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The Journal of Immunology, 2004, 172: 1355-1363.
Copyright © 2004 by The American Association of Immunologists

Mechanistic Insights into Impaired Dendritic Cell Function by Rapamycin: Inhibition of Jak2/Stat4 Signaling Pathway1

Po-Hui Chiang2, Lianfu Wang2, C. Andrew Bonham, Xiaoyan Liang, John J. Fung, Lina Lu and Shiguang Qian3

Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15261


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The suppressive effect of rapamycin on T cells has been extensively studied, but its influence on the function of APC is less clear. The data in this study demonstrated that immunostimulatory activity of B10 (H2b) dendritic cells (DC) exposed to rapamycin (rapa-DC) was markedly suppressed as evidenced by the induction of low proliferative responses and specific CTL activity in allogeneic (C3H, H2k) T cells. Administration of rapa-DC significantly prolonged survival of B10 cardiac allografts in C3H recipients. Treatment with rapamycin did not affect DC expression of MHC class II and costimulatory molecules or IL-12 production. Rapamycin did not inhibit DC NF-{kappa}B pathway, however, IL-12 signaling through Janus kinase 2/Stat4 activation was markedly suppressed. Indeed, Stat4-/- DC similarly displayed poor allostimulatory activity. The Stat4 downstream product, IFN-{gamma}, was also inhibited by rapamycin, but DC dysfunction could not solely be attributed to low IFN-{gamma} production as DC deficient in IFN-{gamma} still exhibited vigorous allostimulatory activity. Rapamycin did not affect DC IL-12R expression, but markedly suppressed IL-18R{alpha} and {beta} expression, which may in turn down-regulate DC IL-12 autocrine activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rapamycin is a potent immunosuppressant used for the prevention of organ transplant rejection. Previous studies have focused on its effects on T and B lymphocytes via diverse pathways, but its influence on the function of APC has not been extensively studied. Inhibition of Ag-presenting function has been shown to be one of the alternative mechanisms by which some immunosuppressants act on T cell responses. Cyclosporine (CSA)4 and tacrolimus, both calcineurin inhibitors, inhibit the expression of costimulatory molecules on in vitro-generated dendritic cells (DC) (1, 2). Mycophenolate mofetil induces a dose-dependent reduction of the expression of MHC class II, CD40, CD80, CD86, and ICAM-1 on DC with a concurrent reduction of IL-12 production (3). Although the molecular structure of rapamycin is similar to that of tacrolimus and rapamycin also targets the same FK-binding protein, the action of rapamycin appears to be distinct from tacrolimus. Recent studies have demonstrated that rapamycin impairs Ag uptake in DC probably via inhibition of macropinocytosis and endocytosis (4, 5). Rapamycin also induces apoptosis in human DC (6).

In this study, we sought to further explore the basis behind rapamycin inhibition of DC function, and demonstrated that rapamycin inhibited DC immune stimulatory activity both in vitro and in vivo. MHC or costimulatory molecule expression on DC was unaffected, and neither was production of IL-12, a pleiotropic cytokine produced by DC. We examined the effect of rapamycin on IL-12 signaling, as DC express high affinity IL-12R, and the IL-12 autocrine signaling pathway has been shown to be an important pathway for DC activation (7, 8). IL-12 signaling has also been shown to lead to DC activation via nuclear localization of NF-{kappa}B (7) and confer DC with increased APC function (9, 10, 11). Our results revealed that DC IL-12R expression and NF-{kappa}B activity was not suppressed, suggesting that it is unlikely that rapamycin acts via interference of the NF-{kappa}B pathway. Rapamycin, however, inhibited IL-12-stimulated IFN-{gamma} expression in DC, leading us to speculate on the possibility that rapamycin may inhibit DC Stat4 pathway. This pathway is crucial to the autocrine activation of DC by IL-12 (12) and it has been shown that Stat4 levels are directly correlated with IL-12-dependent IFN-{gamma} production by DC as well as IFN-{gamma} production by DC during Ag presentation. We showed that IL-12 signaling activation of Stat4 in DC was almost completely blocked by rapamycin. This was associated with inhibition of IL-18R expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Male C57BL/6 (B6; H2b), C57BL/10 (B10; H2b), C3H (H2K), BALB/c (H2d), IFN-{gamma}-/- (H2d), and Stat4-/- (H2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). They were maintained in the specific pathogen-free facility of the University of Pittsburgh Medical Center (Pittsburgh, PA). Animals were fed standard chow ad libitum and used at 8–12 wk of age.

Propagation of DC

Bone marrow (BM) cells were isolated from mouse femurs and prepared in RPMI 1640 media (Life Techonologies, Gaithersburg, MD) supplemented with antibiotics and 10% (v/v) FCS as previously described (13). Recombinant mouse GM-CSF (4 ng/ml) and IL-4 (1000 U/ml, both from Schering-Plough, Kenilworth, NJ) were added to cultures. Cytokine-enriched medium was refreshed 2 days later; after gentle swirling of the plates, half of the original medium was aspirated and replaced with an equivalent volume of fresh, cytokine-supplemented medium. To assess the effect of rapamycin on DC, rapamycin (20 ng/ml, Sigma-Aldrich, St. Louis, MO) was added on day 3 (unless otherwise described) of DC culture (referred to subsequently as rapa-DC). Nonadherent granulocytes were then depleted without dislodging clusters of developing DC attached loosely to a monolayer of plastic-adherent macrophages. Nonadherent cells released spontaneously from the clusters were harvested after culture for 5 days. The purification procedures were similar to those described by Inaba et al. (14). To avoid carry-over of residue rapamycin, DCs were thoroughly washed twice after harvest from culture. DC preparations used in this study contained 85–90% CD11c+ cells. In some studies, DC were further activated by incubation with IL-12 (10 ng/ml) or LPS (10 µg/ml) for 18 h, unless otherwise indicated.

