Tacrolimus: In Vitro Effects on Myelopoiesis, Apoptosis, and CD11b Expression

Abstract

Tacrolimus is a common component of multi-drug immunosuppressive regimens that are used for the prevention of rejection in transplant recipients. Tacrolimus therapy has been associated with anemia after transplantation, and recent clinical evidence in children suggests its association with the development of neutropenia for which an alternative etiology is not apparent. Mechanisms of suspected tacrolimus-related neutropenia have not been previously elucidated. We hypothesized that this variety of neutropenia might be due to a negative effect of tacrolimus on neutrophil production and/or survival. We designed in vitro studies to determine the dose-dependent effect of tacrolimus on myeloid cell production and/or apoptosis. CD34+ cells and neutrophils isolated from umbilical cord blood of term gestations were cultured with tacrolimus (0-1,000 ng/ml). To evaluate apoptosis, cells cultured for 24 hours were stained with annexin V-fluorescein isothiocyanate (V-FITC) and 7-amino-actinomycin D (7-AAD) and analyzed by flow cytometry. For clonal analysis, CD34+ cells cultured in cytokine-enhanced semi-solid media were scored for their myeloid/erythroid mix colony forming units (CFU-Mix) and myeloid (CFU-GM) progenitor cell contents. Tacrolimus induced a dose-dependent enhancement of clonogenesis and survival of CD34+ cells at clinically relevant doses. Conversely, tacrolimus had no effect on the survival of mature neutrophils or on the upregulation of CD11b in response to chemotactic stimulation. In contrast to our initial hypothesis, we observed that tacrolimus at clinically relevant concentrations enhanced clonogenesis of neutrophil progenitors and promoted their survival. Our in vitro studies suggest that tacrolimus alone is unlikely to be a significant factor in the neutropenia observed during immunosuppressive therapy.