A comparative study of cytokines change in bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis before and after treatment with IFNγ-1b and corticosteroids or azathioprine and corticosteroids. A perspective randomized national study

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disorder, the etiology of which is unknown and there is no effective therapy. Combination of corticosteroid and immunosuppressive agents as azathioprine (AZA) or antifibrotic drugs as interferone γ-1b (IFN-γ-1b) have been suggested. Several studies have shown that Th2 cytokines are predominately involved in mediating chronic fibroproliferative disorders such as IPF, whereas Th1 cytokines are downregulated. Moreover,profibrotic mediators such as CTGF and TGFb induce fibroblast activation, collagen synthesis and finally fibrosis. The role of alveolar macrofages in the expression of these cytokines remains unclear. Aim: The aim of the present study was a) to evaluate of the expression of theTh1 cytokines IFN-γ-1b, IL-18 and TNFα, the Th2 cytokine IL-4 and the profibrotic growth factors CTGF and TGFβ in macrophages from bronchoalveolar lavage fluid(BALF) of IPF patients, comparing to control group b) to compare the effectiveness of treatment with interferon g plus low dose prednisone versus azathioprine plus low dose prednisone in patients with IPF, and to investigate whether the two therapeutic options can modulate the cellular apoptotic profile in patients with IPF. Materials & Methods: Bronchoalveolar lavage fluid (BALF) samples derived from 20 newly diagnosed IPF patients before and after six months of treatment with either interferon (IFN-γ-1b) and prednizolone (n=10 patients) or azathioprine (AZA)and prednizolone (n=10 patients) and also BALF samples from 10 normal subjects(control group) were used for the analysis of IL-18, IFNγ, IL-4, CTGF, TGFb and TNFα protein expression in the alveolar macrophages. Furthermore, the effectiveness of these regimens on pulmonary function tests (FEV1, FVC, DLCO), on PaO2 andPaCO2 and on HRCT of the IPF patients was investigated. Results: The two patients groups showed no statistically significant difference regarding the course of the disease after 6 month therapy . In particular, there was no difference referring to FEV1%, FVC%, DLCO% and PaO2, PaCO2 values after treatment with both regimens. Moreover, evaluation of the fibrosis extent in HRCT showed no difference before and after therapy in the two patients groups. Decreased CTGF protein expression was identified in IPF patients correlating to control group(p=0.001) while the two patients groups had similar CTGF expression which remained unchanged after therapy. IL-18, IFNγ, IL-4, TGFb and TNFα, expression inIPF patients did not differ from normal subjects. Neither of the regimens affected those factors’ protein expression. Conclusion: Our findings suggest that the alveolar macrophages are not the main source of CTGF and TNFα in IPF. Moreover, therapeutic intervention with either AZA or IFN-γ-1b did not result in significant change of these factors’ protein expression or the clinical course of the disease.