Flow cytometry

Expression of cell-surface Ags on DC was analyzed by cytofluorography using an EPICS ELITE flow cytometer (Coulter, Hialeah, FL). Cells were stained with FITC or PE-conjugated rat anti-mouse mAbs for H2Kb, IAb, CD40, CD80, or CD86, CD11c (all are IgG2a) after blocking nonspecific binding with 10% v/v normal goat serum. FITC or PE-conjugated isotype-matched irrelevant mAbs were used as negative controls (all from BD PharMingen, San Diego, CA). After staining, the cells were fixed in 2% paraformaldehyde in isotonic saline before analysis.

Apoptosis assay

Apoptotic DC were identified by double staining with anti-CD11c and TUNEL. Cells were fixed in 4% paraformaldehyde after staining with PE-conjugated rat anti-mouse CD11c mAb for 1 h at room temperature. The cells were washed with PBS and resuspended in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. TUNEL reaction mixture (Roche, Mannheim, Germany) was added and 10% goat serum was used to block nonspecific binding. Cells incubated with label solution in the absence of terminal transferase were used as negative controls. Quantitative analysis was performed by flow cytometry (Coulter) with 10,000 events acquired.

Mixed leukocyte reaction

One-way MLR cultures were performed in triplicate in 96-well, round-bottom microculture plates (Corning Glass, Corning, NY). Nylon wool-eluted spleen T cells (2 x 105/well) were used as responders. Graded doses of gamma-irradiated (20 Gy; x-ray source) spleen cells or DC propagated from mouse BM were used as stimulators. Cultures were maintained in RPMI 1640 complete medium for 72 h in 5% CO2 in air. [3H]TdR (1µCi/well) was added for the final 18 h, and incorporation of [3H]TdR into DNA was assessed by liquid scintillation counting. Results were expressed as mean cpm ± 1 SD.

CTL assay

Freshly isolated mouse spleen T cells were cultured with gamma-irradiated (20 Gy) allogeneic DC at a ratio of 20:1 for 5 days, and then used as effectors. EL4 (H2b), (R1.1, H2k) or P815 (H2d) lymphoma cells (both from American Type Culture Collection, Rockville, MD) were labeled with 100 µCi Na251CrO4 (NEN, Boston, MA) and used as donor-specific, syngeneic or third party targets accordingly. They were washed and plated at a concentration of 103 cells/well in 96-well, round-bottom culture plates (Corning Glass). Serial, two-fold dilutions of effector cells were added to give effector: target (E:T) ratios of 100:1, 50:1, 25:1, and 12.5:1 in a total volume of 200 µl/well. The percentage of specific 51Cr release was determined after incubating the plates for 4 h at 37°C in RPMI 1640 complete medium in 5% CO2 in air. An aliquot (100 µl) of supernatant was recovered from each well. Maximum 51Cr release was determined by lysis of the target cells. The percent specific cytotoxicity was calculated using the following formula: % cytotoxicity = 100 x [(experimental cpm) - (spontaneous cpm)]/[(maximum cpm) - (spontaneous cpm)]. The results are expressed as mean ± 1 SD of percentage-specific 51Cr release in triplicate cultures.

Immunoprecipitation and Western blot analysis

Cytoplasmic or nuclear proteins (30–50 µg) were resolved in 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels, transferred onto nitrocellulose, and subjected to Western blot analysis as previously described (16). The primary anti-Stat4, anti-Janus kinase (Jak)2, and anti-tyrosine kinase (Tyk)2 Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phosphorylated Jak2 was purchased from Cell Signaling Technology (Beverly, MA) and anti-phosphorylated Stat4 was obtained from Zymed Laboratories (San Francisco, CA). HRP conjugated anti-rabbit IgG was used as second Ab.

For immunoprecipitation assays, 150 µg of whole cell protein was diluted in 1 ml of PBS, precleared with recombinant protein A-agarose (Santa Cruz Biotechnology), and incubated with anti-Tyk2 Ab. Immune complexes were captured with recombinant protein A-agarose, washed, and resuspended in sample buffer. Samples were run on a polyacrylamide gel, transferred to nitrocellular membranes, and blotted with anti-phosporylated tyrosine Ab. Immunoblots were developed using the SuperSignal system (Pierce, Rockford, IL). Immunoprecipitation assays were repeated to ensure reproducibility.

EMSA and supershift analysis

To determine NF-{kappa}B binding activity in DC, nuclear proteins were extracted according to the method originally described by Andrews and Faller (17). EMSA was performed using a commercial kit (Promega, Madison, WI). Briefly, an NF-{kappa}B oligonucleotide (sense sequence: 5'-AGTTGAGGGGACTTTCCCAGGC-3') end-labeled with [{gamma}-32P]ATP (NEN) was used as probe. Nuclear proteins were incubated with labeled probe and then detected by running the mixture on a 4% acrylamide gel. The specificity of NF-{kappa}B was determined by shifting of the bands in the presence of anti-NF-{kappa}B (p50) mAb.

Cytokine secretion assay

Cell culture supernatants were harvested and analyzed for the presence of cytokines using commercial ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocols.

