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Overexpression of IL-1 Receptor Antagonist Attenuates Obliterative Bronchiolitis in Murine Tracheal Transplant Model
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The Journal of Heart and Lung Transplantation, Vol 32, No 4S, April 2013
rapidly frozen near TLC and Multi detector volumetric computed tomography (MDCT) scans were performed on the intact specimen, which was then cut into 2cm thick slices and cores of tissue removed from each slice were fixed, dried and examined by mCT. Results: The MDCT demonstrated a normal number of airways (BOS vs. control) at each generation when all the branching pathways were examined, but, starting from generation 5, airways o2mm appeared to narrow and become occluded. mCT analysis demonstrated an equal number of terminal bronchioles in BOS (4.5⫾0.5/mL) vs controls (4.9⫾0.3/mL, p¼0.66) and there was no difference in mCT diameter (358⫾15mm vs 375⫾15mm) and area (0.160⫾0.011mm2 vs 0.159⫾0.010mm2) of terminal bronchioles (p¼0.39 and p¼0.93). Two different types of occlusive lesion were identified within more proximal airways: one with complete obliteration of the airway lumen (obliterative bronchiolitis), due to increased collagen within the lamina propria of the wall (1C-D). And a second type where a narrowed lumen is occluded by material that does not appear to be collagen (1A-B). In both cases these lesions reopen into airways with normal morphometry that lead to alveoli after a mean length of 1063⫾157mm. Conclusions: The airway occlusion associated with BOS is located in preterminal bronchioles, re-opening into normal appearing airways before the terminal bronchioles are reached. 259 Overexpression of IL-1 Receptor Antagonist Attenuates Obliterative Bronchiolitis in Murine Tracheal Transplant Model V.Y. Sacher,1 R.C. Levitt,2 P. Ruiz,1 Y. Wei,1 S. Gupta,3 R.I. VazquezPadron,1 S.M. Pham.1 1Surgery, University of Miami, Miami, FL; 2 Anesthesiology, University of Miami, Miami, FL; 3Microbiology and Immunology, University of Miami, Miami, FL. Purpose: Obliterative bronchiolitis (OB) remains an impediment to long-term survival after lung transplantation. IL-1 beta (IL-1b) has been studied as proinflammatory and profibrotic factor in allograft chronic lung tissue rejection. IL-1 receptor antagonist (IL-1rn) is competitive inhibitor to IL-1b and mediates the anti-inflammatory and antifibrotic effects of mesenchymal stem cells during lung injury. We hypothesized that IL-1rn overexpression prevents the progression of OB in an established heterotopic tracheal transplantation model. Methods and Materials: Tracheas from BALB/c mice were implanted and wrapped in the omentum of BALB/c (isografts), C57BL/6
(allografts), IL-1rn overexpression (IL-1rnþ/þ), IL-1rn knock out (IL-1rn-/-) mice. The tracheas explanted after 21 days were evaluated histologically, and real time-PCR for IL-1rn, IL-1b, IL-1 alpha (IL-1a) mRNA levels. Cytokine quantification for these proteins in plasma samples was done by ELISA 21 days after surgery. Results: IL-1rn overexpression mice showed significant reduction of the tracheal luminal occlusion compared to control allograft animals (po0.0001). The gene expression of IL-1rn in tracheal tissue explanted from IL-1rn overexpression animals was increased (50-150 fold) as compared to tissue from control isograft, allograft and IL-1rn knock out animals (po0.0001). There was also significant increase in plasma IL-1rn levels in the IL-1rn þ/þ animals as compared to other groups (po0.0001). Conclusions: The results suggest that IL-1rn overexpression protects against experimental OB. These findings provide new insights into the mechanisms of lung chronic rejection and may lead to new strategy for the treatment of OB. 260 Transbronchial Brush (TBBr) Detection of Activated T Cells Identifies Patients at Risk of Progressive Bronchiolitis Obliterans Syndrome (BOS) D.C. Chambers,1,2 A. Fiene,1 K. Sinclair,1 L. Samson,1 M.E. Tan,1 T. Sladden,1 P.M.A. Hopkins,1,2 S.T. Yerkovich.1,2 1Queensland Lung Transplant Service, The Prince Charles Hospital, Brisbane, QLD, Australia; 2School of Medicine, The University of Queensland, Brisbane, Qld, Australia. Purpose: Lymphocytic bronchiolitis (LB) is one of the strongest risk factors for subsequent BOS, but the operating characteristics of transbronchial biopsy (TBBx) for LB diagnosis are poor. We hypothesised