Phosphorylation of S6 ribosomal protein at serine 235/236 is a marker of human heart transplantation

Abstract

Antibody (Ab) ligation of HLA class I molecules on the surface of endothelial cells (EC) stimulates cell proliferation and survival pathways including activation of the PI3K/Akt pathway. We investigated phosphorylation of S6 ribosomal protein (p-S6RP), a downstream target of the PI3K/Akt/mTOR pathway in EC as a potential marker of Ab mediated-rejection(AMR) of heart allografts. Human aortic EC were treated with anti-HLA class I Ab and cell lysates were studied for phosphorylation of PI3K, Akt and S6 ribosomal protein by Western blot. Endomyocardial biopsies (n 65) from 35 heart transplant recipients (7 with AMR, 7 with acute cellular rejection (ACR)) were tested for the presence of p-S6RP using an anti-phospho serine 235/236 Ab. Diagnosis of AMR was based on histology and immunofluorescence or immunoperoxidase staining of paraffin-embedded tissue (CD68-positive intravascular macrophages). Diagnosis of ACR was based on ISHLT grading. The relationship between p-S6RP and rejection was analyzed by Fisher’s exact test.Treatment of EC with anti-class I Ab resulted in a time and dose dependent increase in phosphorylation of Akt, PI3K and the S6RP. To investigate these results in a relevant in vivo model we determined whether biopsy samples from transplant recipients diagnosed with AMR or ACR showed increased p-S6RP on the graft vasculature.Results showed show a significant association of p-S6RP staining with diagnosis of AMR (p 0.001). Four of 7 patients with ACR showed p-S6RPN staining (p NS). No association between p-S6RP staining with C4d deposition was observed. Study of the kinetics of p-S6RP staining showed that patients diagnosed with AMR display sustained positivity ( 144 days) compared to patients diagnosed with ACR ( 21days). These findings support the role of anti-class I Ab mediated signaling in AMR and indicate that phosphorylation of S6RP is a useful marker of AMR in cardiac allografts. Sustained p-S6RP is observed in patients with AMR supporting its role in chronic allograft vasculopathy.