RNase protection assay

Total RNA was isolated using TRIzol reagent obtained from Invitrogen (Carlsbad, CA). The assay was performed using the RiboQuant Multiprobe RNase Protection Assay kit (BD PharMingen). The purity of RNA was determined from the A260/280 absorbance ratio. Cytokine mRNA was assessed using the RNase Protection Assay Kit (RiboQuant, San Diego, CA). Briefly, probes were synthesized by T7 RNA polymerase with incorporation of [{alpha}-32P]UTP. Five micrograms total RNA was treated overnight with synthesized probes (specific activity: 800 Ci/mM) at 56°C, followed by treatment with RNase A (80 µg/ml) and T1 (250 U/ml) for 45 min at 30°C. The murine L32 and GAPDH riboprobes were used as controls. Protected fragments were submitted for electrophoresis through a 7.0 M urea/5% poly-acrylamide gel, and then exposed to Kodak X-omat film for 72 h.

Semiquantitative RT-PCR

Total RNA was prepared using TRIzol Reagent (Invitrogen) according to the manufacturer’s instruction. The oligonucleotides used for cDNA synthesis and PCR amplification of IL-12R and IL-18R were: IL-12R{beta}1 5': (5'-CCAGCACAGGAACCACACA-3'), IL-12-R{beta}1 3' (5'-CAGAGACGCGAAAATGATG-3'), IL-12R{beta}2 5' (5'-AATTCAGTACCGACGCTCTCA-3'), IL-12R{beta}2 3' (5'-ATCAGGGGCTCAGGCTCTTCA-3'), IL-18R{alpha} 5' (5'-GTGCACAGGAATGAAACAGC-3'), IL-18R{alpha} 3' (5'-ATTTAAGGTCCAATTGCGACGA-3'), IL-18R{beta} 5' (5'-GGAGTGGGAAATGTCAGTAT-3'), and IL-18R{beta} 3' (5'-CCGTGCCGAGAAGGATGTAT-3'). Briefly, 0.2 µg of RNA was reverse transcribed into cDNA for PCR amplification using specific antisense primers and Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen). IL-12R and IL-18R cDNAs were amplified, respectively, at 94°C for 30s, 60°C for 45s, 72°C for 90s, and at 95°C for 30s, 55°C for 30s, 72°C for 1 min at various cycles using the olignucleotides listed above. Fifteen microliters of each reaction was separated by 5% polyacrylamide gel, stained with SYBR Green I (Sigma-Aldrich), and scanned by Amersham Storm 860 (Amersham Pharmacia Biotech, Piscataway, NJ). Amplification at 35 cycles for IL-12R and 28 cycles for IL-18R was performed to ensure Logarithmic PCR amplification.

Heterotopic heart transplantation

Vascularized B10 heart was heterotopically transplanted into the abdomen of C3H mice as described previously (15). The function of the heart was monitored daily after transplantation by abdominal palpation. Rejection was defined as total cessation of cardiac muscle contraction and was confirmed histologically on formalin-fixed, paraffin-embedded tissue. To examine DC function in vivo, DC (2 x 106) were i.v. injected into C3H recipients 7 days before transplantation of B10 cardiac allograft in the absence of immunosuppressive therapy.

Statistical analysis

The parametric data are given as mean ± 1 SD and statistical significance was determined by Student’s t test. Graft survival between groups of transplanted animals was compared using the log-rank test for comparison of survival curves. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rapamycin inhibits allostimulatory activity of DC

To evaluate the effect of rapamycin on immunostimulatory function of DC, rapamycin was added during DC propagation from B10 BM cells in the presence of GM-CSF and IL-4. At the end of culture, DCs were extensively washed. We found that rapamycin added at 20 ng/ml on day 0, 2, or 3 of culture could significantly suppress DC allostimulatory activity. In the subsequent studies, rapamycin was added on day 3 of DC culture unless specifically indicated. Fig. 1A shows that B10 BM-derived rapa-DC induced low proliferative responses of allogeneic C3H T cells compared with normal DC in an MLR assay. C3H T cells stimulated by allogeneic B10 DC that were exposed to rapamycin during DC propagation also displayed notably lower specific CTL activity (Fig. 1B). This was associated with suppressed IFN-{gamma} levels in the culture supernatant (Fig. 1C). No cytotoxic activity against syngeneic (R1.1, H2k) or third-party (P815, H2d) targets was generated in C3H T cells stimulated by either B10 normal or rapa-DC (data not shown). The suppressed allostimulatory effect of rapa-DC was unlikely the result of residue rapamycin carried over from DC cultures. We determined that the minimal concentration of rapamycin to suppress proliferative responses of allogeneic T cells was 0.5 ng/ml. Only trace amounts of rapamycin (<0.05 ng/ml) were detected by HPLC in the supernatants of MLR cultures in which rapa-DC were used as stimulators, and the supernatants did not have any inhibitory effects on T cell proliferative responses (data not shown).



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  		FIGURE 1. Rapamycin inhibits allostimulatory activity of DC. A, In a one way MLR, naive C3H (H2k) spleen T cells (2 x 105) were cultured for 3 days at various ratios with gamma-irradiated DC that were propagated from B10 (H2b) BM cells in the presence or absence of rapamycin (20 ng/ml). Gamma-irradiated B10 and C3H spleen cells were used as control stimulators. DC exposed to rapamycin induced significantly lower T cell proliferative responses compared with normal DC (p < 0.05). B, Naive C3H T cells that were cultured for 5 days with gamma-irradiated B10 bone marrow-derived DC (T:DC ratio of 20:1) in the presence or absence of rapamycin (20 ng/ml) were used as stimulators of CTL induction. Cytotoxicity to EL4 targets (H2b) was determined at various effector:target cell ratios in a 4 h 51Cr release assay. No cytotoxicity was generated against syngeneic EL4 (H2b) or third party R1.1 (H2k) targets (data not shown). T cells stimulated with rapa-DC generated significantly lower specific CTL activity (p < 0.05). C, IFN-{gamma} levels in the supernanants of the 3-day MLR cultures (T:DC ratio of 20:1) were measured by ELISA. T cells cultured with rapa-DC produced markedly lower IFN-{gamma} production (p < 0.05 compared with cultured with normal DC). All data are representative of three separate experiments