Η Ιδιοπαθής Πνευμονική Ίνωση (ΙΠΙ) είναι μια πνευμονική νόσος, αγνώστου αιτιολογίας, με κακή πρόγνωση, για την οποία δεν υπάρχει αποτελεσματική θεραπεία. Συνδυασμοί κυτταροστατικών όπως η αζαθειπρίνη και αντιϊνωτικών παραγόντων όπως η ιντερφερόνη γ-1b με κορτικοειδή έχουν προταθεί. Μελέτες έχουν δείξει πως στη νόσο παρατηρείται αυξημένη έκκριση Th2 και μειωμένη παραγωγή Th1 κυτταροκινών. Αυτή η ανισορροπία στην ανοσιακή απάντηση, μετά από συνεχή έκθεση σε βλαπτικό ερέθισμα ή αντιγόνο, οδηγεί τελικά σε απελευθέρωση προϊνωτικών μεσολαβητών – όπως CTGF και TGFb – από το προσβεβλημένο επιθηλιακό κύτταρο, αυξημένη δραστηριότητα ινοβλαστών, αναδόμηση του παρεγχύματος και τελικά ίνωση. Ο ρόλος των κυψελιδικών μακροφάγων στην έκφραση των κυτταροκινών αυτών παραμένει αδιευκρίνιστος. Σκοπός : Ο σκοπός της παρούσας μελέτης ήταν α) η αξιολόγηση της έκφρασης των Th1 κυτταροκινών IFN-γ και IL-18, TNFα, της Th2 κυτταροκίνης IL-4 και των προϊνωτικών αυξητικών παραγόντων CTGF και TGFβ στα μακροφάγα από το υγρό του βρογχοκυψελιδικού εκπλύματος (BALF) ασθενών με ΙΠΙ συγκριτικά με ομάδα μαρτύρων, β) η επίδραση θεραπείας με ιντερφερόνη γ-1b (IFNγ-1b) ή αζαθειοπρίνη (AZA) στην έκφραση των παραπάνω παραγόντων στα μακροφάγα του BAL των ασθενών, καθώς και στην κλινική πορεία της νόσου. Υλικό & Μέθοδος: Τα δείγματα του βρογχοκυψελιδικού εκπλύματος (BALF) ελήφθησαν από 20 νεοδιαγνωσθέντες ασθενείς με ΙΠΙ προ και μετά από εξάμηνη θεραπεία είτε με IFN-γ-1b και πρεδνιζολόνη (n=10 ασθενείς) ή με AZA καιπρεδνιζολόνη (n=10 ασθενείς). Επίσης, δείγματα BALF από 10 φυσιολογικά άτομα (ομάδα μαρτύρων) χρησιμοποιήθηκαν για την ανάλυση της πρωτεϊνικής έκφρασηςτων IL-18, IFNγ, IL-4, CTGF, TGFb και TNFα στα κυψελιδικά μακροφάγα. Έγινε αξιολόγηση της επίδρασης των θεραπειών αυτών με τις εξετάσεις πνευμονικής λειτουργίας (FEV1, FVC, DLCO), και στα αέρια αίματος (PaO2 και PaCO2) των ασθενών με ΙΠΙ, και με τον απεικονιστικό έλεγχο με HRCT. Αποτελέσματα: Μετά από 6 μήνες θεραπείας, οι δύο ομάδες ασθενών δεν παρουσίασαν στατιστικά σημαντική μεταβολή ως προς την πορεία της νόσου. Συγκεκριμένα, δεν υπήρξε διαφορά στις τιμές FEV1%, FVC%, DLCO% και PaO2, PaCO2 μετά από τη χορήγηση θεραπευτικών σχημάτων. Επίσης, δεν σημειώθηκε στατιστική διαφορά ως προς την έκταση ίνωσης στην HRCT μετά από θεραπεία στις δύο ομάδες. Παρατηρήθηκε μείωση της πρωτεϊνικής έκφρασης του CTGF στους ασθενείς με ΙΠΙ συγκριτικά με την ομάδα μαρτύρων (p=0.001) και η έκφραση αυτή παρέμεινε μειωμένη μετά από θεραπεία και στις δύο ομάδες. Επιπλέον, οι παράγοντες TNFα, TGFβ, IL-4, IL-8 και IFNγ δεν διέφεραν στατιστικά μεταξύ ασθενών και ομάδας ελέγχου ούτε διέφεραν προ και μετά θεραπεία στις δύο ομάδες ασθενών. Συμπέρασμα: Τα ευρήματα της μελέτης υποδηλώνουν ότι τα κυψελιδικά μακροφάγα δεν είναι η κύρια πηγή των παραγόντων CTGF, TGFb, TNFα, IL-18, IL-4 και IFNγ στην ΙΠΙ. Επιπλέον, φάνηκε ότι η θεραπευτική παρέμβαση με AZA ή με IFN-γ-1b δεν είχε σαν αποτέλεσμα σημαντική αλλαγή της πρωτεϊνικής έκφρασης των παραγόντων αυτών, ούτε βελτίωση της πορείας της νόσου ή της αναπνευστικής λειτουργίας των ασθενών με ΙΠΙ

