)

30 50 (22–58) 8,625,000 (4,850,000–1.75e+07) 186,666 (105,000–340,000) 68.5 (47–76) 14.25 (9–19) 9.5 (4–17) 6 (3–10.75) 1.25 (0.81–2.16)

12 62.3 (53.8–70.8) 1.46e+07 (6,900,000–1.96e+07) 53,889 (40,000–103,157) 84.5 (78.5–92.5) 13.5 (6.5–19.5) 3 (2–4) 1.25 (0.5–1.25) 1.95 (1.34–2.37)

NS NS 0.0077 0.0002 NS 0.0031 0.0408 NS

E. Bargagli et al. / Respiratory Physiology & Neurobiology 167 (2009) 261–267

263

Fig. 1. Position of MIF spot in two-dimensional electrophoretic gel of IPF patient.

linear gradient gels. Gels were stained with ammoniacal silver nitrate (Hochstrasser et al., 1988). Electrophoretogram images were obtained with a computing densitometer (Molecular Dynamics 300S; Sunnyvale, CA, USA) and processed with the program Melanie III.

substrate solution was added to the wells and colour developed in proportion to the amount of MIF bound in the initial step. Colour development was stopped and its intensity measured. MIF levels in BAL were expressed in pg/ml and pg/mg BAL protein. Total protein concentrations in BAL were determined with the Bio-Rad Protein assay, based on the Bradford’s method (Bradford, 1976).

2.4. MIF assay 2.5. Immunohistochemistry MIF concentrations were determined by ELISA (R&D Systems, Minneapolis, USA). A monoclonal antibody specific for MIF was pre-coated onto a microplate. Standards and samples were pipetted into the wells and any MIF present was bound by the immobilized antibody. After washing away any unbound substances, an enzymelinked polyclonal antibody specific for MIF was added to the wells. Following a wash to remove unbound antibody–enzyme reagent, a

Immunohistochemistry was performed using the streptavidin–biotin method. Sections (4 ␮m) were dewaxed, rehydrated, and washed in Tris-buffered saline (TBS; 20 mM Tris–HCl and 150 mM NaCl pH 7.6). Antigen retrieval was carried out by incubating sections in sodium citrate buffer (10 mM pH 6.0) in a microwave oven at 750 W for 5 min. Slides were then

264

E. Bargagli et al. / Respiratory Physiology & Neurobiology 167 (2009) 261–267

Table 3 MIF expression analysed by immunohistochemistry in bronchiolar and alveolar epithelium, fibroblastic foci and lymphoid nodules in IPF patients and controls (absent: absence of fibroblastic foci or lymphoid nodules in field/sample). Bronchiolar epithelium

Alveolar epithelium

Fibroblastic foci

Lymphoid nodule

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8

++ ++ ++ ++ + ++ +++ +++

+ ++ ++ ++ ++ ++ +++ +++

++ ++ ++ ++ ++ ++ +++ ++

Absent Absent ++ ++ + + ++ ++

Control 1 Control 2 Control 3 Control 4 Control 5

++ − + + +

− − − − −

Absent Absent Absent Absent Absent

Absent Absent Absent Absent Absent

washed three times with TBS for 5 min, and incubated with a rabbit anti-goat antibody labelled with biotin (MIF, Dako, Copenhagen, Denmark) at a dilution of 1:5000 for 60 min. The reaction was revealed using the streptavidin–biotin complex (Dako). Sections were weakly counterstained with haematoxylin. Slides were mounted and examined under a light microscope. For each case, a negative control was obtained by replacing the specific antibody with non-immune serum immunoglobulins at the same concentration as the primary antibody. 2.6. Statistical analysis All results were expressed as mean ± standard deviation for continuous variables having normal distribution. Median and range (25th–75th percentiles) were used for data, such as BAL MIF levels, having non-normal distribution. Group comparisons for categorical data were made by the Pearson 2 -test. For comparison of continuous data between two groups, the unpaired t-test was used for normally distributed data and the Mann–Whitney two sample test for non-normally distributed data. Spearman’s rank correlation was used to express correlations between MIF levels and BAL cell pattern. Statistical analysis was performed with the program STATA 9.0 for Windows.