 
Administration of rapa-DC prolongs cardiac allograft survival

To examine the allostimulatory activity of rapa-DC in vivo, B10 DC (2 x 106) that were propagated with GM-CSF and IL-4 in the presence or absence of rapamycin were injected i.v. into C3H recipients 7 days before transplantation of a B10 cardiac allograft. We have previously demonstrated that this was the optimal protocol to determine the effect of in vivo DC administration on allograft rejection (18). In contrast to the administration of normal DC that significantly accelerated rejection of B10 cardiac allografts (p < 0.05, compared with non DC-treated controls), administration of rapa-DC from B10 mice markedly prolonged survival of B10 cardiac allografts (p < 0.05, compared with non DC-treated controls). The immunosuppressive effect of rapa-DC is donor-specific, as they failed to prolong survival of allografts from third party (BALB/c) mice (Table I).


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  		Table I. Survival of B10 (H2b) or BALB/c (H2d, third party) cardiac allografts in C3H (H2K) recipients treated with B10 DCa

 
Effect of rapamycin on DC phenotype and viability

To address the mechanisms whereby DC allostimulatory activity was inhibited by rapamycin, we first examined whether rapamycin affected DC maturation. The phenotype of DC propagated from B10 BM cells in the presence of rapamycin was analyzed by flow cytometry. As shown in Fig. 2A, rapamycin slightly inhibited DC expression of MHC class I, but had little effect on the expression of MHC class II or the costimulatory molecules CD80, CD86, and CD40. We next examined the influence of rapamycin on DC viability using trypan blue exclusion and TUNEL staining. DC viability by trypan blue exclusion assay was unaffected by rapamycin (data not shown), and intracellular TUNEL staining revealed that exposure to rapamycin did not enhance apoptotic activity in CD11c positive cell population (Fig. 2B). These results collectively indicate that the reduced stimulatory capacity of rapa-DC is unlikely due to a negative effect on DC maturation or a reduction in DC viability.



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  		FIGURE 2. Effect of rapamycin treatment on DC phenotype and apoptotic death. DC were propagated from B10 (H2b) BM cells in the presence or absence of rapamycin (20 ng/ml). A, Surface Ag presentation on DC was determined by staining with mAbs against H2Kb, IAb, CD40, CD80, and CD86 and analyzed by flow cytometry. Rapamycin did not suppress expression of MHC class II and costimulatory molecules and only slightly inhibited MHC class I expression on DC. B, DC apoptotic activity was determined by double staining with anti-CD11c and TUNEL and analyzed by flow cytometry. DC apoptosis was not enhanced by rapamycin. Data are representative of three separate experiments.

 
Effect of rapamycin on DC cytokine expression

IL-12 is one of the most important cytokines secreted by DC and macrophages, and it plays a crucial role in priming Th1 T cell responses (19, 20, 21, 22) and allograft rejection (2). To address the effect of rapamycin on DC IL-12 production, DC propagated from B10 BM in the presence or absence of rapamycin were purified using CD11c positive selection beads (99% CD11c+) and then incubated with LPS (10 µg/ml) for 18 h. Cytokine levels in the supernatant were assessed by ELISA, and cytokine mRNA expression in DC was determined by RNase protection assays. Without LPS stimulation, DC produced minimal amounts of IL-12 (data not shown) as previously reported (16). LPS stimulation induced DC expression of IL-12 that was not significantly inhibited by exposure to rapamycin either at the mRNA or protein level (Fig. 3, A and B). This suggests that the effect of rapamycin on inhibition of DC immune stimulatory function is not mediated by down-regulation of IL-12 production.



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  		FIGURE 3. Effect of rapamycin on DC cytokine expression. DC propagated from B10 (H2b) BM cells in the presence or absence of rapamycin (20 ng/ml) were purified using CD11c positive selection beads before exposure to LPS (10 µg/ml) for 18 h. A, Levels of IL-12 in culture supernatant were determined by ELISA. Rapa-DC produced identical levels of IL-12 as that of normal DC. B, mRNA expression of IL-12 p35 and p40 in DC was determined by RNase protection assay. IL-12 mRNA expression in DC was not inhibited by rapamycin. C, To determine the effect of rapamycin on DC secretion of IFN-{gamma}, DCs were exposure to various concentrations of rapamycin during culture. For activation, DCs were then incubated with IL-12 (10 ng/ml) for 18 h. IFN-{gamma} levels in culture supernatants were evaluated by ELISA. Dose dependent inhibition of IFN-{gamma} secretion by rapamycin was demonstrated. D, Expression of cytokine mRNA in DC was determined using RNase protection assays. Expression of IL-10, IL-6, IFN-{gamma}, and TNF-{alpha} was induced by IL-12 and inhibited by exposure to rapamycin. IL-1 and IL-1R{alpha} expression not inducible by IL-12 was also markedly inhibited by rapamycin. All data are representative of three separate experiments.

