Extracellular signal-regulated kinases 1/2 (ERK1/2 ) (p42/p44 MAPK) are serine/threonine protein kinases belonging to the family of mitogen-activated protein kinase (MAPK), in which three major subfamilies have been identified: ERK1/2 , c-Jun N-terminal kinase (JNK), and p38 MAP kinase. Each MAPK subfamily has capable of responding to different stimuli, such as cellular stress and growth factors (1–3 ERK1/2 depends on the interaction of several signaling proteins, including Ras, Raf, MEK1/2, and ERK1/2 (4, 5 6, 7
It has been documented that ERK1/2 signaling plays a crucial role in T-cell activation, which is initially mediated by the Syk family tyrosine kinase ZAP70, followed by two signal pathways. One is the calcium/calcineurin/NFAT pathway via activation of phospholipase c-γ. The other is Grb/SOS/Ras pathway, which consequently stimulates MEK1/2-ERK1/2 signaling. Activation of ERK1/2 results in transcription of c-Fos and JunB , which protein products heterodimerize to form the AP-1 complex and bind to gene enhancers to stimulate transcription of interleukin (IL)-2 and many other genes (8–11 ERK1/2 signaling plays a critical role in Th1/Th2 polarization. Stimulation with high concentrations of peptide resulted in the failure to produce early IL-4 production, which can be reversed by blockade of the ERK1/2 signaling leading to subsequent Th2 differentiation (12 ERK1/2 signaling by PD98059 switches T-cell phenotype to Th2 (13
In this study, we report the immunosuppressive effects of ERK1/2 signaling blockade using a small molecular compound PD98059, a specific ERK1/2 inhibitor (14 ERK1/2 signaling inhibitor may allow CsA minimization without permitting rejection in clinical transplantation. Finally, the effect of ERK1/2 inhibition with PD98059 appears to be novel in that intragraft Th1 responses were attenuated while Th2 responses were augmented.
MATERIALS AND METHODS
Animals and Drugs
Male C57BL/6 (B6) (H-2b ) and Balb/c (H-2d ) mice, weighing 25–30 g, were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were housed in the animal care and use facility at the University of Western Ontario (London, Ontario, Canada), and were cared and used in accordance with the guidelines established by the Canadian Council on Animal Care.
PD98059 [2′-animo-3′-methoxyflavone; 2-(2′-amino-3′-methoxypheny)-oxanaphthalen-4-one] (LC Labs, Woburn, MA) were prepared in dimethyl sulfoxide (DMSO; 33 mg/mL). Prior to injection both CsA (Novartis, Basel, Switzerland) and PD98059 were diluted in saline. Drugs were given to the recipient mice daily until end of experiment or for 20 days. PD98059 and vehicle (DMSO in saline) were intraperitoneally injected, and CsA was subcutaneously injected.
Heterotopic Cardiac Transplantation
Intra-abdominal heterotopic cardiac transplantation was performed as previously described (15
Determination of Intragraft Leukocyte Infiltration
After phosphate-buffered saline (PBS) perfusion histological analysis and direct cell count of intragraft leukocyte infiltration were performed in a blinded fashion. Tissue samples were removed at necropsy and fixed in 10% buffered formaldehyde. Specimens were then embedded in paraffin, and sectioned for the hematoxylin and eosin (H&E) staining. The microscopic sections were examined for pathological markers for rejection and severity of leukocyte infiltration, which were scored as: 0: no infiltrates; 1: 0–25% of viewed area with infiltrates; 2: 25–50% of viewed area with infiltrates; 3: 50–75% of viewed area with infiltrates; or 4: 75–100% of viewed area with infiltrates. The scores of leukocyte infiltration in histological analysis were confirmed by direct cell count of infiltrated leukocytes. Intragraft leukocytes were isolated as described previously (16
Preparation of Activated T Cells
Concanavalin (Con) A-activated T cells were prepared by stimulation of mouse splenocytes with Con A (Sigma- Aldrich) in RPMI 1640 medium containing 10% fetal bovine serum (FBS) as described previously (17
Mixed Leukocyte Reactions (MLR)
MLR assay was performed in triplicate in 96-well U-bottom microculture plates (Corning Inc, Corning, NY). Splenocytes of B6 mice (1×105 /well, 3000 rad γ-irradiated) were used as stimulators, and splenocytes from Balb/c mice (2×105 /well) were used as responders. The controls for basal levels of responder proliferation were the cultures without stimulator cells. Cultures were maintained in RPMI-1640 complete medium for four days in 5% CO2 . [3 H] thymidine (1 μCi/well) was added for the final 18 hr, and its incorporation as an index of cell proliferation was assessed by liquid scintillation counting. Results were expressed as mean counts per minute (cpm)±standard deviation (SD).
