Rapamycin inhibits macropinocytosis and mannose receptor–mediated endocytosis by bone marrow–derived dendritic cells

Abstract

IntroductionDendritic cells (DCs) arise from CD34 ϩ bone marrow (BM) stem cells and represent a heterogenous population of ubiquitously distributed antigen-presenting cells (APCs) that play critical roles as initiators and modulators of immune responses. 1,2 Among the most striking features underlying the efficiency of DCs as APCs is their unsurpassed capacity to take up antigens via constitutive macropinocytosis and mannose receptor-mediated endocytosis 3 and to subsequently process and present major histocompatibility complex (MHC)-antigen complexes on their surface. 1 The capacity of DCs to endocytose and to present antigens is under tight developmental control: immature DCs are excellent at internalizing antigens but express low surface levels of MHC class II molecules, whereas mature DCs down-regulate endocytotic activity and up-regulate MHC class II and costimulatory molecules (CD40, CD80, CD86) that promote T-cell activation. 1 Anti-inflammatory drugs such as corticosteroids 4 or salicylates 5 suppress DC maturation and as a consequence enhance their endocytotic activity. Recent reports point toward Rho family proteins Cdc42 6 and Rac, 7 as well as aquaporins, 8 as important factors that regulate DC endocytosis. Rapamycin is a potent immunosuppressive macrolide, isolated from Streptomyces hygroscopicus, that inhibits downstream signaling from the targets of rapamycin proteins (TORs) by forming a complex with its intracellular receptor FK506-binding protein 12 (FKBP12) and TORs. 9 It is used clinically to prevent and treat allograft rejection. [10][11][12] Interaction of the rapamycin-FKBP12 complex with TORs results in inhibition of multiple biochemical pathways (eg, p70 S6 kinase, cyclin-dependent kinases, translational effector proteins) that are critical for cytokine/growth factor-induced cellular proliferation, ribosome biosynthesis, translation initiation, and cell cycle progression into S phase. 9,13 In view of the paucity of information concerning the influence of rapamycin on APC function and its well-documented inhibitory effects on protein synthesis, we analyzed the impact of rapamycin on DC endocytosis. Our results indicate that rapamycin is an inhibitor of DC endocytosis in vitro and in vivo and that molar excess of the structurally related immunophilin ligand FK506 partially reverses its inhibitory effects. Study design Generation of BM-derived DCsMale C57BL/10J (I-A b ) mice, 8 to 12 weeks old, were purchased from the Jackson Laboratory (Bar Harbor, ME). BM-derived DCs were generated and characterized as described, 5 with minor modifications. Briefly, BM cells were cultured for 7 days in RPMI-1640 with 10% heat-inactivated fetal calf serum (FCS), L-glutamine, nonessential amino acids, sodium pyruvate, penicillin-streptomycin, N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid (HEPES), 2-mercaptoethanol (2-ME) (all from Life Technologies, Gaithersburg, MD), 1000 U/mL murine granulocytemacrophage colony-stimulating factor (GM-CSF; Schering-Plough, Kenilworth, NJ) Ϯ 1000 U/mL murine interleuki...

Immunosuppression using the mTOR inhibition mechanism affects replacement of rat liver with transplanted cells

“…11 In this context, Rapa can inhibit dendritic cell activity as well as chemokine-dependent leukocyte migration, both of which are major components of the inflammatory response. 25,26 We found that Rapa impaired hepatic cytokine expression at the mRNA level after cell transplantation, including that of monocyte chemotactic protein 1 mRNA, which mediates monocyte/macrophage chemotaxis, as well as mRNAs of macrophage inflammatory protein 2 and cytokine-in- duced neutrophil chemoattractant, both of which are potent neutrophil chemoattractants. Cell engraftment in Rapa-treated rats most likely benefited from these perturbations, similar to the depletion of Kupffer cells in earlier studies, 11 although our studies did not verify changes in cytokines at the protein level.…”

“…Experiments were repeated at least twice for reproducing results.Tx, transplantation. (25,50, and 100 mg/kg/d) were used either alone or in combination (n ϭ 3 DPPIV Ϫ rats each). By themselves, Tacro, Rapa, or MMF in these dosages did not yield allograft survival to 7 days.…”

The Selective Estrogen Receptor Modulators, Tamoxifen and Raloxifene, Impair Dendritic Cell Differentiation and Activation

“…fluorescein (DexFITC) were obtained from Molecular Probes. The conditions for Ag uptake were chosen based upon methods used in similar studies (37,38). At day 7, BMDC were harvested from DEF medium cultures and washed with PBS.…”

Correlation of dendritic cell maturation and the formation of aggregates of poly-ubiquitinated proteins in the cytosol

“…In the cell, Rapamycin binds to FKBPs (FK506 binding proteins), a speciWc family of cytosolic proteins (immunophilins) [17,18]. It is known that mTOR inhibition in APCs during the time of culturing attenuates cytokine production and surface marker expression as well as it prevents macropinocytosis and receptor mediated endocytosis [16]. Additionally, in higher doses Rapamycin induces apoptosis.…”

“…Rapamycin, the potent inhibitor for the mammalian target of Rapamycin (mTOR), is previously described as immunosuppressive drug [16]. This macrolide antibiotic, produced by Streptomyces hygroscopicus, was introduced to prevent allograft rejection.…”