 
Although IFN-{gamma} is predominantly produced by T and NK cells, recent studies have shown that DC produce significant amounts of IFN-{gamma} upon stimulation by infection or IL-12 (23). To examine the influence of rapamycin on DC IFN-{gamma} production, DC propagated from B10 BM in the presence or absence of rapamycin were purified using CD11c positive selection beads, resuspended in RPMI medium (106/well), and stimulated with IL-12 (10 ng/ml) for 18 h. Minimal amounts of IFN-{gamma} were detected in culture supernatants without IL-12 stimulation (data not shown), while DC released significant amounts of IFN-{gamma} after the addition of IL-12. This IL-12-induced IFN-{gamma} production was inhibited by rapamycin in a dose dependent manner (Fig. 3C). Data from RNase protection assays showed that mRNA expression of IFN-{gamma}, TNF-{alpha}, IL-6, and IL-10 induced by IL-12 stimulation were inhibited by rapamycin. Indeed, other cytokines that were constitutively expressed in DC including IL-1{alpha} and {beta} were also markedly inhibited by rapamycin. Expression of IL-15 was slightly up-regulated by IL-12, but was not affected by exposure to rapamycin (Fig. 3D).

DC allostimulatory activity is not inhibited by deficiency in IFN-{gamma} expression

To ascertain whether inhibition of DC IFN-{gamma} expression by rapamycin contributes to DC dysfunction, we examined the allostimulatory activity of DC propagated from IFN-{gamma}-/- mice (H2d). DC deficient in IFN-{gamma} stimulated significantly higher proliferative responses in allogeneic (H2k) T cells compared with DC derived from normal controls (Fig. 4A). In addition, T cells stimulated by DC deficient in IFN-{gamma} expression consistently generated higher specific cytotoxic activity than those stimulated by DC from wild-type animals (Fig. 4B). These data suggest that IFN-{gamma} produced by DC may be a negative regulator of DC function, and rapamycin suppression of DC function is unlikely related to DC down-regulation of IFN-{gamma} production.



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  		FIGURE 4. DC deficient in IFN-{gamma} induce high levels of allogeneic T cell responses. A, In a one way MLR, B6 (H2b) spleen T cells (2 x 105) were cultured for 3 days with number-graded gamma-irradiated DC that were propagated from IFN-{gamma} -/- (H2d) BM cells. DC propagated from normal BALB/c (H2d) mice in the absence or presence of rapamycin (20 ng/ml) were used as control stimulators. DC deficient in IFN-{gamma} induced higher T cell proliferative responses (*, p < 0.05, IFN-{gamma} -/- DC vs other groups at stimulator number of 104) (B) Naive B6 spleen T cells that were cultured for 6 days with gamma-irradiated DC propagated from IFN-{gamma} -/- mice or normal BALB/c mice, at T:DC ratio of 20:1, in the presence or absence of rapamycin (20 ng/ml) were used as stimulators of CTL induction. Cytotoxicity to P815 (H2d) lymphoma cells was determined at various E:T ratios in a 4 h 51Cr release assay (*, p < 0.05, IFN-{gamma}-/- DC vs other groups at E:T ratios of 50:1 and 100:1). No cytotoxicity was generated against syngeneic EL4 (H2b) or third party R1.1 (H2k) targets (data not shown). All data are representative of three separate experiments.

 
Rapamycin does not alter NF-{kappa}B activity in DC

DC are not only main producers of IL-12, they also express high-affinity receptors for IL-12 (8). IL-12 signaling has been shown to be critical in the activation of DC by IL-12 autocrine activity (12). There are two distinct signal transduction pathways involved in DC IL-12 autocrine stimulated activation. One is mediated by NF-{kappa}B, in which IL-12 acts directly through an IL-12R on DC to promote activation of NF-{kappa}B and prime DC for IL-12 production (7). To investigate the effect of rapamycin on NF-{kappa}B in DC, nuclear proteins extracted from DC were assessed for the DNA binding activity of NF-{kappa}B by EMSA using a radiolabeled NF-{kappa}B consensus sequence-specific DNA probe. The specificity of the NF-{kappa}B band was determined by supershifting assay using anti-NF-{kappa}B p50 mAb. The results demonstrated that NF-{kappa}B binding capacity in DC was not inhibited by treatment with rapamycin (Fig. 5A), suggesting that rapamycin does not affect the NF-{kappa}B pathway in DC.



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  		FIGURE 5. Effect of rapamycin on IL-12 signaling pathways. A, DC were propagated from B10 (H2b) BM cells in the presence or absence of rapamycin (20 ng/ml). For further activation, purified DC were incubated with IL-12 (10 ng/ml) for 18 h. Nuclear proteins were extracted and NF-{kappa}B DNA binding activity was determined by ESMA as described in Materials and Methods. Specificity of NF-{kappa}B bands was ascertained by gel-super shifting assay using specific anti- NF-{kappa}B p50 mAb. NS = nonspecific band. Rapamycin showed no inhibitory effect on NF-{kappa}B binding capacity. B and C, Proteins were isolated from DC, and expression of Jak2, Tyk2, Stat4, phosphorylated Stat4 and Jak2 was analyzed by Western blotting. Expression of phosphorylatedf Tyk2 was determined by immunoprecipitation assay as described in Materials and Methods. Activation of Jak2 and Stat4 was induced by IL-12 stimulation. Rapamycin markedly inhibits expression of both phosphorylated Jak2 and Stat4, but did not affect phosphorylated Tyk2 expression. D, Proteins were isolated from B10 mouse naive or activated (Con A 25 mg/ml for 48 h) spleen T cells cultured in the presence or absence of rapamycin (20 ng/ml, added at beginning the culture). For further activation, T cells were exposed to IL-12 (10 ng/ml) for 20 min or 18 h. Expression of phosphorylated Jak2 and Stat4 was analyzed by Western blotting. Rapamycin markedly inhibits expression of phosphorylated Jak2, but not phosphorylated Stat4. All data are representative of three separate experiments.