Induction of T Cell Differentiation
Naïve splenic T cells from Balb/c mice were enriched by Nylon wool-eluted (Robbins Scientific Co., Sunnyvale, CA) and were differentiated into Th1 or Th2 by an established procedure described previously (17 5 cells/mL, 4 mL/well) were stimulated in anti-CD3 antibody-coated plate in the presence of PD980559 in the culture medium supplemented with either 10 ng/mL rIL-4 (BD Biosciences) and 5 μg/ml anti-interferon (IFN)-γ antibody (clone R4-6A2) for Th2 differentiation, or 10 ng/mL rIL-12 (BD Biosciences) for Th1 differentiation. After seven days of incubation, the differentiated T cells were washed several times with culture medium to remove exogenous cytokines, and then an equal number of these cells (5×105 cells/mL) from each sample were restimulated with plate-bound anti-CD3 antibody for 24 hr. The level of differentiation (Th1 or Th2) in each culture was determined by amount of the marker cytokine (Th2: IL-4; Th1: IFN-γ) production measured by enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA).
Phenotype Analysis of Graft Infiltrating T Cells
The population of Th1 (producing IFN-γ) or Th2 (producing IL-4) was determined by fluorescence-activated cell sorting (FACS) analysis with intracellular cytokine staining. Graft-infiltrating cells were isolated as described above and stimulated with phorbol myristate acetate (PMA; 50 ng/mL, Sigma-Aldrich) and ionomycin (500 ng/mL, Sigma- Aldrich) for four hours at 37°C, to which monensin (1:1000, eBioscience) added for the last two hours. Cell surface staining using anti-CD4-allophycocyanin (APC; BD Biosciences) was performed in PBS/fetal calf serum for 30 min. After washed and fixed in 4% paraformaldehyde for 20 min, the cells were washed with permeabilization buffer (eBioscience) twice, then preincubated with permeabilization buffer for 5 min. The intracellular cytokine (IFN-γ or IL-4) was labeled with either anti-IFN-γ-FITC (clone XMG1.2, 5.0 μg/mL, eBioscience) or anti-IL-4-PE (clone 11B11, 5.0 μg/mL, eBioscience) for 20 min. After wash with permeabilization buffer, the cells were washed in PBS to allow membrane closure. Samples were analyzed by FACS analysis as compared to control immunoglobulin (Ig) G staining. Results were analyzed using CellQuest software (BD Biosciences).
Cytokine Secretion
The cell culture supernatants were harvested and analyzed for the presence of cytokines (IFN-γ and IL-4) using commercial ELISA kits (eBioscience) according to the manufacturer’s protocols. All experiments are performed at least in triplicate samples. A standard curve using recombinant cytokine was generated for each assay.
Western Blot
Protein levels of phosphorylated ERK1/2 (p-ERK1/2 ), phosphorylated STAT6 (p-STAT6) and c-Fos were determined using Western blots. Briefly, whole cell lysates were homogenized in lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM ethylenediamine tetraacetic acid (EDTA), 0.1 mM ethylene glycol tetraacetic acid, 0.1% NP 40, 1 mM dithiothreitol, and a protease inhibitor cocktail (Roche, Mannheim, Germany)], followed by mixing with an equal volume of 2× sodium dodecyl sulfate (SDS) sample buffer [20 mM Tris-HCl (pH 6.8), 5% (w/v) SDS, 10% (v/v) mercaptoethanol, 2 mM EDTA, and 0.02% bromophenol blue] and then boiled for 5 min. The protein content of cell lysates was determined by Bio-Rad assay (Bio-Rad Lab, Hercules, CA). Then 100–150 μg of total protein from each sample was fractionated by 10% (for p-ERK) or 7% (for p-STAT6) SDS and transferred to nitrocellulose membranes (Bio-Rad Lab), blocked with 5% fat-free milk (Carnation) in tris-buffered saline (TBS)-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for one hour, and then probed with either anti-p-ERK1/2 or anti-c-Fos antibody (Santa Cruz Biotech, CA) in TBS containing 2.5% of milk or anti-p-STAT6 antibody (Cell Signaling Technology, MA) in TBS containing 5% bovine serum albumin at 4o C overnight. The p-ERK1/2 , c-Fos, or p-STAT6 protein binds recognized by the antibodies on the membrane were visualized by an enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech, Buckinghamshire, England). Blots were reprobed using anti-ERK1/2 (Stressgen, Victoria, BC) or anti-STAT6 antibody (Chemicon International, Temecula, CA) for total ERK1/2 or STAT6 proteins and confirmation of equal protein loading.