that combining transbronchial brush (TBBr) with flow cytometric phenotyping of epithelial lymphocyte populations would better assess allograft health. Methods and Materials: TBBr and bronchial brushings were performed to obtain small and large airway epithelium respectively at surveillance and diagnostic bronchoscopy. Cells were stained with cytokeratin, CD3, CD8, CD103 (intraepithelial T cells) and granzyme B (GrB) before flow cytometry was performed. Data are presented as median (interquartile range) percentage of total cells. Results: 67 patients (23 CF, 21 COPD, 13 IPF, 91% bilateral, median age 49.7 yrs, 51% male, median 14.3 (3.9-49.5) months post-transplant) were assessed. Bronchial CD3þ(p¼0.006), CD8þ (p¼0.044), CD3þCD103þ (p¼0.022) and CD3þGrBþ (p¼0.009) cells were increased in those undergoing diagnostic bronchoscopy (n¼23) and those with A grade rejection (n¼5 A1, n¼3 A2, n¼1 A3) compared to those having a surveillance bronchoscopy (n¼35). In contrast, only the CD3þGrBþ subset was increased in the bronchiolar region (1.44 (0.93.0) vs 0.73 (0.4-1.5), p¼0.005). There was no correlation between any subset and immunosuppressant dose or level, however treatment of A grade rejection with augmented immunosuppression led to a reduction in the bronchial CD3þ (p¼0.043) and bronchiolar CD3þGrBþ (p¼0.012) subsets. Only the bronchiolar CD3þGrBþ fraction was increased (1.92 (1.0-4.1) vs 1.0 (0.4-1.7), p¼0.028) in the 18% of the cohort who experienced progressive BOS in the following 13.3 (5.818.3) months. TBBx did not predict outcome. Conclusions: Infiltration of the bronchiolar region by activated CD3þGrBþ T cells is associated with subsequent allograft dysfunction. As this subset is responsive to augmented immunosuppression, vigilant immune monitoring using TBBr could prevent BOS. 261 Chronic Rejection Lesions Are Decreased in IL-17 KO Mice after Orthotopic Lung Transplantation A. Vaneylen,1 E. Vandermeulen,1 J. Somers,2 S.E. Verleden,1 D. Ruttens,1 R. Vos,3 D.E. Van Raemdonck,2 W. Jungraithmayr,4 B.M. Vanaudenaerde,1 G.M. Verleden.3 1Lab of Pneumology, KU Leuven, Leuven, Belgium; 2Lab of Experimental Thoracic Surgery, KU Leuven, Leuven, Belgium; 3Lung Transplant Unit, University Hospital, Leuven, Belgium; 4Division of Thoracic Surgery, University Hospital, Zurich, Switzerland.
The Journal of Heart and Lung Transplantation, Vol 32, No 4S, April 2013
rapidly frozen near TLC and Multi detector volumetric computed tomography (MDCT) scans were performed on the intact specimen, which was then cut into 2cm thick slices and cores of tissue removed from each slice were fixed, dried and examined by mCT. Results: The MDCT demonstrated a normal number of airways (BOS vs. control) at each generation when all the branching pathways were examined, but, starting from generation 5, airways o2mm appeared to narrow and become occluded. mCT analysis demonstrated an equal number of terminal bronchioles in BOS (4.5⫾0.5/mL) vs controls (4.9⫾0.3/mL, p¼0.66) and there was no difference in mCT diameter (358⫾15mm vs 375⫾15mm) and area (0.160⫾0.011mm2 vs 0.159⫾0.010mm2) of terminal bronchioles (p¼0.39 and p¼0.93). Two different types of occlusive lesion were identified within more proximal airways: one with complete obliteration of the airway lumen (obliterative bronchiolitis), due to increased collagen within the lamina propria of the wall (1C-D). And a second type where a narrowed lumen is occluded by material that does not appear to be collagen (1A-B). In both cases these lesions reopen into airways with normal morphometry that lead to alveoli after a mean length of 1063⫾157mm. Conclusions: The airway occlusion associated with BOS is located in preterminal bronchioles, re-opening into normal appearing airways before the terminal bronchioles are reached. 259 Overexpression of IL-1 Receptor Antagonist Attenuates Obliterative Bronchiolitis in Murine Tracheal Transplant Model V.Y. Sacher,1 R.C. Levitt,2 P. Ruiz,1 Y. Wei,1 S. Gupta,3 R.I. VazquezPadron,1 S.M. Pham.1 1Surgery, University of Miami, Miami, FL; 2 Anesthesiology, University of Miami, Miami, FL; 3Microbiology and Immunology, University of Miami, Miami, FL. Purpose: Obliterative bronchiolitis (OB) remains an impediment to long-term survival after lung transplantation. IL-1 beta (IL-1b) has been studied as proinflammatory and profibrotic factor in allograft chronic lung tissue rejection. IL-1 receptor antagonist (IL-1rn) is competitive inhibitor to IL-1b and mediates the anti-inflammatory and antifibrotic effects of mesenchymal stem cells during lung injury. We hypothesized that IL-1rn overexpression prevents the progression of OB in an established heterotopic tracheal transplantation model. Methods and Materials: Tracheas from BALB/c mice were implanted and wrapped in the omentum of BALB/c (isografts), C57BL/6