 
Influence of rapamycin on Stat4 signal transduction pathway

IL-12 also activates a signal transduction cascade family consisting of specific Jaks and Stats that play pivotal roles in cytokine-induced gene expression (24, 25, 26, 27), and interacts directly with DNA sequences in the IFN-{gamma} promoter to increase gene transcription, providing one mechanism for IL-12-induced IFN-{gamma} expression in T cells (24, 25, 26, 27, 28, 29). Its role in regulating DC signaling activity has also been reported (30). To further explore the mechanisms underlying the down-regulation of IFN-{gamma} production by rapamycin, we studied the effects of rapamycin on IL-12-induced Jak2, Tyk2, and Stat4 expression. Proteins were extracted from normal or rapa-DC with or without stimulation with IL-12. Expression of whole and phosphorylated (activated) Jak2, Tyk2, and Stat4 was analyzed by Western blotting. The levels of phosphorylated Tyk2 were determined by immunoprecipitation because anti-phosphorylated Tyk2 Ab was not available. Control DC expressed Jak2, Tyk2, Stat4, and phosphorylated Tyk2, whereas phosphorylated Jak2 and Stat4 levels were low. IL-12 markedly enhanced expression of phosphorylated Jak2 and Stat4 in both early (20 min, data not shown) and late (18 h) phases of IL-12 stimulation. Rapamycin almost completely inhibited expression of both phosphorylated Jak2 and Stat4, but did not affect phosphorylated Tyk2 expression (Fig. 5, B and C). These data clearly indicate a strong inhibitory effect of rapamycin on Jak2 and Stat4 activation in DC. To determine whether the inhibition of Stat4 activation by rapamycin was specifically occurred in DC, we examined the effect of rapamycin on stat4 phosphorylation in T cells. As shown in Fig. 5D, naive spleen T cells did not express phosphorylated Stat4, expression of which was however, slightly upreguated in T cells following activation by Con A or IL-12 alone. Exposure of Con A activated T cells to IL-12 either for 20 min or 18 h dramatically enhanced activation of Stat4, which appeared not to be inhibited by rapamycin. Phosphorylated Jak2 was only expressed in Con A activated T cells that were stimulated by IL-12 for 18h, which was markedly inhibited by rapamycin. These data suggest that rapamycin inhibits Stat4 activation more specifically in DC.

Inhibited allostimulatory activity of DC deficient in Stat 4

To address whether inhibition of Stat4 activation was responsible for rapa-DC dysfunction, allostimulatory activity of DC propagated from Stat4 deficient mice was examined in MLR and CTL assays. Compared with DC derived from normal control mice, DC from Stat4-/- mice stimulated significantly lower proliferative responses in allogeneic T cells to the levels comparable to that of rapa-DC from normal mice (Fig. 6A). Generation of specific CTL activity by stimulation of Stat4 deficient DC was also significantly suppressed, but the suppression was not as profound as that induced by rapa-DC (Fig. 6B). T cells stimulated by Stat4-/- DC produced markedly reduced amounts of IFN-{gamma} but increased levels of IL-10 (Fig. 6C), suggesting a Th1 to Th2 shift. These data suggest that suppression of Stat4 activation appears to, at least partially, mediate rapamycin impairment of DC.



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  		FIGURE 6. Suppressed allostimulatory activity of DC deficient in Stat4 expression. A, In a one way MLR, B6 (H2b) spleen T cells (2 x 105) were cultured for 3 days with number-graded gamma-irradiated DC that were propagated from Stat4-/- (H2d) bone marrow cells. DC propagated from normal BALB/c (H2d) mice in the presence or absence of rapamycin (20 ng/ml) were used as control stimulators. DC deficient in Stat4 and rapa-DC from normal BALB/c mice induced significantly lower T cell proliferative responses compared with normal DC (both p < 0.05). B, Naive B6 spleen T cells that were cultured for 6 days with gamma-iradiated DC propagated from Stat4-/- or normal BALB/c at T:DC ratio of 20:1 in the presence or absence of rapamycin (20 ng/ml) were used as stimulators of CTL induction (BALB/c spleen cells were used as syngeneic control stimulators). Cytotoxicity to P815 targets (H2d) was determined at various effector:target cell ratios in a 4 h 51Cr release assay. No cytotoxicity was generated against syngeneic EL4 (H2b) or third party R1.1 (H2k) targets (data not shown). T cells stimulated with Stat4-/- or rapa-DC from normal BALB/c generated significantly lower specific CTL activity compared with normal control DC (both p < 0.05). C, IFN-{gamma} and IL-10 levels in the supernanants of the 3 day MLR cultures (T:DC ratio of 20:1) were measured by ELISA. T cells cultured with Stat4-/- DC produced markedly lower levels of IFN-{gamma}, but higher IL-10 (*, p < 0.05, compared with cultured with normal DC). All data are representative of three separate experiments

 
Rapamycin inhibits expression of IL-18R on DC

It has been demonstrated in T cells that IL-12-induced expression of IL-12R{beta} and IL-18R is Stat4-dependent (31). We next examined the effect of rapamycin on IL-12-induced expression of IL-12R{beta} and IL-18R in DC. mRNA expression of IL-12R{beta}1, IL-12R{beta}2, IL-18R{alpha}, and IL-18R{beta} in DC and rapa-DC before and after IL-12 stimulation were assessed by semiquantitative PCR. IL-12 stimulated an increase in both IL-12R{beta} and IL-18R expression (Fig. 7). Interestingly, although rapamycin blocked the activation of Stat4, a dual effect was noted: IL-12R{beta} expression was not affected, but IL-18R{alpha} and IL-18R{beta} expression were both markedly suppressed. Because IL-12 and IL-18 have been shown to have synergistic effects (32), down-regulation of IL-18R expression by rapamycin may result in the suppression of IL-12 autocrine activation in DC.



