Sequential analysis of multidrug resistance protein-1 expression and function in cardiac transplant patients: A possible mechanism of therapy-resistant acute rejection

Abstract

Multidrug resistance protein-1 (mdr1) is a well characterized membrane protein, expressed in a variety of cell types. In cancer cells, an overexpression of mdr1 has the function of conferring drug-resistance. The exact physiological function of mdr1 constitutively expressed in normal cells still remains unclear. A goal of this work was to determine if there is an increase in expression of mdr1, above constitutive levels, on CD4+ and CD8+ T-lymphocytes, following cardiac transplantation. The expression of mdr1 was correlated with episodes of acute cardiac rejection in order to determine if mdr1 has the functional ability to confer immunosuppressive drug resistance. Drug resistant CD4+ and CD8+ T-lymphocytes could explain episodes of acute allograft rejection observed in a number of immunosuppressed patients. Immunochemical techniques measuring mdr1 were performed on endomyocardial biopsy specimens and peripheral blood mononuclear cells (PBMCs) isolated from cardiac transplant patients. Functional mdr1 assays and flow cytometry were performed on PBMCs isolated from cardiac transplant patients prior to transplantation and at longitudinal time points post-transplantation. The expression and function of mdr1 was correlated to the histological diagnosis of acute rejection. Immunochemical analysis of endomyocardial biopsy samples showed mdr1 expression localized on the plasma membrane of graft infiltrating mononuclear cells. Immunochemical analysis of PBMC samples showed a constitutive mdr1 expression on normal volunteer PBMCs and a increased expression of mdr1 on cardiac transplant PBMCs during episodes of acute rejection. Doxorubicin cytotoxicity assays showed an increased drug resistance profile in transplant patients compared to normal individuals. An mdr1 efflux assay demonstrated an increase efflux function in PBMCs during episodes of acute rejection. Flow cytometric analysis showed significant increases in the intensity of mdr1 expression on CD4+ and CD8+ T-lymphocytes in transplant patients compared to normal individuals. Flow cytometry also confirmed a significant increase in both the number of CD4+ and CD8+ T-lymphocytes expressing mdr1 and the intensity of mdr1 on these subtypes for those patients that had an episode of acute rejection compared to those transplant patients with no episodes of acute rejection. Thus, this study demonstrates an overexpression of a functionally active multidrug resistant protein-1 during episodes of acute rejection.