Reverse-Transcriptase Polymerase Chain Reaction
Reverse-transcriptase (RT) polymerase chain reaction (PCR) kits were used to determine relative differences in cytokine transcript (mRNA) levels following the provided protocol (Invitrogen, Carlsbad, CA). Briefly, total RNA was obtained from cells or tissues using TRIzol Reagent (Invitrogen), and 5 μg of total RNA were used in RT, followed by PCR amplification of the target using appropriate cycle number and primers (IFN-γ, sense, 5′-GCTGTTTCTGGCTGTTACTG and antisense, 5′-CTGTGGGTTGTTGACCTCA; IL-4, sense, 5′-CATCCTGCTCTTCTTTCTCG and antisense, 5′-GGAAGTCTTTCAGTGATGTGG). PCR products were visualized in 1% agarose in TAE buffer containing 0.5 μg/mL of ethidium bromide. Analysis of levels was semiquantitative with comparison to glyseraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
Statistical Analysis
Data were collected from separate experiments (at least three experiments if results were consistent) in each study for statistical test. Statistical significance between groups was determined by t test (one-tailed distribution). Graft survival between groups of transplanted animals was compared using the Kaplan-Meier Analysis (Statview statistical software, Cary, NC). Values of P ≤0.05 were considered statistically significant.
RESULTS
PD98059 Inhibits T Cell Proliferative Response to Alloantigen and Prolongs Cardiac Allograft Survival in Mice
An early study has demonstrated that the presence of ERK1/2 inhibitor U0126 inhibits T-cell proliferation in response to anti-CD3 plus CD28 antibodies cross-linking via reduction of IL-2 production but not IL-2R signaling (9 ERK1/2 inhibitor (18 19 Figure 1A and B , PD98059 inhibited TCR/CD28-mediated T cell proliferation as well as ERK1/2 activation, the same effects of U0126 as previously reported. PD98059 was further tested in T-cell proliferation and ERK1/2 activation stimulated by alloantigen in MLR. As shown in Figure 1C and D , PD98059 inhibited alloantigen-stimulated T-cell proliferation in MLR in a dose-response manner. Proliferation was decreased from 27,095±1,493 cpm in vehicle treated cultures to 12,194±2,942 cpm in 7.5 μM PD98059-treated (46% of decrease), 6,978±886 cpm in 15 μM PD98059-treated (70.4% of decrease), or 2,914±45 cpm in 30 μM PD98059-treated (94.3% of decrease). The basal levels of proliferation in the cultures without stimulator cells were 2,047±50 cpm. At the same time the ERK1/2 activation and the downstream c-Fos expression were inhibited by the presence of PD98059 (Fig. 1D ).
FIGURE 1.:
PD98059 inhibits T cell proliferation and ERK1/2 activation in vitro. (A) Naïve splenocytes from naïve Balb/c mice (0.2×105 cells/well in 96-well plates) were stimulated with anti-CD3/CD28 antibodies (2 μg/ml, each) in the absence or presence of PD98059 for 48 h. [3 H] thymidine (1 μCi/well) was added for the final 18 hr. Data are presented as mean±SD of triplicate determinants in a typical experiment, which was repeated twice with consistent results; P <0.001 (7.5 μM PD98059-treated vs. vehicle cultures, n=3). (B) Naïve splenocytes (12×106 cell/ml) isolated from Balb/c mice were pretreated with PD98059 or DMSO for 30 min in RPMI 1640 complete medium at 37o C under 5% CO2 , followed by stimulation with anti-CD3 and/or CD28 antibodies (5 μg/ml each) for 30 min. Phosphorylation of ERK1/2 (p-ERK1/2 ) was analyzed by Western blot with anti-phos-ERK1/2 antibody, and total ERK1/2 protein was detected by reprobing the blot with anti-ERK1/2 antibody. Data are representative of three separate experiments. (C) MLR cultures were established by adding γ-irradiated splenocytes (1×105 cells/well) from B6 mice to naïve splenocytes (2×105 cells/well) from Balb/c mice in U-bottom 96-well plates, and treated with different concentrations of PD98059. Data are presented as mean±SD of triplicate determinants in a typical experiment, which was repeated twice with consistent results; P <0.01 (PD98059 vs. vehicle controls, n=3). (D) MLR cultures were incubated in the absence or presence of 30 μM PD98059 for 24 hr. The protein levels of p-ERK1/2 , c-Fos and total ERK1/2 were examined by Western blot. Basal: Nonstimulated splenocytes from Balb/c mice. Data are representative of three separate experiments.