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  		FIGURE 7. Effect of rapamycin on expression of IL-12R and IL-18R mRNA in DC. DC were propagated from B10 (H2b) BM cells in the presence or absence of rapamycin (20 ng/ml). For further activation, purified DC were incubated with IL-12 (10 ng/ml) for 18 h. Expression of IL-12R{beta}1, {beta}2, IL-18R{alpha} and {beta} mRNA was determined by semiquantitative PCR. IL-18R{alpha} and {beta} mRNA expression was induced by IL-12, which was markedly suppressed by rapamycin. Rapamycin had no inhibitory effect on expression of IL-12R{beta}1 or {beta}2. All data are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies of the pharmacological mechanisms of the immunosuppressive effects of rapamycin have mostly focused on its direct influence on T and B cells (33, 34). Its influence on the function of APC is less clear. The present study provides both in vitro and in vivo evidence that rapamycin substantially suppresses DC immune stimulatory function as shown by a reduction in proliferative responses and specific cytotoxic activity in alloreactive T cells and inhibition of Th1 cell differentiation (Figs. 1 and 2). In contrast to the effect of in vivo administration of normal DC that accelerated cardiac allograft rejection, injection of rapa-DC significantly prolonged graft survival in a donor-specific manner (Table I). The underlying mechanisms have not been closely examined. Several studies have demonstrated that rapamycin impairs DC Ag presentation capacity probably by inhibition of macropinocytosis and endocytosis (4, 5), and rapamycin has also been shown to induce apoptosis in human DC (6). In this study, we investigated the mechanisms involved in the suppressive effect of rapamycin on DC immune stimulatory activity. Exposure to rapamycin during DC propagation from mouse BM cells did not enhance DC apoptotic activity as indicated by TUNEL staining (Fig. 2B), suggesting that suppression of DC function by rapamycin is unlikely a result of enhanced DC apoptotic death. This is inconsistent with previous reports of apoptosis in human DC (6) and may reflect species variability.

Our findings indicate that the inhibition of DC immune activity by rapamycin is likely mediated by mechanisms distinct to that of the calcineurin inhibitors, CSA and tacrolimus. For instance, although these two agents inhibit DC expression of MHC class II and costimulatory molecules (1, 2), the results of this study (Fig. 2A) as well as others (2) illustrate that rapamycin does not significantly decrease the expression of these molecules in DC. Another marked difference between rapamycin and calcineurin inhibitors involves DC IL-12 signaling pathways. IL-12 is known to be the most important proinflammatory cytokine produced by DC that promotes differentiation of Th1 effector cells (35, 36). IL-12 production is dependent on the activation of the transcription factor NF-{kappa}B (37, 38); NF-{kappa}B has also been shown to regulate immune responses by promoting DC costimulatory molecule expression (39, 40). CSA and tacrolimus significantly suppress DC production of IL-12 by blocking the NF-{kappa}B pathway (41). In contrast, our results demonstrated that rapamycin does not interfere with NF-{kappa}B DNA binding activity (Fig. 5) or IL-12 expression in murine DC (Figs. 3, A and B). However, IL-12-induced IFN-{gamma} production in DC culture is significantly down-regulated by rapamycin (Fig. 3, C and D). The importance of IFN-{gamma} production by activated DC has been controversial. IFN-{gamma} is mainly produced by T and NK cells, whereas, there are several reports demonstrating that the amounts of IFN-{gamma} produced by DC are even greater than those by NK cells (23). It has been proposed that IFN-{gamma} from DC serves as an adjuvant to IL-12 in the initiation of immune responses. Thus, T cell-derived IFN-{gamma} usually appears 2–4 days after the initiation of a response, while DC are a source of early IFN-{gamma} generated in response to a cascade of signaling events that facilitates early polarization of T cell responses (42). We posited that the decrease in DC IFN-{gamma} production brought about by rapamycin may result in the abrogation of early IFN-{gamma} production during the interaction of DC and T cells, thus leading to a decrease in Th1 lymphocyte differentiation by lowering T cell responsiveness to IL-12 and accounting for rapamycin’s immunosuppressive effect. However, we found that DC propagated from IFN-{gamma}-/- mice stimulated even higher proliferative responses and CTL activity in allogeneic T cells compared with DC derived from normal controls (Fig. 4, A and B), suggesting that DC-produced IFN-{gamma} may actually play the role of a negative regulator of DC function. Therefore, inhibition of IFN-{gamma} in DC by rapamycin is unlikely attributed to impaired DC function. RNase protection assays revealed that mRNA expression of other cytokines such as TNF-{alpha}, IL-6, and IL-10 induced by IL-12 stimulation were also markedly inhibited by rapamycin (Fig. 3D). Cytokines constitutively expressed in DC such as IL-1{alpha} and IL-1{beta} were also markedly down-regulated by rapamycin. The suppression of these proinflammatory cytokines may play a role in rapamycin-induced DC dysfunction.

