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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Hackstein, H. Articles by Thomson, A. W. Search for Related Content PubMed PubMed Citation Articles by Hackstein, H. Articles by Thomson, A. W. Related Collections Immunobiology Phagocytes Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 August 2002, Vol. 100, No. 3, pp. 1084-1087 BRIEF REPORT Rapamycin inhibits macropinocytosis and mannose receptor-mediated endocytosis by bone marrow-derived dendritic cells Holger Hackstein, Timucin Taner, Alison J. Logar, and Angus W. Thomson From the Thomas E. Starzl Transplantation Institute and Department of Surgery and the Department of Molecular Genetics and Biochemistry, University of Pittsburgh, PA, and the Institute of Clinical Immunology and Transfusion Medicine, Justus-Liebig University, Giessen, Germany.     Abstract Top Abstract Introduction Study design Results and discussion References Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that use 2 major pathways for antigen uptake: constitutive macropinocytosis and mannose receptor-mediated endocytosis. Efficient endocytosis is critical for DCs to fulfill their sentinel function in immunity. We investigated the influence of the immunosuppressive macrolide rapamycin on macropinocytosis of fluorescein isothiocyanate (FITC)-albumin and mannose receptor-mediated endocytosis of FITC-dextran by murine bone marrow-derived DCs by flow cytometry. The data show that (1) at a low, physiologically relevant concentration (1 ng/mL), rapamycin impairs macropinocytosis and mannose receptor-mediated endocytosis; (2) the effects are independent of DC maturation and can be demonstrated specifically in immature CD11c+ major histocompatibility complex (MHC) class IIlo DCs by 3-color flow cytometry; (3) inhibition of endocytosis is not related to apoptotic cell death; and (4) molar excess of the structurally related molecule FK506 inhibits the actions of rapamycin. The inhibitory effects of rapamycin on DC endocytosis were confirmed in vivo. To our knowledge, this is the first report that a clinically relevant immunosuppressant inhibits DC endocytosis. (Blood. 2002;100:1084-1087) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Dendritic cells (DCs) arise from CD34+ 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 endocytosis3 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 corticosteroids4 or salicylates5 suppress DC maturation and as a consequence enhance their endocytotic activity. Recent reports point toward Rho family proteins Cdc426 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.10-12 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 Top Abstract Introduction Study design Results and discussion References Generation of BM-derived DCs Male C57BL/10J (I-Ab) 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-N'-2-ethanesulfonic acid (HEPES), 2-mercaptoethanol (2-ME) (all from Life Technologies, Gaithersburg, MD), 1000 U/mL murine granulocyte-macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Kenilworth, NJ) ± 1000 U/mL murine interleukin 4 (IL-4) (R&D Systems, Minneapolis, MN). At day 2, 1-100 ng/mL rapamycin (Sigma, St Louis, MO) ± 20-250 ng/mL FK506 (Prograf for intravenous use, Fujisawa Healthcare, Deerfield, IL) was added. Every 2 days, 75% supernatant was replaced with fresh cytokine-containing medium (± rapamycin, FK506). On day 4, nonadherent cells were removed; on day 7,  50% of the nonadherent cells expressed CD11c. In vivo administration of rapamycin Rapamycin (Wyeth-Ayerst, Princeton, NJ) was dissolved in 51% wt/vol polyethylene glycol 300 (PEG300), 2.5% wt/vol polysorbate 80, 10% vol/vol ethanol (vehicle; reagents from Sigma). Chinese hamster ovary (CHO) cell-derived rhuman Flt3 ligand (10µg/kg/d, intraperitoneally; Immunex, Seattle, WA) was used to expand DCs in mice over a period of 10 days.14 Animals were injected with rapamycin (0.5 mg/kg/d, intraperitoneally) or vehicle at the same times. Spleens were disaggregated with fine scissors and passed through a nylon cell strainer (Falcon Becton Dickinson, NJ) to obtain a single-cell suspension. Erythrocytes were lysed with 0.83% wt/vol NH4Cl. Endocytosis Quantitative analysis of endocytosis was performed as described,5 with minor modifications. Cells (5 × 105) were incubated with 5 µg/mL FITC-albumin (Sigma) or 0.1 mg/mL FITC-dextran (MW 42000, Sigma) at either 37°C or 4°C for 60 minutes (for in vivo-expanded DCs, 500 µg/mL FITC-albumin and 1 mg/mL FITC-dextran for 40 minutes). Endocytosis was stopped by 2 washes in ice-cold 0.1% sodium azide/1% FCS/phosphate-buffered saline (PBS). Cells were stained for CD11c (HL3) and, in some experiments, for MHC class II expression (IAb -chain, 25-9-17) as described (monoclonal antibodies from