(841 pg/ml, range 326–1552) (Fig. 2b). A positive correlation was found between MIF concentrations (expressed in pg/ml) in patients with IPF and neutrophil percentages in BAL (r = 0.465, p = 0.0095). MIF concentrations were also expressed as pg/mg protein of BAL (Fig. 3) and were 108.43 ± 92.89 pg/mg (M ± DS) in IPF patients and 43.85 ± 23.18 pg/mg in controls (p < 0.001), confirmed also by calculation of median (p < 0.001) (IPF patients: 53.87 pg/mg, range 28.64–83.33; controls 20.32 pg/mg, range 11.62–33.44). Table 3 shows the quantification and distribution of MIF expression by immunohistochemistry in lung tissue of a subgroup of IPF patients with histologically demonstrated diagnosis and controls. The table indicates the presence/absence of MIF in fibroblastic foci, lymphoid nodules, and alveolar/bronchiolar epithelium, quantified by a scale ranging from 0 (absence) to +++ (highly expressed). The main results were that MIF was sharply up-regulated in fibroblastic foci and bronchiolar and alveolar epithelium of IPF patients (especially case 7 who had very severe disease) (Table 3 and

3. Results Table 1 shows the demographic data, smoking habits, blood gas analysis values and lung function test parameters of the study population. Statistically significant differences (p < 0.001) were found between FEV1, FVC and DLCO percentages in patients with IPF and controls. Likewise, macrophage percentages in BAL of IPF patients were significantly lower than in controls (p = 0.0002, Table 2), while BAL eosinophil (p = 0.04) and neutrophil (p = 0.003) percentages were higher in patients than in controls (Table 2). In 2-DE gels, MIF was identified by gel matching with a reference gel from the breast tissue map 2D database in SWISS-2DPAGE and confirmed by immunoblot (Fig. 1). MIF was found in all gels of IPF patients. This protein (accession number P-14174) was resolved in two spots with isoelectric points (pI) of 6.99 and 7.84 and molecular weights of 10.3 kDa and 10.5 kDa. Spot relative volume was calculated for IPF and control BAL and expressed as percentage volume. IPF relative volume was significantly higher than that of controls (p < 0.05). Fig. 2a shows MIF relative volume percentages (detected by 2-DE) in BAL of IPF patients and controls. MIF levels quantified by ELISA were reported as median because they had not a normal distribution. MIF concentrations (expressed in pg/ml) in BAL from IPF patients were significantly higher (p < 0.001; 5078 pg/ml, range 2669–10,000) than in controls

Fig. 2. MIF relative volumes analysed by 2-DE in BAL of IPF patients and controls (median and range) (p < 0.05) (a). MIF concentrations expressed as pg/ml of BAL recovered, evaluated by ELISA in IPF patients and healthy controls (median and range) (p < 0.001) (b).

E. Bargagli et al. / Respiratory Physiology & Neurobiology 167 (2009) 261–267

265

In a genetic study of IPF, Zuo et al. (2002) recently reported upregulation of the MIF gene in patients with idiopathic pulmonary fibrosis and its increased expression (8.9-fold induction) in lung tissue of patients with IPF (UIP), determined using oligonucleotide microarrays. This observation may be a further confirmation of the increase of MIF in IPF patients, as observed by us. Hypoxia is a common feature of IPF. A very recent study by Oda et al. (2008) on the involvement of MIF in the pathogenesis of IPF found over-expression of MIF under hypoxic conditions and that MIF played a critical role as activator of hypoxia inducible factor (HIF-1) with a p-53 dependent mechanism. They concluded that

Fig. 3. MIF concentrations (by ELISA) expressed as pg/mg protein in BAL of IPF patients and controls (p < 0.001).