(allografts), IL-1rn overexpression (IL-1rnþ/þ), IL-1rn knock out (IL-1rn-/-) mice. The tracheas explanted after 21 days were evaluated histologically, and real time-PCR for IL-1rn, IL-1b, IL-1 alpha (IL-1a) mRNA levels. Cytokine quantification for these proteins in plasma samples was done by ELISA 21 days after surgery. Results: IL-1rn overexpression mice showed significant reduction of the tracheal luminal occlusion compared to control allograft animals (po0.0001). The gene expression of IL-1rn in tracheal tissue explanted from IL-1rn overexpression animals was increased (50-150 fold) as compared to tissue from control isograft, allograft and IL-1rn knock out animals (po0.0001). There was also significant increase in plasma IL-1rn levels in the IL-1rn þ/þ animals as compared to other groups (po0.0001). Conclusions: The results suggest that IL-1rn overexpression protects against experimental OB. These findings provide new insights into the mechanisms of lung chronic rejection and may lead to new strategy for the treatment of OB. 260 Transbronchial Brush (TBBr) Detection of Activated T Cells Identifies Patients at Risk of Progressive Bronchiolitis Obliterans Syndrome (BOS) D.C. Chambers,1,2 A. Fiene,1 K. Sinclair,1 L. Samson,1 M.E. Tan,1 T. Sladden,1 P.M.A. Hopkins,1,2 S.T. Yerkovich.1,2 1Queensland Lung Transplant Service, The Prince Charles Hospital, Brisbane, QLD, Australia; 2School of Medicine, The University of Queensland, Brisbane, Qld, Australia. Purpose: Lymphocytic bronchiolitis (LB) is one of the strongest risk factors for subsequent BOS, but the operating characteristics of transbronchial biopsy (TBBx) for LB diagnosis are poor. We hypothesised that combining transbronchial brush (TBBr) with flow cytometric phenotyping of epithelial lymphocyte populations would better assess allograft health. Methods and Materials: TBBr and bronchial brushings were performed to obtain small and large airway epithelium respectively at surveillance and diagnostic bronchoscopy. Cells were stained with cytokeratin, CD3, CD8, CD103 (intraepithelial T cells) and granzyme B (GrB) before flow cytometry was performed. Data are presented as median (interquartile range) percentage of total cells. Results: 67 patients (23 CF, 21 COPD, 13 IPF, 91% bilateral, median age 49.7 yrs, 51% male, median 14.3 (3.9-49.5) months post-transplant) were assessed. Bronchial CD3þ(p¼0.006), CD8þ (p¼0.044), CD3þCD103þ (p¼0.022) and CD3þGrBþ (p¼0.009) cells were increased in those undergoing diagnostic bronchoscopy (n¼23) and those with A grade rejection (n¼5 A1, n¼3 A2, n¼1 A3) compared to those having a surveillance bronchoscopy (n¼35). In contrast, only the CD3þGrBþ subset was increased in the bronchiolar region (1.44 (0.93.0) vs 0.73 (0.4-1.5), p¼0.005). There was no correlation between any subset and immunosuppressant dose or level, however treatment of A grade rejection with augmented immunosuppression led to a reduction in the bronchial CD3þ (p¼0.043) and bronchiolar CD3þGrBþ (p¼0.012) subsets. Only the bronchiolar CD3þGrBþ fraction was increased (1.92 (1.0-4.1) vs 1.0 (0.4-1.7), p¼0.028) in the 18% of the cohort who experienced progressive BOS in the following 13.3 (5.818.3) months. TBBx did not predict outcome. Conclusions: Infiltration of the bronchiolar region by activated CD3þGrBþ T cells is associated with subsequent allograft dysfunction. As this subset is responsive to augmented immunosuppression, vigilant immune monitoring using TBBr could prevent BOS. 261 Chronic Rejection Lesions Are Decreased in IL-17 KO Mice after Orthotopic Lung Transplantation A. Vaneylen,1 E. Vandermeulen,1 J. Somers,2 S.E. Verleden,1 D. Ruttens,1 R. Vos,3 D.E. Van Raemdonck,2 W. Jungraithmayr,4 B.M. Vanaudenaerde,1 G.M. Verleden.3 1Lab of Pneumology, KU Leuven, Leuven, Belgium; 2Lab of Experimental Thoracic Surgery, KU Leuven, Leuven, Belgium; 3Lung Transplant Unit, University Hospital, Leuven, Belgium; 4Division of Thoracic Surgery, University Hospital, Zurich, Switzerland.