Targeting ERK1/2 signaling as a therapeutic strategy was tested in a murine model of cardiac allograft transplantation using PD98059. As shown in Figure 2 , lower doses of PD98059 (20 mg/kg) alone did not markedly improve cardiac allograft survival, but administration of higher doses PD98059 (100 mg/kg) significantly prolonged graft survival to 12.6±1.3 days from 8.3±0.5 days in vehicle-treated control group (log-rank, P <0.0001). Mice were able to tolerate PD98059 for this period of treatment. Isografts (Balb/c heart to balb/c) survived more than 100 days. These data demonstrate that blockade of ERK1/2 signaling might be considered as a novel immunosuppressive strategy, although longer-term studies would be required.
FIGURE 2.:
PD98059 therapy prolongs cardiac allograft survival in mice. Balb/c mice received B6 heart donors and were treated daily by ip injection of vehicle DMSO (○) (n=10), 20 mg/kg PD98059 (▪) (vs. DMSO, log-rank P =0.7445, n=10) or 100 mg/kg PD98059 (•) (vs. DMSO, log-rank P <0.0001, n=10).
Additional Effect of PD98059 on CsA Immunosuppression in the Prevention of Cardiac Allograft Rejection
PD98059 and CsA block different signaling pathways required for T-cell activation (14, 20 Figure 3A , CsA at 10 ng/mL alone suppressed 35% of lymphocyte proliferation (from 33,535±3,256 cpm in controls to 22,195±1,292 cpm in CsA-treated), which was further decreased to 69% (11,412±926 cpm versus controls) by 7.5 μM PD98059 treated cultures and to 93% (3,563±587 cpm versus controls) by 15 μM PD98059. The basal proliferation was 1,363±853 cpm. The additional effect of PD98059 on CsA in the inhibition of alloantigen-stimulated proliferation in MLR was statistically significant (P <0.001).
FIGURE 3.:
PD98059 increases CsA-mediated suppression of T cell proliferation in vitro and prolongation of cardiac allograft survival in vivo. (A) Increase of CsA-mediated inhibition of T cell proliferation by PD98059 in the responses to alloantigen in MLR. MLR cultures were established as described in
Figure 1C , and treated with different concentrations of PD98059 (PD) in the presence of CsA (10 nM). Data are presented as mean±SD of triplicate determinants in a typical experiment, which was repeated twice with consistent results;
P <0.001 (combination of CsA and PD98059 vs. CsA alone, n=3). (B) Additional prolongation of allograft survival by the combination therapy of CsA and PD98059 in a mouse cardiac transplantation model. Recipient Balb/c mice received B6 heart donors and were treated with vehicle DMSO (○) (n=10), 15 mg/kg CsA monotherapy (▴) (vs. DMSO, log-rank
P <0.0001, n=9) or combination therapy of 15 mg/kg CsA and 100 mg/kg PD98059 for 20 days (▪) (vs. CsA monotherapy, log-rank
P <0.0001, n=9).
The therapeutic effect of combination therapy of PD98059 and CsA was tested in vivo. As indicated in Figure 3B , combination therapy of PD98059 (100 mg/kg) with CsA (15 mg/kg for 20 days) further enhanced graft survival (34.4±1.2 days) compared to CsA (15 mg/kg) monotherapy (14.9±1.1 days; log-rank, P <0.0001). The survival of grafts in the combination therapy-treated recipients was also considerably longer than those treated with PD98059 (100 mg/kg) monotherapy (P <0.0001), shown in Figure 2 . One has to acknowledge that intestine adhesion was noted in the mice receiving the combination therapy. Taken together, our data demonstrate that PD98059 provides additional inhibition of T-cell proliferation in CsA-treated T cells in the responses to alloantigen stimulation, which may contribute to additional prolongation of allograft in combination therapy.