BD PharMingen, San Diego, CA).5 Apoptotic cell death Apoptosis was analyzed by staining of phosphatidylserine translocation with FITC-annexin-V in combination with the vital dye 7-AAD (BD PharMingen) and detection of DNA fragmentation by terminal deoxynucleotidyl transferase-mediated fluorescein-2'-deoxyuridine 5'-triphosphate (dUTP) labeling (TUNEL assay; Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions. Positive controls included deoxyribonuclease (DNase) treatment and ultraviolet C (UVC) irradiation (60 J/m2) in the TUNEL and annexin-V assay, respectively. Cells were costained for CD11c expression and analyzed by means of an EPICS XL flow cytometer (Beckman Coulter, Hialeah, FL). Statistics Statistical analysis was performed with a 2-tailed Student t test. Normal distribution of values was proved by the Kolomogorov-Smirnov test.     Results and discussion Top Abstract Introduction Study design Results and discussion References Analysis of GM-CSF+IL-4-expanded, BM-derived DCs harvested at day 7 of culture revealed a reduced capacity of cells exposed to rapamycin to exhibit macropinocytosis of FITC-albumin and mannose receptor-mediated endocytosis of FITC-dextran. This was evident with respect to both the incidence (Figure 1A, B) and the mean fluorescence intensity (MFI; Figure 1D) of CD11c+ cells. CD11c is a very reliable marker for murine DCs15 and is not expressed in significant amounts by murine macrophages.5,16 Because DC maturation is associated with marked down-regulation of endocytotic capacity,3 we questioned whether these effects might be related to a stimulatory effect of rapamycin on DC maturation. However, rapamycin-exposed CD11c+ DCs displayed significantly decreased surface MHC class II (Figure 1C) and costimulatory molecules (data not shown), indicating that they were more immature than untreated, control cells. It could be argued that the analysis of a fixed number of DCs by fluorescence-activated cell sorting (FACS) might mask an inhibitory effect of rapamycin on the differentiation of precursor cells into DCs. As shown in Figure 1E, however, rapamycin inhibited total cell expansion in a dose-dependent manner (antagonizable by a molar excess of FK506) but did not significantly block the differentiation of precursor cells into CD11c+ DCs, as evidenced by the comparable yield of CD11c+ DCs. Achievement of similar DC yields was due to consistently increased percentages of CD11c+ DCs with characteristic DC morphology in the rapamycin-treated cultures. View larger version (57K): [in this window] [in a new window]   Figure 1. Rapamycin inhibits endocytosis by GM-CSF+IL-4-expanded DC. (A-D) BM-derived DCs were expanded for 7 days as described in "Study design"; rapamycin (Rapa) was added at day 2 (± FK506) at the concentrations indicated. FITC-albumin and FITC-dextran internalization at 37°C and 4°C (negative control), MHC class II (IAb -chain), and CD11c expression were analyzed by flow cytometry. In all experiments, CD11c+ cells were analyzed to determine endocytosis and MHC class II expression specifically in DCs. (A-C) Numbers indicate the percentage of CD11c+ cells that were positive for the marker indicated. (D) Data represent mean values (± SE) of CD11c+ cells after subtraction of background fluorescence (4°C). (E) Comparison of total cell and DC numbers on day 7 of culture. (D, E) Differences between paired cultures were compared by 2-tailed Student t test for paired samples (*P < .05 vs control;  P < .05 vs Rapa 1). (A-D) Results are representative of 4 (FITC-dextran) and 6 (FITC-albumin) separate experiments. (E) Results are representative of 6 separate experiments. Because additional experiments indicated that the inhibitory effect of rapamycin on DC maturation was IL-4 dependent (H.H., T.T., unpublished observations, October 2001), we expanded DCs with GM-CSF only. Generation of murine DCs in GM-CSF is a well-established culture method.17 Additionally, to determine more precisely the endocytotic activity of homogenous DCs at the same stage of maturation, we specifically analyzed immature MHC IIlo DCs. These experiments confirmed the inhibitory effects of rapamycin on DC endocytosis and indicated that they were not IL-4 related (Figure 2A-C). Again, low concentrations of 1 ng/mL rapamycin (1.1 nmol) were sufficient to significantly and markedly suppress endocytotic activity. When 1 ng/mL rapamycin was used, the relative MFI of immature MHC class IIlo CD11c+ DCs compared with controls was < 42% and < 32% with respect to FITC-albumin and FITC-dextran, respectively (Figure 2C). Given that mean trough whole blood levels of rapamycin in patients are 17.3 ng/mL (5 mg rapamycin/d)18 and that the free plasma fraction is 8%, these concentrations are clinically highly relevant. When rapamycin