Fig. 4), whereas in controls there was only a weak expression of MIF in bronchiolar epithelium (obviously fibroblastic foci were not present). Moreover positive immunostaining for MIF was observed in the cells infiltrating lung parenchyma (macrophages and neutrophils). 4. Discussion In a previous proteomic study (Rottoli et al., 2005b), the observation of increased expression of MIF in BAL of IPF patients with respect to sarcoidosis and systemic sclerosis patients prompted us to compare percentage volumes in BAL between IPF and controls. Significantly increased percentage volumes of MIF were observed in IPF patients than controls by 2-DE analysis. Therefore these findings were further validated by ELISA and immunohistochemistry in a larger cohort of patients. MIF concentrations were found to be significantly elevated whether expressed in pg/ml of BAL recovered or in pg/mg protein. This increase may be due to enhanced production at single cell level and/or increased numbers of MIF-producing bronchoalveolar cell populations in IPF. It is known from the literature that MIF-producing cells include activated macrophages, eosinophils, neutrophils and epithelial cells, regarded as crucial in the pathogenesis of IPF (Selman et al., 2001; Ohno-Matsui et al., 2003; Noble and Homer, 2005; Strieter, 2005; Van Molle and Libert, 2005; Baugh et al., 2006; Kim et al., 2007). Interestingly, we found a positive correlation between MIF concentrations and neutrophil percentages in BAL. Several reports in the literature indicate that neutrophils are enhanced in BAL of IPF patients as observed also by us and it has been hypothesized that they play a major role in pathogenesis of IPF mainly by oxidative stress mediated tissue damage (Lue et al., 2002; Rottoli et al., 2005a). By immunohistochemistry we also observed that MIF expression was particularly enhanced in bronchiolar and alveolar epithelium of lung regions subjected to remodelling and active fibrosis (i.e. fibroblastic foci). Strongly positive tissues from our IPF patients resembled that of certain solid tumours. It has been demonstrated that MIF expression is correlated with tumour aggressiveness and metastatic potential (Lue et al., 2002). It promotes fibroblast, endothelial and tumour cell growth suggesting a potential role in the abnormal proliferative processes present also in IPF. Our results indicate that different cell populations, including epithelial cells and neutrophils, may have a crucial role in MIF secretion in IPF. The broad range of MIF activities make possible its potential involvement in the pathogenesis of IPF: MIF regulates apoptosis and cytokine expression by macrophages (Morand, 2005), it induces matrix metalloproteinases, it promotes proliferation of fibroblasts (Amin et al., 2006) and it reinforces collagen deposition and lung fibrotic remodelling (Baugh et al., 2006).

Fig. 4. Immunohistochemistry of MIF in the lung tissue from a patient with idiopathic pulmonary fibrosis. MIF positive immunostaining in fibroblastic foci (*), alveolar epithelium (#) and diffuse cellular infiltration (**) (a; original magnification 100×), in lymphoid nodule (#) (b; original magnification 100×) and in fibroblastic foci (c; original magnification 100×).