PD98059 Reduces Intragraft Leukocyte Infiltration and IFN-γ Production
The in vivo mechanisms by which PD98059 prolonged allograft survival were investigated. We first confirmed ERK signaling pathway was blocked by PD98059 in vivo. Graft tissues were harvested on day seven posttransplantation from vehicle- or PD98059 (100 mg/kg)-treated recipient mice. As shown in Figure 4 , the intragraft p-ERK1/2 , particularly ERK1 (p42), was markedly increased in vehicle control group, and was reduced by PD98059 treatment to the levels found in naïve tissue in Western blot analysis. Even these data could not provide exactly in which cell type(s) ER1/2 are activated, but at least they suggest that PD98059 therapy can achieve effective ERK1/2 -mediated signaling pathway blockade in transplanted grafts.
FIGURE 4.:
PD98059 therapy inhibits
ERK1/2 activation in grafts. The transplanted recipients as described in
Figure 2 were treated with PD98059 (100 mg/kg) or DMSO (vehicle) for seven days after transplantation. After perfusion with PBS, graft tissue was immediately homogenized in lysis buffer and
ERK1/2 activation, indicated by the level of p-ERK, was analyzed by Western blot. The total
ERK1/2 proteins as control were reprobed in the same blot. Naïve: naïve heart tissue; Vehicle: grafts from vehicle DMSO-treated group; PD98059: grafts from PD98059-treated group. Data are presented as a typical image of p-
ERK1/2 vs. internal control total
ERK1/2 of three separate analyses.
The inhibitory effect of PD98059 on alloimmunity (particularly T cells) in vivo was directly verified by examination of leukocyte infiltration in cardiac grafts. Grafts from recipients treated with PD98059 (100 mg/kg) or vehicle were examined on day seven after transplantation. As shown in Figure 5A , in histological analysis more infiltrates and hemorrhage were obviously observed in vehicle-treated grafts, whereas PD98059 treatment resulted in reduction of infiltrates and absence of hemorrhage. The leukocyte infiltration was significantly reduced by PD98059 therapy, with scores of 1.5±0.25 in PD98059-treated groups compared to 2±0.41 in vehicle-treated group (P =0.029; Fig. 5B ). These data were further supported by a direct count of infiltrated leukocytes. As shown in Figure 5C , the total leukocytes isolated from each graft in vehicle-treated group were 4.2±1.3 (×106 ), which was reduced to 2.5±0.6 (×106 ) in PD98059-treated group (P =0.047).
FIGURE 5.:
PD98059 therapy reduces leukocyte infiltration to the grafts. The recipient mice with cardiac allografts were treated with vehicle DMSO or PD98059 as described in
Figure 3 . Grafts were harvested after PBS perfusion on day seven posttransplantation. (A) Reduction of histological rejection (infiltrates and hemorrhage) by PD98059 treatment. Naïve heart or graft tissue was fixed in 10% buffered formaldehyde, embedded in paraffin, and then sectioned for H&E staining. Naïve: naïve heart tissue; Vehicle: grafts from vehicle DMSO-treated group; PD98059: grafts from PD98059-treated group. Data are presented as a representative of microscopic images in each group. (B) The leukocyte infiltration in the sections of grafts was scored by histological analysis in a blinded fashion. Data are presented as mean±SD of four grafts;
P =0.029 (vehicle vs. PD98059, n=4). (C) The intragraft leukocytes in each graft were counted using a hemacytometer. Data are presented as mean±SD of four grafts (
P =0.047; vehicle vs. PD98059, n=4).
