Chronic Rejection Pathology after Orthotopic Lung Transplantation in Mice: The Development of a Murine BOS Model and Its Drawbacks

Almost all animal models for chronic rejection (CR) after lung transplantation (LTx) fail to resemble the human situation. It was our attempt to develop a representative model of CR in mice. Orthotopic LTx was performed in allografts receiving daily immunosuppression with steroids and cyclosporine. Controls included isografts and mice only undergoing thoracotomy (SHAM). Allografts were sacrificed 2, 4, 6, 8, 10 or 12 weeks after LTx. Pulmonary function was measured repeatedly in the 12w allografts, isografts and SHAM mice. Histologically, all allografts demonstrated acute rejection (AR) around the blood vessels and airways two weeks after LTx. This decreased to 50–75% up to 10 weeks and was absent after 12 weeks. Obliterative bronchiolitis (OB) lesions were observed in 25–50% of the mice from 4–12 weeks. Isografts and lungs of SHAM mice were normal after 12 weeks. Pulmonary function measurements showed a decline in FEV0.1, TLC and compliance in the allografts postoperatively (2 weeks) with a slow recovery over time. After this initial decline, lung function of allografts increased more than in isografts and SHAM mice indicating that pulmonary function measurement is not a good tool to diagnose CR in a mouse. We conclude that a true model for CR, with clear OB lesions in about one third of the animals, but without a decline in lung function, is possible. This model is an important step forward in the development of an ideal model for CR which will open new perspectives in unraveling CR pathogenesis and exploring new treatment options

Neutrophilic Reversible Allograft Dysfunction (NRAD) and Restrictive Allograft Syndrome (RAS)

Lung transplantation is currently considered as an ultimate live-saving treatment for selected patients suffering from end-stage pulmonary disease. Long-term survival, however, is hampered by chronic rejection, or chronic lung allograft dysfunction (CLAD). Recently, various phenotypes within CLAD have been identified, challenging the established clinical definition of bronchiolitis obliterans syndrome (BOS). Some patients with presumed BOS, for instance, demonstrate an important improvement in forced expiratory volume in the first second of expiration (FEV1) after treatment with azithromycin. These patients are characterized by the presence of excess (≥ 15%) bronchoalveolar lavage (BAL) neutrophils, in absence of concurrent infection. This phenotype of CLAD has been redefined as neutrophilic reversible allograft dysfunction (NRAD), and these patients generally have a very good prognosis after diagnosis. Another group of patients with CLAD develop a restrictive rather than an obstructive pulmonary function defect (defined as a decline in total lung capacity of at least 10%) and demonstrate persistent interstitial and ground-glass opacities on chest computed tomographic (CT) scan. This phenotype is called restrictive allograft syndrome (RAS), and patients with RAS have a much worse prognosis after diagnosis. This review further discusses both of these CLAD phenotypes that do not fit the classical definition of BOS. Potential pathophysiological mechanisms, etiology, diagnosis, prognosis, and treatments are discussed.status: publishe

Involvement of interleukin-17 during lymphocytic bronchiolitis in lung transplant patients

BACKGROUND: Interleukin-17 (IL-17) is involved in autoimmune and chronic pulmonary diseases and linked with neutrophilic inflammation. Azithromycin reduces and prevents broncholaveolar lavage (BAL) neutrophilia after lung transplantation (LTx). In this investigation we assessed the involvement of IL-17 in different post-transplant complications in human LTx biopsies. METHODS: Immunohistochemical staining against IL-17A was performed on biopsies of LTx patients with either chronic rejection, acute A-grade rejection (A > 2B0), lymphocytic bronchiolitis (LB), infection, and stable patients. Biopsies of 7 patients with LB were stained before and after azithromycin treatment. IL-17+ cells were quantified as number per square millimeter of lamina propria. Double staining with CD4/CD8 was performed to determine the origin of IL-17. RESULTS: In the LB group, biopsies showed a significant presence of IL-17+ cells/mm2 of lamina propria compared with the stable, acute A-grade/chronic rejection and infection groups (p < 0.0001). The number of IL-17+ cells on biopsy correlated with percent BAL (%BAL) neutrophilia (r = 0.34, p = 0.0056). Azithromycin reduced both %BAL neutrophilia and IL-17+ cells (both p = 0.016) in the LB group. CD8+ cells were the major source of IL-17. CONCLUSIONS: IL-17+ / CD8+ cells are present in LB after LTx but not in acute A-grade/chronic rejection nor during infection. Moreover, azithromycin significantly decreased the number of IL-17+ cells in the airway wall, which may further explain its effect on BAL neutrophilia.status: publishe