was added at day 6 of culture, it still inhibited DC endocytosis, but the overall effect was weaker (relative MFI 69% and 53% for FITC-albumin and FITC-dextran, respectively, at 5 ng/mL rapamycin). View larger version (42K): [in this window] [in a new window]   Figure 2. Rapamycin inhibits endocytosis by GM-CSF-expanded immature BM-derived DCs. (A-E) BM-derived DCs were expanded for 7 days with GM-CSF only, as described in "Study design"; rapamycin (Rapa) was added at day 2 (± FK506) at the concentrations indicated. (A-C) FITC-albumin and FITC-dextran internalization at 37°C and 4°C (negative control), MHC class II (IAb -chain), and CD11c expression were analyzed by 3-color flow cytometry. CD11c+ MHC class IIlo cells were gated to determine endocytosis specifically in immature DCs. Regions R1 and R2 show endocytotic activity of immature MHC class IIlo and mature MHC class IIhi DCs, respectively. (A, B) Numbers indicate the percentage of CD11c+ MHC class IIlo cells positive for the marker indicated. (B, C) Data represent mean values (± SE) of CD11c+ MHC class IIlo cells after subtraction of background fluorescence (4°C). Differences between paired cultures were compared by the 2-tailed Student t test for paired samples (*P  .01 vs control;  P < .05 vs Rapa 1). (D, E) Apoptosis of CD11c+ DC was determined by annexin-V/7-AAD staining (D) and the TUNEL assay (E). Numbers represent mean values (± SE). Incidences of apoptotic (annexin-V positive/7-AAD negative or TUNEL positive) or secondary necrotic (annexin-V positive/7-AAD positive) cells were consistently lower than 10%. There was a trend toward decreased apoptosis/cell death in rapamycin-treated cultures, but the differences were not statistically significant. (A-C) Results are representative of 4 separate experiments. (D, E) Results are representative of 2 (TUNEL assay) and 3 (annexin-V/7-AAD assay) separate experiments. Binding of rapamycin to FKBP12 and TOR inhibition can be antagonized in vitro by the structurally related immunophilin ligand FK506.19,20 Whereas FK506 alone (20 ng/mL) did not interfere with DC endocytosis (data not shown), addition of 20 ng/mL FK506 to 1 ng/mL rapamycin (22.3-fold molar excess) antagonized the inhibitory effects of rapamycin, indicating that these were related to FKBP12-mediated TOR inhibition (Figures 1A, B; 2A-C). However, the antagonistic effect of FK506 was incomplete, especially with respect to mannose receptor-mediated endocytosis. Similar results were obtained over a wide range of drug concentrations (1-20 ng/mL rapamycin, 10-250 ng/mL FK506) and at different FK506/rapamycin ratios (10-55 molar). This suggested that other FKBPs might also be involved in endocytosis inhibition. One candidate is FKBP25, which has > 100 times greater binding affinity for rapamycin than FK506.21 Having established that rapamycin inhibited DC endocytosis, we analyzed whether this effect was due to increased apoptotic cell death. In contrast to a recent report,22 the incidence of apoptosis at day 7 of culture was consistently low (< 10%) and was not affected significantly by rapamycin, even at a suprapharmacological dose of 100 ng/mL, as determined independently by annexin-V/7-AAD and TUNEL staining (Figure 2D, E). Similar results were obtained with GM-CSF+IL-4-expanded DCs and at day 4 of culture (data not shown). To investigate the in vivo relevance of DC endocytosis inhibition, we analyzed endocytotic activity in splenic DCs of animals that were injected with rapamycin (0.5 mg/kg/d for 10 days, ip) or vehicle and in which DCs were expanded with Flt3 ligand as we have described.14 After 10 days, the animals were killed and FITC-albumin and FITC-dextran uptake by freshly isolated splenic CD11c+ DCs was analyzed. The phenotype of these DCs was immature in both treatment groups. As shown in Figure 3, CD11c+ DC of rapamycin-treated animals displayed significantly reduced macropinocytotic activity, with respect to both the number of positive cells and the relative MFI (P = .001 and P = .002, respectively). Similar findings were obtained with respect to FITC-dextran uptake (41.5% positive cells vs 24.8% in rapamycin-injected animals, P < .05; relative MFI 74.7%, P < .05). View larger version (44K): [in this window] [in a new window]   Figure 3. In vivo administration of rapamycin inhibits endocytosis by Flt3 ligand-expanded splenic DCs. (A-C) DCs were expanded in vivo with Flt-3 ligand and animals were injected with either rapamycin or vehicle, as described in "Study design." Endocytotic activity was measured by analyzing FITC-albumin uptake at 37°C and 4°C (negative control) and relative MFI in comparison with animals injected with vehicle only. Results are representative of 8 animals per group and were obtained in 5 independent experiments. Differences