The regulation of T cell phenotypes by PD98059 in grafts was tested by analysis of intragraft mRNA cytokine profiles (IFN-γ versus IL-4) using RT-PCR as well as enumerating IFN-γ or IL-4 expressing cells by FACS. As shown in Figure 6A , in three out of four grafts from the PD98059-treated group, IFN-γ mRNA level was reduced while IL-4 mRNA was increased as compared vehicle-treated controls. To further confirm whether PD98059 therapy could bias towards potentially beneficial Th2 immune responses inside the grafts, infiltrates were isolated and characterized for cytokine expression (IFN-γ producing versus IL-4 producing) by FACS analyses. As shown in a representative graft from each group (Fig. 6B ), in a vehicle-treated graft 21.99% expressed IFN-γ in 64.2% of total CD4+ T cell, while 12.43% expressed IL-4 in 61.49% of total CD4+ T cells. In contrast, in a PD98059-treated graft IFN-γ producing cells decreased to 11.81% in a total of 63.55% of CD4+ T cells, and IL-4 producing cells increased to 25.7% in a total of 58.86% of CD4+ T cells. Overall, the percentage of IFN-γ producing T cells (Th1 type) in CD4+ cell population was significantly reduced from 46.4±9.5% in vehicle group to 26.6±12.7% in PD98059 group (P =0.0422; Fig. 6C ), while the percentage of IL-4 producing T cells (Th2 type) in CD4+ cell population was increased from 18.9±3.6% in vehicle group to 30.4±10.3% in PD98059 group (P =0.0338). Taken together, these data suggest that that PD98059 therapy reduces leukocyte infiltration but can also shift Th1 to Th2 immune responses inside the grafts even its effect on alloantibody levels or isotypes remains elusive.
FIGURE 6.:
PD98059 therapy reduces inflammatory Th1 immune responses inside the grafts. The recipient mice with cardiac allografts were treated with vehicle DMSO or PD98059 as described in
Figure 3 . Naïve hearts or grafts were harvested after PBS perfusion on day seven posttransplantation. (A) Total RNA of graft or heart tissue was isolated, and the mRNA levels of IFN-γ, IL-4, and GAPDH were determined by RT-PCR with appropriate PCR cycle numbers. Naïve: naïve heart tissue; Vehicle: grafts from vehicle DMSO-treated group; PD98059: grafts from PD98059-treated group. Data are presented as a typical image of IFN-γ vs. IL-4 of three separate RT-PCR analyses. The GAPDH mRNA was included as an internal control for amount of total RNA in each sample. (B) Infiltrates were isolated, and the IFN-γ -producing CD4
+ cells (top panel) or IL-4-producing CD4
+ cells (bottom panel) were counted by FACS analysis with intracellular cytokine staining. Data are presented as a typical profile of IFN-γ-producing or IL-4-producing CD4
+ cells from one graft in vehicle or PD98059-treated group. (C) The percentage of IFN-γ-producing CD4
+ cells in total CD4
+ infiltrates was calculated in each graft. Data are presented as mean±SD of four grafts (
P =0.0422; vehicle vs. PD98059, n=4). (D) The percentage of IL-4-producing CD4
+ cells in total CD4
+ infiltrates was calculated in each graft. Data are presented as mean±SD of four grafts (
P =0.0338; vehicle vs. PD98059, n=4).
PD98059 Promotes Development of Th2 Cells by Stimulation of IL-4 Production
To understand the molecular mechanisms by which PD98059 might regulate Th differentiation, we examined the effect of PD98059 on Th cell differentiation. As shown in Figure 7A , PD98059 did not change IFN-γ production under conditions that favored Th1 response but increased IL-4 producing Th2 cells. IL-4 production in the presence of 1 μM PD98059 increased from 6.06±0.44 (basal levels) to 33.98±0.89 ng/mL (5.6-fold) at 24 hours (Fig. 7A ), indicating that inhibition of ERK1/2 signaling enhances Th2 differentiation.