Azithromycin Attenuates Fibroblast Growth Factors Induced Vascular Endothelial Growth Factor Via p38(MAPK) Signaling in Human Airway Smooth Muscle Cells

The airways in asthma and COPD are characterized by an increase in airway smooth muscle (ASM) mass and bronchial vascular changes associated with increased expression of pro-angiogenic growth factors, such as fibroblast growth factors (FGF-1 and FGF-2) and vascular endothelial growth factor (VEGF). We investigated the contribution of FGF-1/-2 in VEGF production in ASM cells and assessed the influence of azithromycin and dexamethasone and their underlying signaling mechanisms. Growth-synchronized human ASM cells were pre-treated with MAPK inhibitors, U0126 for ERK1/2(MAPK) and SB239063 for p38(MAPK) as well as with dexamethasone or azithromycin, 30 min before incubation with FGF-1 or FGF-2. Expression of VEGF (VEGF-A, VEGF121, and VEGF165) was assessed by quantitative PCR, VEGF release by ELISA and MAPK phosphorylation by Western blotting. Both FGF-1 and FGF-2 significantly induced mRNA levels of VEGF-A, VEGF121, and VEGF165. The VEGF protein release was increased 1.8-fold (FGF-1) and 5.5-fold (FGF-2) as compared to controls. Rapid transient increase in ERK1/2(MAPK) and p38(MAPK) phosphorylation and subsequent release of VEGF from FGF-1 or FGF-2-treated ASM cells were inhibited by respective blockers. Furthermore, azithromycin and dexamethasone significantly reduced both the VEGF release and the activation of p38(MAPK) pathway in response to FGF-1 or FGF-2 treatment. Our Results demonstrate that FGF-1 and FGF-2 up-regulate VEGF production via ERK1/2(MAPK) and p38(MAPK) pathways. Both azithromycin and dexamethasone elicited their anti-angiogenic effects via p38(MAPK) pathway in vitro, thereby suggesting a possible therapeutic approach to tackle VEGF-mediated vascular remodeling.status: publishe

Weight evolution of isografts, allografts and SHAM mice.

<p>POD: post-operative day; LTx: lung transplantation; SHAM: only thoracotomy.</p

Distribution of the histological lesions of the airways at the different time points after lung transplantation.

<p>Perivascular lesions were not taken into consideration.</p

Study design.

<p>Allograft: from BALB/C to C57BL6, isografts from C57BL6 to C57BL6. ☆ represents the time point of lung function measurement, at the end of each time bar animals were sacrificed and BAL and histology were performed.</p

Dose-response curve of Cyclosporine in healthy animals after daily subcutaneous injection during two weeks.

<p>Dose-response curve of Cyclosporine in healthy animals after daily subcutaneous injection during two weeks.</p

Histology of allografts showing lymphocytic bronchiolitis lesions.

<p>A) early after transplantation (2w) and B) late after transplantation (12w). Left side: HE staining, right side: Sirius Red (SR) staining. 2w after transplantation allografts showed enlargement of broncho-vascular axes with infiltration of lymphocytes around airways (Aw) and blood vessels (Bv). Sirius Red staining shows collagen both perivascular as peribronchial. After 12w, lungs completely recovered with some leftover damage seen in pigmented macrophages (arrow).</p