The inhibition of IL-12-stimulated IFN-{gamma} expression by rapamycin prompted us to more extensively investigate the effect of rapamycin on IL-12 signaling and autocrine activity in DC. The possibility of IL-12 autocrine activity was raised by data showing that not only are DC a major cell type that produces IL-12, but they also express a high affinity receptor for IL-12 (43). Although it had long been assumed that the only cells responding to IL-12 were T, B, NK, and NKT cells (35), it became evident that IL-12 released by DC not only acted on bystander T cells and facilitated Th1 cell differentiation but also primed DC to enhance their Ag-presenting capacity (4, 7, 43). Signaling through IL-12R on DC appears to involve at least two distinct pathways. One pathway involves the activation of NF-{kappa}B which leads to enhanced expression of MHC class II molecules, costimulatory molecules, and IL-12 production, ultimately resulting in more effective presentation of Ags by DC (9, 10, 11). The results of this study suggest that rapamycin does not affect the NF-{kappa}B pathway of IL-12 signaling in DC (Fig. 5A). Another IL-12 activated signal transduction pathway that has been characterized recently involves Jaks and Stats (24, 25, 26, 27, 44). The transcription factor Stat4 has been shown to mediate the IL-12 autocrine pathway (12, 30) and Stat 4 is definitely required for IL-12-dependent IFN-{gamma} production as demonstrated in studies using Stat4 knockout mice (46). IL-12 exerts its effect by inducing phosphorylation of Jak2 and Tyk2, which in turn phosphorylate Stat4 (46, 47). Recent studies have demonstrated that DC are capable of producing significant amounts of IFN-{gamma} in response to IL-12 (7, 23, 48), and Stat4 levels directly correlate with IL-12-dependent IFN-{gamma} production by DC (12). The data in this study demonstrated that control DC propagated from mouse BM in GM-CSF and IL-4 did not express activated (phosphorylated) Stat4 (Fig. 5C). IL-12 stimulation induced expression of phosphorylated Jak2 and Stat 4 in DC, and this was almost completely inhibited by rapamycin (Fig. 5B). These observations are consistent with previous reports that DC and macrophages from Stat4-/- mice failed to produce IFN-{gamma}, suggesting that the control of Stat4 expression is an important regulator of high-level IFN-{gamma} production (47). The functional importance of Stat4 blockade is evident in Stat4-/- DC, which failed to stimulate allogeneic T cell proliferation as well as generate specific CTL activity (Fig. 6, A and B). Stat4 is induced in DC during the maturation and activation processes, and this can be suppressed by the Th2 cytokines IL-4 and IL-10 (12). This suggests that complex regulatory mechanisms of Stat4 signal transduction pathway exists in DC. Although IFN-{gamma} is a downstream product of IL-12/Stat4 signaling and exposure to rapamycin markedly blocks the production of IFN-{gamma} in DC (Fig. 3C), the data obtained from DC derived from IFN-{gamma}-/- mice suggest that inhibition of IFN-{gamma} is not related to DC functional impairment by rapamycin (Fig. 4, A and B). This indicates that a Stat4 downstream effector factor(s) other than IFN-{gamma} may be involved in rapamycin-induced DC dysfunction. Interestingly, IL-12 induced activation of Stat4 in T cells appeared not to be inhibited by rapamycin, despite rapamycin at the same dose suppressed expression of phosphorylated Jak2 (Fig. 5D). Taken together, these data suggest that inhibition of Stat4 activation by rapamycin occurs more specifically in DC.

IL-18R expression, but not IL-12R expression in DC was markedly inhibited by rapamycin (Fig. 7). Expression of IL-18 has been shown to be Stat4 dependent (31). This raises the possibility that blockade of DC immune stimulatory activity by rapamycin may be also mediated by the suppression of IL-18R, because IL-12 and IL-18 have been shown to have many synergistic effects (32). Therefore, down-regulation of IL-18R expression by rapamycin may negatively affect DC autocrine activation mediated by IL-12 signaling, thus resulting in impaired DC immune stimulatory function. Inhibition of the proinflammatory cytokines IL-1, TNF-{alpha}, and IL-6 by rapamycin (Fig. 3D) may also contribute to suppressed DC function.

In summary, the data in this study demonstrates that rapamycin inhibits Stat4 phosphorylation but does alter NF-{kappa}B activation in murine DC. Rapamycin decreased DC IL-18R expression and IL-12 autocrine activation, and this may be a principal mechanism by which rapamycin inhibits DC allostimulatory activity. Selective inhibition of the Stat4 signal transduction pathway might constitute a novel approach to the regulation of DC immune responses. To our knowledge, this is the first study that has demonstrated a pharmacological mechanism of action of rapamycin that involves blockade of the Stat4 pathway. These findings should encourage further investigation into the interactions of immunosuppressive drugs and the Stat pathway and future work may potentially provide the basis for a novel and attractive immunotherapeutic approach.


   Acknowledgments
 
We thank Alison Logar for assistance with flow cytometry, and Dr. Michael De Vera for helpful review.


   Footnotes
 
1 This study was supported by funds from National Institutes of Health Grant DK58316 and Juvenile Diabetes Foundation International Grant P1893135. P.-H.C. was a fellow from Chang Gung Memorial Hospital, Kaohsiung, Taiwan. Back

2 P.-H.C and L.W. contributed equally to this study. Back

3 Address correspondence to Dr. Shiguang Qian, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh Medical Center, E1540 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. E-mail address: qiansl{at}msx.upmc.edu Back

4 Abbreviations used in this paper: CSA, cyclosporine; DC, dendritic cell; BM, bone marrow; Jak, Janus kinase; Tyk, tyrosine kinase. Back

Received for publication May 30, 2003. Accepted for publication November 5, 2003.


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