FIGURE 7.:
PD98059 promotes Th2 differentiation by stimulation of IL-4 production. (A) Naïve T cells Balb/c mice were differentiated into Th1 or Th2 in the presence of PD98059. Th1 differentiation was indicated by IFN-γ production (top), and Th2 differentiation was reflected by IL-4 production (bottom). Data are presented as mean±SD of triplicate determinants in a typical experiment, which was repeated twice with consistent results. No significant difference was found in the levels of IFN-γ production between PD98059-treated and -untreated cells, while PD98059 significantly increased IL-4 production (Th2 differentiation) as compared to untreated controls (PD98059 vs. untreated control, P <0.001, n=3). (B) Activated T cells were pretreated with various concentrations of PD98059 (PD) for 30 min, then stimulated by 10 ng/mL of IL-12 or IL-4. Total RNA was isolated after two or six hours of treatment. The mRNA levels of IFN-γ (IL-12 stimulated T cells) and IL-4 (IL-4-stimulated T cells) were analyzed by RT-PCR with appropriate PCR cycle number and determined by the density ratio to the internal control of GAPDH. N: basal mRNA of cytokines in nonstimulated activated T cells. Data are representative of three separate experiments, in which results were consistent. No significant difference was found in the levels of IL-12-induced IFN-γ mRNA between PD98059-treated and -untreated cells, while PD98059 at 3–10 μM significantly increased IL-4-induced IL-4 transcript in cells as compared to untreated controls (PD98059 vs. untreated control, P <0.01, n=3). (C) Cells were pretreated with various concentrations of PD98059 (PD), and then stimulated by 25 ng/mL of IL-4 for 15 min. Proteins were harvested from whole cells. The activation of IL-4 stimulated ERK1/2 or STAT6 was analyzed by Western blot with anti-p-ERK1/2 or anti-p-STAT6 antibody, and total ERK1/2 or total STAT6 protein was detected by reprobing the same blot with anti-ERK1/2 or anti-STAT6 antibody. The data are representative of four independent experiments.
To further understand the mechanisms by which PD98059 increases Th2 development, the regulation of cytokine production (IFN-γ versus IL-4) following inhibition of ERK1/2 signaling was tested in vitro. Activated T cells were stimulated with either 10 ng/mL of IL-12 (for IFN-γ production) or 10 ng/mL of IL-4 (for IL-4 production) in the absence or presence of PD98059. As shown in Figure 7B , in six hour-cultures the density ratio of IFN-γ mRNA to GAPDH mRNA was largely unchanged being 1.22 in 1 μM PD98059-treated cultures, 1.32 in 3 μM PD98059-treated cultures, and 1.29 in 10 μM PD98059-treated cultures, compared to 0.99 in vehicle (0 μM PD98059) cultures. However, the density ratio of IL-4 mRNA to GAPDH mRNA was 0.48 in vehicle cultures, which was significantly increased to 0.82 in 1 μM PD98059-treated cultures, 1.23 in 3 μM PD98059-treated cultures (2.6-fold increase), and 1.53 in 10 μM PD98059-treated cultures (3.2-fold increase; P <0.05).Similar results were also seen in the two hour-cultures. These data suggest that inhibition of ERK1/2 signaling enhances IL-4-stimulated IL-4 production but not IL-12-stimulated IFN-γ production in activated T cells.
In Th2 cells, IL-4R leads two signaling pathways, JAK1/3-STAT6 and IRS/Ras/ERK1/2 (21 ERK1/2 in activated cells reached a maximum at 15 min after stimulation with IL-4 (25 ng/mL; data not shown). As shown in Figure 7C , IL-4 activated both ERK1/2 and JAK/STAT signaling pathways with phosphorylation of ERK1/2 (p-ERK1/2 ) and STAT6 (p-STAT6). However, addition of PD98059 exclusively inhibited IL-4-induced phosphorylation of ERK1/2 but not STAT6. Collectively, our data suggest that PD98059 specifically inhibits IL-4-stimulated ERK1/2 signaling, resulting in increase of IL-4 production and promotes Th2 differentiation, which may not be regulated by JAK/STAT signaling pathway.
DISCUSSION
In this study, we demonstrated that administration of ERK1/2 signaling inhibitor PD98059 significantly prolonged cardiac allograft survival with marked reduction of leukocyte infiltration as well as an increase in intragraft Th2 immune responses. PD98059 enhanced IL-4-stimulated IL-4 production without attenuating STAT6 signaling in activated T cells.
It has been documented that specific inhibition of ERK1/2 signaling reduces T-cell proliferation/activation in response to antigens including alloantigen in MLR through reduction of IL-2 production, and addition of exogenous IL-2 can rescue the inhibited T-cell proliferation (8, 9, 22 ERK1/2 inhibitors has not been tested in vivo. Our data for the first time demonstrate that PD98059 treatment results in a significant reduction of alloantigen-stimulated leukocyte (including T cells) infiltration in cardiac allograft (Fig. 5 ) and delays graft rejection (Fig. 2 ).