Expression of CTGF and TNFa in alveolar macrophages of patients with idiopathic pulmonary fibrosis before and after treatment with azathioprine or interferon-γ-1b

SUMMARY.Backgrou nd: Idiopathic pulmonary fibrosis (IPF) is a fatal lungdisorder the aetiology of which is unknown and for which there isno effective therapy. Connective tissue growth factor (CTGF) andtumour necrosis factor alpha (TNFα) have been reported to participatesignificantly in the pathogenesis of the disease. The role of alveolarmacrophages in the expression of these cytokines remains unclear.Materials and Methods: Samples of bronchoalveolar lavagefluid (BALF) derived from 20 newly diagnosed patients with IPF beforeand after 6 months of treatment with either interferon (IFN-γ-1b) andprednisolone (10 patients) or azathioprine (AZA) and prednisolone(10 patients) and from 10 normal subjects (control group) were usedfor the analysis of CTGF and TNFα protein expression in the alveolarmacrophages. The effectiveness of the two drug regimes on thepulmonary function tests (FEV1, FVC, DLCO) and PaO2 and PaCO2 ofthe patients with IPF was investigated. Results: Decreased CTGFprotein expression was detected in the patients with IPF comparedwith the control group (p=0.001). TNFα expression in IPF patientsdid not differ from that of the normal control subjects. Neither ofthe drug regimes affected the protein expression of these factorsorthe pulmonary function parameters. Co nclusion: These findingssuggest that the alveolar macrophages are not the main source ofCTGF and TNFα in IPF. Treatment with either AZA or IFN-γ-1b did notresult in any significant change in the protein expression of thesefactors. Pneumon 2011, 24(2):149-156

Comparative study of azathioprine-interferon g dispensing to patients with idiopathic pulmonary fibrosis

SUMMARY. Introduction: Idiopathic pulmonary fibrosis (IPF) is characterized by progressive deterioration of lung function, leading ultimately to death. No pharmacological treatment has been found to stabilize the evolution of the disease, but interferon-g and azathioprine have been used as therapeutic options. Aim: To compare the effectiveness of treatment with interferon-g plus low dose prednisone or azathioprine plus low dose prednisone in patients with IPF. Materials and methods: Patients newly diagnosed with IPF were recruited, 22 in total, of whom 10 received azathioprine plus prednisone and 12 patients received interferon-g plus prednisone for six months. Clinical evaluation, lung function tests, HRCT, bronchoscopy and bronchoalveolar lavage (BAL) were performed at baseline and after six months of treatment. Results: All patients were alive after six months of treatment. No statistically significant difference between the two groups was detected regarding clinical deterioration, inflammatory biomarkers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and BAL cell sub-populations. There was a trend, not statistically significant, towards a greater reduction in forced vital capacity and diffusing capacity for carbon monoxide in the interferon-g group. Conclusion: Interferon-g does not offer any therapeutic advantage over azathioprine as regards the clinical course, lung function tests and BAL cell counts of patients with IPF. Pneumon 2009, 22(3):240-253

Decreased Apoptotic Rate of Alveolar Macrophages of Patients with Idiopathic Pulmonary Fibrosis

Introduction. Increased apoptosis of epithelial cells and decreased apoptosis of myofibroblasts are involved in the pathogenesis of IPF. The apoptotic profile of alveolar macrophages (AMs) in IPF is unclear. Aim. To investigate whether AMs of patients with IPF exhibit a different apoptotic profile compared to normal subjects. Methods. We analyzed, by immunohistochemistry, the expression of the apoptotic markers fas, fas ligand , bcl-2, and bax in AM obtained from bronchoalveolar lavage fluid (BALF) of 20 newly diagnosed, treatment-naive IPF patients and of 16 controls. Apoptosis of AM was evaluated by Apoptag immunohistochemistry. IPF patients received either interferon-g and corticosteroids or azathioprine and corticosteroids for six months. Results. BALF AMs undergoing apoptosis were significantly less in IPF patients. No difference was found in the expression of fas or fas ligand, bcl-2 and bax between IPF and control group. No difference was found between the respiratory function parameters of the two treatment groups after six months. A positive correlation was found between the number of bcl-2 positive stained macrophages and DLCO after treatment. Conclusions. The decreased apoptotic rate of AM of patients with IPF is not associated with decreased expression of apoptosis mediators involved in the external or internal apoptotic pathway