0041-1337/01/7107-966/0 TRANSPLANTATION Vol. 71, 966–976, No. 7, April 15, 2001 Copyright © 2001 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. ACTIVATION OF HUMAN AIRWAY EPITHELIAL CELLS BY NON- HLA ANTIBODIES DEVELOPED AFTER LUNG TRANSPLANTATION: A POTENTIAL ETIOLOGICAL FACTOR FOR BRONCHIOLITIS OBLITERANS SYNDROME 1 ANDR ´ ES JARAMILLO, 2 BASHOO NAZIRUDDIN, 2 LEIYING ZHANG, 2 SCOTT I. REZNIK, 2 MICHAEL A. SMITH, 2 AVIVA A. ALOUSH, 2 ELBERT P. TRULOCK, 3 G. ALEXANDER PATTERSON, 2 AND T. MOHANAKUMAR 2,4,5 Departments of Surgery, Medicine, and Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110-1093 Background. The main cause of morbidity and mor- tality after lung transplantation (LT) is bronchiolitis obliterans syndrome (BOS). Anti-HLA antibodies de- velopment after LT has been shown to play an impor- tant role in BOS pathogenesis. However, the nature of non-HLA antibodies developed after LT and their role in BOS pathogenesis have not been determined. Methods. Sera from 16 BOS patients and 11 BOS patients were collected at 12, 24, 36, and 48 months after LT. Anti-HLA class I and class II antibodies were absorbed with pooled human platelets and pooled hu- man lymphoblastoid cell lines, respectively. Then, the presence of non-HLA antibodies against several cell lines from different origin was determined by flow cytometric analysis. Antibody-positive samples were tested for induction of proliferation and growth factor production in two selected airway epithelial cell (AEC) lines. Results. Five of 16 BOS patients (31.2%) and 0 of 11 BOS- patients (0%) developed anti-AEC antibodies af- ter LT (P0.05). No reactivity against endothelial cells, lymphocytes, monocytes, or granulocytes was detected. Further analysis of two selected sera demon- strated the development of reactivity against a 60-kDa antigen expressed by 60% of AEC lines and only 12% of cell lines from other tissues. Antibody binding to this antigen induced intracellular Ca  influx, tyrosine phosphorylation, proliferation, and up-regulation of transforming growth factor- and heparin-binding epidermal growth factor mRNA transcription in AECs. Conclusions. These results indicate that anti-AEC antibodies may play a role in the immunopathogenesis of BOS in the absence of anti-HLA antibodies. INTRODUCTION The main cause of long-term morbidity and mortality after lung transplantation (LT) is the development of bronchiolitis obliterans syndrome (BOS), which is considered to represent chronic lung allograft rejection (1). BOS is a fibroproliferative process identified histologically by inflammation and fibrosis of the lamina propria and lumen and clinically by progressive airflow obstruction (2). The specific etiology and pathogene- sis of BOS are still unclear. However, a growing body of evidence suggests that BOS is caused by the immune re- sponse developed against antigenic determinants expressed by airway epithelial cells (AEC) and endothelial cells (EC) of the lung allograft (3). In fact, it has been shown that en- hanced immunosuppression slows the progression of the dis- ease (4, 5). Previous studies have shown that AECs are immunological targets during lung allograft rejection (6–8). Interestingly, multiple studies have shown that, upon activation, epithelial cells from different tissues have the ability to produce high quantities of several growth factors including epidermal growth factor (EGF) (9), heparin-binding EGF (HB-EGF) (10), endothelin (ET)-1 (11), basic fibroblast growth factor (bFGF) (10, 12), granulocyte-monocyte colony-stimulating factor (GM-CSF) (13), insulin-like growth factor (IGF)-1 (14), platelet-derived growth factor (PDGF) (10, 12), and trans- forming growth factor (TGF)- (10, 12). All these growth factors are involved in the activation of proliferation of fibro- blasts and smooth muscle cells (14 –22). Growth factor-mediated proliferation of fibrous tissue, pre- sumably in response to acute and chronic lung allograft in- jury, leads to airway constriction and luminal obliteration as BOS develops. The specific growth factors and their levels of expression involved in the regulation of the tissue remodel- ing observed during the course of BOS are still unknown. Heterotopic allogeneic transplantation of mouse or rat tra- cheas reveal fibroproliferation with pathology identical to BOS although similar isograft developed the same lesion only when exposed to exogenous PDGF or bFGF (23, 24). Only PDGF and TGF- have been found to be increased during the process of BOS development after LT in humans (25–27). ET-1 has been found to be steadily increased in serial bron- choalveolar lavage samples collected from LT patients (28). However, there is no information regarding the levels of this growth factor before or at the time of BOS development. 1 This work was supported by the National Institute of Health Grant No. HL56643. AJ is a recipient of the National Research Service Award AI07163; SIR is a recipient of the National Research Service Award HL07776; MAS is a recipient of the National Re- search Service Award HL09557; and TM is a recipient of the Jac- queline G. and William E. Maritz endowed Professorship in Immu- nology and Oncology. 2 Department of Surgery. 3 Department of Medicine. 4 Department of Pathology and Immunology. 5 Address correspondence to: T. Mohanakumar, PhD, Department of Surgery, Washington University School of Medicine, Box 8109- 3328.CSRB, 660 South Euclid Avenue, St. Louis, MO 63110-1093. 966