Abstract

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.

Introduction

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

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 β-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 Studentt test. Normal distribution of values was proved by the Kolomogorov-Smirnov test.

Results and discussion

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 inhibitedtotal 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.

Fig. 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 β-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.

Fig. 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 β-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 (Figure2A-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 IIloCD11c+ DCs compared with controls was < 42% and < 32% with respect to FITC-albumin and FITC-dextran, respectively (Figure2C). 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).

Fig. 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 β-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 towarddecreased 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.

Fig. 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 β-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 towarddecreased 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 Figure3, 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 andP = .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).

Fig. 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.”

Fig. 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.

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.

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.

References

References
1
Banchereau
J
Briere
F
Caux
C
et al
Immunobiology of dendritic cells.
Annu Rev Immunol.
18
2000
767
811
2
Hackstein
H
Morelli
AE
Thomson
AW
Designer dendritic cells for tolerance induction: guided not misguided missiles.
Trends Immunol.
22
2001
437
442
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.
182
1995
389
400
4
Piemonti
L
Monti
P
Allavena
P
et al
Glucocorticoids affect human dendritic cell differentiation and maturation.
J Immunol.
162
1999
6473
6481
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.
166
2001
7053
7062
6
Garrett
WS
Chen
LM
Kroschewski
R
et al
Developmental control of endocytosis in dendritic cells by Cdc42.
Cell.
102
2000
325
334
7
West
MA
Prescott
AR
Eskelinen
EL
Ridley
AJ
Watts
C
Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation.
Curr Biol.
10
2000
839
848
8
de Baey
A
Lanzavecchia
A
The role of aquaporins in dendritic cell macropinocytosis.
J Exp Med.
191
2000
743
748
9
Sehgal
SN
Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression.
Clin Biochem.
31
1998
335
340
10
Saunders
RN
Metcalfe
MS
Nicholson
ML
Rapamycin in transplantation: a review of the evidence.
Kidney Int.
59
2001
3
16
11
Hong
JC
Kahan
BD
Sirolimus rescue therapy for refractory rejection in renal transplantation.
Transplantation.
71
2001
1579
1584
12
Shapiro
AM
Lakey
JR
Ryan
EA
et al
Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.
N Engl J Med.
343
2000
230
238
13
Raught
B
Gingras
AC
Sonenberg
N
The target of rapamycin (TOR) proteins.
Proc Natl Acad Sci U S A.
98
2001
7037
7044
14
Morelli
AE
Antonysamy
MA
Takayama
T
et al
Microchimerism, donor dendritic cells, and alloimmune reactivity in recipients of Flt3 ligand-mobilized hemopoietic cells: modulation by tacrolimus.
J Immunol.
165
2000
226
237
15
Grabbe
S
Kampgen
E
Schuler
G
Dendritic cells: multi-lineal and multi-functional.
Immunol Today.
21
2000
431
433
16
Metlay
JP
Witmer-Pack
MD
Agger
R
Crowley
MT
Lawless
D
Steinman
RM
The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies.
J Exp Med.
171
1990
1753
1771
17
Inaba
K
Inaba
M
Romani
N
et al
Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.
J Exp Med.
176
1992
1693
1702
18
MacDonald
A
Scarola
J
Burke
JT
Zimmerman
JJ
Clinical pharmacokinetics and therapeutic drug monitoring of sirolimus.
Clin Ther.
22(suppl B)
2000
B101
B121
19
Kuo
CJ
Chung
J
Fiorentino
DF
Flanagan
WM
Blenis
J
Crabtree
GR
Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase.
Nature.
358
1992
70
73
20
Dumont
FJ
Melino
MR
Staruch
MJ
Koprak
SL
Fischer
PA
Sigal
NH
The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells.
J Immunol.
144
1990
1418
1424
21
Jin
YJ
Burakoff
SJ
Bierer
BE
Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25.
J Biol Chem.
267
1992
10942
10945
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.
98
2001
174
180
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.
97
2000
14650
14655
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.
105
2000
1194
1201
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.
85
1996
573
583
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.
344
1999
803
812

Author notes

Angus W. Thomson, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, W1544 BST, 200 Lothrop St, Pittsburgh, PA 15213; e-mail: thomsonaw@msx.upmc.edu.