266

E. Bargagli et al. / Respiratory Physiology & Neurobiology 167 (2009) 261–267

activation of HIF-1 can be induced by the over-expression of MIF under hypoxic conditions (Oda et al., 2008). Anti-MIF monoclonal antibody treatment has been tested in an animal model of bleomycin-induced fibrosis (Tanino et al., 2002). Interestingly, this approach obtained a reduction in neutrophil accumulation in the alveolar spaces and in mice mortality, although anti-MIF antibody did not affect the content of lung hydroxyproline or the histopathological lung fibrosis score at 21 days post-bleomycin exposure in their experimental model. The anti-MIF antibody effect on neutrophil accumulation seems interesting considering the increase of neutrophils observed in IPF. It has been reported that antioxidant activity in the lungs may be induced by anti-MIF antibody and that it increased effectiveness of steroid therapy (counteracting the negative feedback of MIF towards glucocorticoids) (Van Molle and Libert, 2005). In conclusion, MIF is a pleiotropic cytokine that could be involved in the pathogenesis of IPF being particularly abundant in BAL of these patients, significantly related to neutrophils and mainly expressed in lung regions subject to remodelling and active fibrosis. Conflict of interest The authors declare that they have no conflict of interest to disclose. References Amano, T., Nishihira, J., Miki, I., 2007. Blockade of macrophage migration inhibitory factor (MIF) prevents the antigen-induced response in a murine model of allergic airway inflammation. Inflamm. Res. 56 (1), 24–31. American Thoracic Society, 2000. Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS) and the European Respiratory Society (ERS). Am. J. Respir. Crit. Care Med. 161 (2 (Pt. 1)), 646–664. Amin, M.A., Haas, C.S., Zhu, K., Mansfield, P.J., Kim, M.J., Lackowski, N.P., Koch, A.E., 2006. Migration inhibitory factor up-regulates vascular cell adhesion molecule1 and intercellular adhesion molecule-1 via Src, PI3 kinase and Nf kappa B. Blood 107 (6), 2252–2261. Baugh, J.A., Gantier, M., Li, L., Byrne, A., Buckley, A., Donnelly, S.C., 2006. Dual regulation of MIF expression in hypoxia by CREB and HIF-1. Biochem. Biophys. Res. Commun. 347 (4), 895–903. Beishuizen, A., Thijs, L.G., Haanen, C., Vermes, I., 2001. Macrophage migration inhibitory factor and hypothalamo-pituitary-adrenal function during critical illness. J. Clin. Endocrinol. Metab. 86 (6), 2811–2816. Bernhagen, J., Bacher, M., Calandra, T., Metz, C.N., Doty, S.B., Donnelly, T., Bucala, R., 1996. An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction. J. Exp. Med. 183 (1), 277–282. Bjellqvist, B., Pasquali, C., Ravier, F., Sanchez, J.C., Hochstrasser, D., 1993. A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale. Electrophoresis 14 (12), 1357–1365. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Calandra, T., Bernhagen, J., Metz, C.N., Spiegel, L.A., Bacher, M., Donnelly, T., Cerami, A., Bucala, R., 1995. MIF as a glucocorticoid-induced modulator of cytokine production. Nature 377 (6544), 68–71. Donnelly, S.C., Haslett, C., Reid, P.T., Grant, I.S., Wallace, W.A., Metz, C.N., Bruce, L.J., Bucala, R., 1997. Regulatory role for macrophage migration inhibitory factor in acute respiratory distress syndrome. Nat. Med. 3 (3), 320–323. Haslam, P.L., Baughman, R.P., 1999. Report of ERS Task Force: guidelines for measurement of acellular components and standardization of BAL. Eur. Respir. J. 14 (2), 245–248. Hizawa, N., Yamaguchi, E., Takahashi, D., Nishihira, J., Nishimura, M., 2004. Functional polymorphisms in the promoter region of macrophage migration inhibitory factor and atopy. Am. J. Respir. Crit. Care Med. 169 (9), 1014–1018. Hochstrasser, D.F., Harrington, M.G., Hochstrasser, A.C., Miller, M.J., Merril, C.R., 1988. Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal. Biochem. 173 (2), 424–435. Kibiki, G.S., van der Ven, A.J., Geurts-Moespot, A., Shao, J., Calandra, T., Sweep, F.C., Dolmans, W.M., 2007. Serum and BAL macrophage migration inhibitory factor levels in HIV infected Tanzanians with pulmonary tuberculosis or other lung diseases. Clin. Immunol. 123 (1), 60–65. Kim, H.R., Park, M.K., Cho, M.L., Yoon, C.H., Lee, S.H., Park, S.H., Leng, L., Bucala, R., Kang, I., Choe, J., Kim, H.Y., 2007. Macrophage migration inhibitory factor upregulates angiogenic factors and correlates with clinical measures in rheumatoid arthritis. J. Rheumatol. 34 (5), 927–936.