between the groups were compared by the 2-tailed Student t test for independent samples. Similar findings were obtained with respect to FITC-dextran uptake, as described in "Results and discussion." The results of this study suggest that rapamycin can interfere with immune responses at a very early stage by inhibiting DC endocytosis. Thus, rapamycin targets a unique function of DCs that influences the induction of immunity against microbial pathogens (eg, Salmonella23) and allergens.24 This effect may also suppress indirect alloantigen processing following transplantation. The precise mechanisms by which rapamycin inhibits DC endocytosis remain to be determined. In this context, it is important to note that the Rho GTPases CDC42 and Rac that interfere with the endocytotic activity of DCs7,8 complex with and activate the p70 S6 kinase25 that belongs to the central signaling pathway disrupted by rapamycin.9 In addition, rapamycin's inhibition of TOR signaling down-regulates protein translation and has been demonstrated to suppress actin synthesis.26 Because, to our knowledge, no clinical immunosuppressant has been reported to inhibit DC endocytosis, these findings may provide further incentive for trials of rapamycin in clinical settings other than transplantation, for example, in autoimmune disease.     Acknowledgments We thank Jan Urso for excellent technical assistance, Wyeth-Ayerst for providing rapamycin and advice, Immunex for providing Flt3 ligand, and Dr Venkataramanan for helpful discussion.     Footnotes Submitted October 29, 2001; accepted April 2, 2002. Supported by the National Institutes of Health (grants R01 DK49745 and R01 AI41011 to A.W.T.). H.H. is supported by a scholarship from the Stiftung Hämotherapie-Forschung, Bonn, Germany. H.H. and T.T. contributed equally to this work. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Angus W. Thomson, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, W1544 BST, 200 Lothrop St, Pittsburgh, PA 15213; e-mail: thomsonaw{at}msx.upmc.edu.     References Top Abstract Introduction Study design Results and discussion References 1. Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767-811[CrossRef][Medline] [Order article via Infotrieve]. 2. Hackstein H, Morelli AE, Thomson AW. Designer dendritic cells for tolerance induction: guided not misguided missiles. Trends Immunol. 2001;22:437-442[CrossRef][Medline] [Order article via Infotrieve]. 3. Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med. 1995;182:389-400[Abstract/Free Full Text]. 4. Piemonti L, Monti P, Allavena P, et al. Glucocorticoids affect human dendritic cell differentiation and maturation. J Immunol. 1999;162:6473-6481[Abstract/Free Full Text]. 5. Hackstein H, Morelli AE, Larregina AT, et al. Aspirin inhibits in vitro maturation and in vivo immunostimulatory function of murine myeloid dendritic cells. J Immunol. 2001;166:7053-7062[Abstract/Free Full Text]. 6. Garrett WS, Chen LM, Kroschewski R, et al. Developmental control of endocytosis in dendritic cells by Cdc42. 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The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells. J Immunol. 1990;144:1418-1424[Abstract]. 21. Jin YJ, Burakoff SJ, Bierer BE. Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25. J Biol Chem. 1992;267:10942-10945[Abstract/Free Full Text]. 22. Woltman AM, de Fijter JW, Kamerling SW, et al. Rapamycin induces apoptosis in monocyte- and CD34-derived dendritic cells but not in monocytes and macrophages. Blood. 2001;98:174-180[Abstract/Free Full Text]. 23. Niedergang F, Sirard JC, Blanc CT, Kraehenbuhl JP. Entry and survival of Salmonella typhimurium in dendritic cells and presentation of recombinant antigens do not require macrophage-specific virulence factors. Proc Natl Acad Sci U S A. 2000;97:14650-14655[Abstract/Free Full Text]. 24. Noirey N, Rougier N, Andre C, Schmitt D, Vincent C. Langerhans-like dendritic cells generated from cord blood progenitors internalize pollen allergens by macropinocytosis, and part of the molecules are processed and can activate autologous naive T lymphocytes. J Allergy Clin Immunol. 2000;105:1194-1201[CrossRef][Medline] [Order article via Infotrieve]. 25. Chou M, Blenis J. The 70 kDa S6 kinase complexes with and is activated by the Rho Family G proteins Cdc42 and Rac1. Cell. 1996;85:573-583[CrossRef][Medline] [Order article via Infotrieve]. 26. Miyamoto S, Safer B. Immunosuppressants FK506 and rapamycin have different effects on the biosynthesis of cytoplasmic actin during the early period of T cell activation. Biochem J. 1999;344:803-812[CrossRef][Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: Y. Huang, M. Parker, C. Xia, R. Peng, C. Wasserfall, T. Clarke, L. 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