It has been suggested that PD98059 stimulates anti- inflammatory Th2 immune responses by upregulation of Th2 cytokine production in T cells. In anti-CD3 antibody/PMA or anti-CD3/CD28 antibodies stimulated human T cells PD98059 profoundly upregulates secretion of IL-4 and other Th2 cytokines (IL-5 and IL-13) (8 8 Fig. 7 ). The discrepancy of these two studies may be due to the difference of experimental systems and stimuli. We stimulated activated T cells with IL-12, whereas in anti-CD3 antibody/PMA or anti-CD3/CD28 antibodies stimulated T cells PD98059 blocks TCR/CD28-ERK signaling resulting in decrease in IFN-γ and other T-cell cytokines such as IL-2 (8 ERK1/2 but not STAT6 signaling pathway by PD98059 increases IL-4 production (Fig. 7 ), implying that ERK1/2 signaling may act as a negative signaling pathway in IL-4-driven Th2 development and IL-4 production in Th2 cells. Taken together, the immunosuppressive efficacy of pharmacologic intervention of ERK1/2 signaling could be mediated by two ways. One is to reduce alloantigen-ERK signaling leading to inhibition of T-cell proliferation and activation by reducing secretion of IL-2 or other T-cell growth cytokines (e.g., IFN-γ). The other is to promote intragraft anti-inflammatory Th2 immune responses by increasing IL-4-producing Th2 cells through blocking negative IL-4R-ERK signaling. As a result, increase in Th2 suppresses inflammatory Th1 immune responses within grafts. Although the therapeutic effects of PD98059 have conflicting aspects; inhibition of T cell proliferation or infiltration versus increase in Th2 population in the grafts of PD98059-treated recipients, it is possible that PD98059 therapy has much stronger impact on Th1-prone T-cell proliferation as demonstrated previously that a high affinity peptide-stimulated T-cell proliferation requires ERK activation (13 Fig. 6 ) allows PD98059 therapy to increase intragraft IL-4 production and Th2 population. Other immunosuppressive actions of PD98059 may include inhibition of effector function of natural killer cells (23 24
As compared to CsA monotherapy combination therapy of CsA with PD98059 provides additional prolongation of graft survival (Fig. 3 ), in which CsA inhibits calcineurin/NFAT and MM7/MM6/NF-κB while PD98059 blocks ERK1/2 /AP-1 (14, 20 10 Fig. 3 ). Our data suggest that this combination therapy may allow to reducing dosage of calcineurin inhibitors without the loss of immunosuppression in patients respectively.
ERK1/2 signaling pathway presents in diverse types of cells including hematopoietic cells and is activated by a wide variety of extracellular stimuli, including growth factors, hormone, cytokines as well as proto-oncogenes, leading to alterations in cell proliferation, differentiation, survival, and apoptosis (25, 26 ERK1/2 signaling as an immunosuppressive strategy has a potential toxicity. However, the strength of ERK1/2 activation in response to different stimuli and in different types of cell is different (26 ERK1/2 activation to the inhibitor (e.g., PD98059) is not always the same. Indeed, our unpublished data shows that in T cells lower dosages of PD98059 (less than 15 μM) can significantly inhibit TCR/CD28-ERK but not IL-2R-ERK-mediated cell proliferation (27 ERK1/2 activation (28 ERK1/2 signaling has different sensitivity to PD98059. Therefore, it will be possible that a given dosage of ERK1/2 inhibitor may suppress ERK1/2 signaling dependent immune responses, such as alloimmunity, but not tissue homeostasis (i.e., normal cell growth or differentiation) leading to adverse toxicity. Recent studies show that CI-1040 (PD184352), a highly selective and potent inhibitor of ERK1/2 signaling, does not have obvious adverse side effects over several months of treatment in preclinical models and clinical trials (29, 30 ERK1/2 signaling could be avoided or minimized by development of more specific inhibitors or through the monitor of drug dosage in clinical practice.
In conclusion, we for the first time demonstrate pharmacological interference of ERK1/2 signaling using PD98059 as a novel immunosuppressive strategy, which prolongs allograft survival through a mechanism that involves reduced graft infiltration as well as a beneficial skewing of Th1/Th2 T cell subsets. Although further studies are needed to demonstrate the therapeutic potential of inhibiting ERK1/2 signaling as an immunosuppressive strategy using new generation of ERK1/2 inhibitors (e.g., CI1040), the present results clearly show a preclinial proof of interference of ERK1/2 signaling as a therapeutic strategy for transplanted organ rejection, particularly in combination with CsA.
ACKNOWLEDGMENTS
We thank Ms. Pamela Gardner for secretarial support and Dr. Hao Wang for assistance in transplantation.
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