Kobayashi, M., Nasuhara, Y., Kamachi, A., Tanino, Y., Betsuyaku, T., Yamaguchi, E., Nishihira, J., Nishimura, M., 2006. Role of macrophage migration inhibitory factor in ovalbumin-induced airway inflammation in rats. Eur. Respir. J. 27 (4), 726–734. Leng, L., Metz, C.N., Fang, Y., Xu, J., Donnelly, S., Baugh, J., Delohery, T., Chen, Y., Mitchell, R.A., Bucala, R., 2003. MIF signal transduction initiated by binding to CD74. J. Exp. Med. 197 (11), 1467–1476. Lenzini, L., Rottoli, P., Rottoli, L., Sestini, P., 1973. Leucocyte-migration-inhibition tests with kveim antigen in sarcoidosis. Lancet 2, 1087. Lue, H., Kleemann, R., Calandra, T., Roger, T., Bernhagen, J., 2002. Macrophage migration inhibitory factor (MIF): mechanisms of action and role in disease. Microbes Infect. 4 (4), 449–460. Magi, B., Bini, L., Perari, M.G., Fossi, A., Sanchez, J.C., Hochstrasser, D., Paesano, S., Raggiaschi, R., Santucci, A., Pallini, V., Rottoli, P., 2002. Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study. Electrophoresis 23 (19), 3434–3444. Magi, B., Bargagli, E., Bini, L., Rottoli, P., 2006. Proteome analysis of bronchoalveolar lavage in lung diseases. Proteomics 6 (23), 6354–6369. Magalhaes, E.S., Mourao-Sa, D.S., Vieira-de-Abreu, A., Figueiredo, R.T., Pires, A.L., Farias-Filho, F.A., Fonseca, B.P., Viola, J.P., Metz, C., Martins, M.A., Castro-FariaNeto, H.C., Bozza, P.T., Bozza, M.T., 2007. Macrophage migration inhibitory factor is essential for allergic asthma but not for Th2 differentiation. Eur. J. Immunol. 37 (4), 1097–1106. Makita, H., Nishimura, M., Miyamoto, K., Nakano, T., Tanino, Y., Hirokawa, J., Nishihira, J., Kawakami, Y., 1998. Effect of anti-macrophage migration inhibitory factor antibody on lipopolysaccharide-induced pulmonary neutrophil accumulation. Am. J. Respir. Crit. Care Med. 158 (2), 573–579. Mizue, Y., Ghani, S., Leng, L., McDonald, C., Kong, P., Baugh, J., Lane, S.J., Craft, J., Nishihira, J., Donnelly, S.C., Zhu, Z., Bucala, R., 2005. Role for macrophage migration inhibitory factor in asthma. Proc. Natl. Acad. Sci. U.S.A. 102 (40), 14410–14415. Morand, E.F., 2005. New therapeutic target in inflammatory disease: macrophage migration inhibitory factor. Intern. Med. J. 35 (7), 419–426. Nunez, C., Rueda, B., Martinez, A., Lopez-Nevot, M.A., Fernandez-Arquero, M., de la Concha, E.G., Martin, J., Urcelay, E., 2007. Involvement of macrophage migration inhibitory factor gene in celiac disease susceptibility. Genes Immun. 8 (2), 168–170. Noble, P.W., Homer, R.J., 2005. Back to the future: historical perspective on the pathogenesis of idiopathic pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol. 33 (2), 113–120. Oda, S., Oda, T., Nishi, K., et al., 2008. Macrophage migration inhibitory factor activates hypoxia-inducible factor in a p-53 dependent manner. PLoS ONE 3, 2215–2225. Ohno-Matsui, K., Uetama, T., Yoshida, T., Hayano, M., Itoh, T., Morita, I., Mochizuki, M., 2003. Reduced retinal angiogenesis in MMP-2-deficient mice. Invest. Ophthalmol. Vis. Sci. 44 (12), 5370–5375. Pakozdi, A., Amin, M.A., Haas, C.S., Martinez, R.J., Haines III, G.K., Santos, L.L., Morand, E.F., David, J.R., Koch, A.E., 2006. Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis. Arthritis Res. Ther. 8 (4), R132. Plant, B.J., Ghani, S., O’Mahony, M.J., Morgan, L., O’Connor, C.M., Morgan, K., Baugh, J.A., Donnelly, S.C., 2007. Sarcoidosis and MIF gene polymorphism: a case–control study in an Irish population. Eur. Respir. J. 29 (2), 325–329. Rodriguez-Sosa, M., Rosas, L.E., David, J.R., Bojalil, R., Satoskar, A.R., Terrazas, L.I., 2003. Macrophage migration inhibitory factor plays a critical role in mediating protection against the helminth parasite Taenia crassiceps. Infect. Immun. 71 (3), 1247–1254. Rossi, A.G., Haslett, C., Hirani, N., Greening, A.P., Rahman, I., Metz, C.N., Bucala, R., Donnelly, S.C., 1998. Human circulating eosinophils secrete macrophage migration inhibitory factor (MIF). Potential role in asthma. J. Clin. Invest. 101 (12), 2869–2874. Rottoli, P., Magi, B., Cianti, R., Bargagli, E., Vagaggini, C., Nikiforakis, N., Pallini, V., Bini, L., 2005a. Carbonylated proteins in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis. Proteomics 5 (10), 2612–2618. Rottoli, P., Magi, B., Perari, M.G., Liberatori, S., Nikiforakis, N., Bargagli, E., Cianti, R., Bini, L., Pallini, V., 2005b. Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis. Proteomics 5 (5), 1423–1430. Satoskar, A.R., Bozza, M., Rodriguez Sosa, M., Lin, G., David, J.R., 2001. Migrationinhibitory factor gene-deficient mice are susceptible to cutaneous Leishmania major infection. Infect. Immun. 69 (2), 906–911. Selman, M., King, T.E., Pardo, A., 2001. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann. Intern. Med. 134 (2), 136–151. Strieter, R.M., 2005. Pathogenesis and natural history of usual interstitial pneumonia: the whole story or the last chapter of a long novel. Chest 128 (5 (Suppl. 1)), 526S–532S. Tanino, Y., Makita, H., Miyamoto, K., Betsuyaku, T., Ohtsuka, Y., Nishihira, J., Nishimura, M., 2002. Role of macrophage migration inhibitory factor in bleomycin-induced lung injury and fibrosis in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 283 (1), L156–L162. Thiele, M., Bernhagen, J., 2005. Link between macrophage migration inhibitory factor and cellular redox regulation. Antioxid. Redox Signal. 7 (9–10), 1234–1248. Van Molle, W., Libert, C., 2005. How glucocorticoids control their own strength and the balance between pro- and anti-inflammatory mediators. Eur. J. Immunol. 35 (12), 3396–3399.

E. Bargagli et al. / Respiratory Physiology & Neurobiology 167 (2009) 261–267 Yamada, G., Shijubo, N., Takagi-Takahashi, Y., Nishihira, J., Mizue, Y., Kikuchi, K., Abe, S., 2002. Elevated levels of serum macrophage migration inhibitory factor in patients with pulmonary tuberculosis. Clin. Immunol. 104 (2), 123–127. Yu, X., Lin, S.G., Huang, X.R., Bacher, M., Leng, L., Bucala, R., Lan, H.Y., 2007. Macrophage migration inhibitory factor induces MMP-9 expression in macrophages via the MEK-ERK MAP kinase pathway. J. Interferon Cytokine Res. 27 (2), 103–109.

267

Zuo, F., Kaminski, N., Eugui, E., Allard, J., Yakhini, Z., Ben-Dor, A., Lollini, L., Morris, D., Kim, Y., DeLustro, B., Sheppard, D., Pardo, A., Selman, M., Heller, R.A., 2002. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc. Natl. Acad. Sci. U.S.A. 99 (9), 6292–6297.