Lipid raft facilitated ligation of K-α1-tubulin by specific antibodies on epithelial cells: Role in pathogenesis of chronic rejection following human lung transplantation

Research highlights: → Addition of KAT Abs (+) sera to NHBE culture causes upregulation of growth factors. → Cholesterol depletion causes down regulation of growth factor expression. → Cholesterol depletion is accompanied by loss of membrane bound caveolin. → Thus, we demonstrate lipid raft are critical for efficient ligation of the KAT Abs. -- Abstract: Long term function of human lung allografts is hindered by development of chronic rejection manifested as Bronchiolitis Obliterans Syndrome (BOS). We have previously identified the development of antibodies (Abs) following lung transplantation to K-α1-tubulin (KAT), an epithelial surface gap junction cytoskeletal protein, in patients who develop BOS. However, the biochemical and molecular basis of the interactions and signaling cascades mediated by KAT Abs are yet to be defined. In this report, we investigated the biophysical basis of the epithelial cell membrane surface interaction between KAT and its specific Abs. Towards this, we analyzed the role of the lipid raft-domains in the membrane interactions which lead to cell signaling and ultimately increased growth factor expression. Normal human bronchial epithelial (NHBE) cells, upon specific ligation with Abs to KAT obtained either from the serum of BOS(+) patients or monoclonal KAT Abs, resulted in upregulation of growth factors VEGF, PDGF, and bFGF (6.4 ± 1.1-, 3.2 ± 0.9-, and 3.4 ± 1.1-fold increase, respectively) all of which are important in the pathogenesis of BOS. To define the role for lipid raft in augmenting surface interactions, we analyzed the changes in the growth factor expression pattern upon depletion and enrichment with lipid raft following the ligation of the epithelial cell membranes with Abs specific for KAT. NHBE cells cultured in the presence of β-methyl cyclodextran (βMCD) had significantly reduced growth factor expression (1.3 ± 0.3, vs βMCD untreated being 6.4 ± 1.1-fold increase) upon stimulation with KAT Abs. Depletion of cholesterol on NHBE cells upon treatment with βMCD also resulted in decreased partitioning of caveolin in the membrane fraction indicating a decrease in raft-domains. In conclusion, our results demonstrate an important role for lipid raft-mediated ligation of Abs to KAT on the epithelial cell membrane, which results in the upregulation of growth factor cascades involved in the pathogenesis of BOS following human lung transplantation.