The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Singh, R. A. K.
Right arrow Articles by Zhang, J. Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Singh, R. A. K.
Right arrow Articles by Zhang, J. Z.
The Journal of Immunology, 2004, 173: 7299-7307.
Copyright © 2004 by The American Association of Immunologists

Differential Activation of ERK, p38, and JNK Required for Th1 and Th2 Deviation in Myelin-Reactive T Cells Induced by Altered Peptide Ligand1

Rana A. K. Singh* and Jingwu Z. Zhang2,*,{dagger}

* Department of Neurology, Baylor College of Medicine, Houston, TX 77030; and {dagger} Health Science Center, Chinese Academy of Sciences-Shanghai Second Medical University, Shanghai, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Autoreactive T cells can be induced by altered peptide ligands to switch Th1 and Th2 phenotypes. The underlying molecular mechanism is critical for understanding of activation of autoreactive T cells and development of novel therapeutic strategies for autoimmune conditions. In this study, we demonstrated that analog peptides of an immunodominant epitope of myelin basic protein (residues 83–99) with alanine substitution at Val86 and His88 had a unique partial agonistic property in the induction of Th1 or Th2 deviation in MBP83–99-reactive T cell clones typical of Th0 phenotype. The observed phenotypic switch involved differential activation of ERK, p38, and JNK MAPKs. More specifically, Th1 deviation induced by peptide 86V->A (86A) correlated with enhanced p38 and JNK activities, while Th2 deviation by peptide 88H->A (88A) was associated with up-regulated ERK activity and a basal level of p38 and JNK activity. Further characterization revealed that a specific inhibitor for ERK selectively prevented Th2 deviation of MBP83–99-specific T cells. Conversely, specific inhibitors for p38 and JNK blocked Th1 deviation in the same T cell preparations induced by peptide 86A. The findings have important implications in our understanding of regulation of ERK, p38, and JNK by altered peptide ligands and their role in cytokine regulation and phenotype switch of autoreactive T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The activation of protein-tyrosine kinases constitutes one of the initial steps for the induction of signaling cascades, which ultimately results in the activation of T cell effector function (see Ref.1 and references therein). p56 Lck kinase is a lymphoid-specific cytoplasmic protein tyrosine kinase that mediates initial events in TCR/CD3 signaling, such as phosphorylation of the TCR complex within amino acid sequences known as immunoreceptor-based tyrosine activation motifs. These serve as docking sites for Src homology domain 2-containing molecules, predominantly ZAP-70 and Syk, and activation of MAPKs (see Refs.1 and 2 and references therein). The MAPK cascade represents another key signaling pathway, critical for the linking of membrane receptors to cytoplasmic and nuclear effectors.

In mammalian cells, three parallel MAPK pathways have been identified. ERK, stress-activated protein kinase/JNK, and p38 are serine/threonine kinases, which constitute major components of this inducible signaling pathway and regulate many intracellular events, including cell proliferation and differentiation (2, 3). The ERK pathway, which is also the 42-/44-kDa MAPK pathway, is activated in response to signals from a variety of cell surface receptors and plays an important role in the transmission of many proliferative and differentiative signals involved in normal development (4, 5). In contrast, p38 and JNK pathways are primarily activated by cellular stress signals such as proinflammatory cytokines, heat shock, or UV light, and have therefore also been described as stress-activated protein kinases (3). Upon activation, the MAPKs phosphorylate various cytoplasmic effector proteins and are translocated to the nucleus, where they participate in the regulation of the gene expression by acting on transcription factors (2, 6).

There is recent evidence indicating that the TCR signal-transducing complex is able to sense structural differences in its ligands (= altered peptide ligands (APLs))3 and transduces different signaling patterns, resulting in qualitatively and quantitatively distinct sequela on subsequent activation and function of T cells (7, 8, 9). Certain structural modifications in TCR ligand render them as antagonist that transduces distinct signals and leads to the suppression of all ligand-induced activities such as T cell proliferation and cytokine production. Other APLs act as partial agonist and may uncouple proliferation and cytokine production or may alter the cytokine production profile of T cells (7, 8, 9, 10). Dynamic deviation or phenotype switch of memory Th0 cells into Th1 or Th2 cells is critical to immune tolerance, and its deficiency may be attributable to autoimmune abnormalities. In particular, the balance between Th1 and Th2 immune responses plays an important role in the pathogenesis of autoimmune diseases. For example, in addition to the recognition of encephalitogenic epitopes, the ability to produce Th1 cytokines is an important functional requirement by which myelin basic protein (MBP)-reactive T cells mediate experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (MS) (11). In contrast, there is an increasing body of evidence suggesting Th2 cells secreting IL-10 can suppress autoimmune inflammation in various experimental animal models (12, 13, 14). Similar roles of Th1 and Th2 cytokines have been implicated in the pathogenesis of MS, in which T cell responses to MBP are potentially involved in the disease process (15). The beneficial effect of IFN-{beta} on the clinical course of MS is at least partially attributable to its ability to promote the production of Th2 cytokines, such as IL-10 (16). These findings provide a rationale for further investigations into the molecular mechanisms responsible for Th1 and Th2 deviations and their implication for design of an effective peptide-based immunotherapy for MS.

Recently, we have shown that T cells specific for an immunodominant peptide (residues 83–99) of MBP derived from patients with MS produce Th0-specific cytokines (IL-4, IL-5, IL-10, IL-2, IFN-{gamma}, and TNF-{alpha}), and that APLs of MBP83–99 transduce a different signaling pattern that leads to the uncoupling of proliferation and cytokine production (10). Using a complete panel of 17 APL spanning the immunodominant region (residues 83–99) of MBP, in which each amino acid was sequentially replaced with an alanine, we demonstrated that single alanine substitution at certain positions not only leads to complete inhibition or partial activation of human MBP83–99-specific Th0 cell clones (producing IL-4, IL-10, IFN-{gamma}, and TNF-{alpha}) derived from MS patients, but also alters their ability to produce Th1 cytokines (i.e., IFN-{gamma}, TNF-{alpha}, IL-2) or Th2 cytokines (IL-4, IL-5, IL-10) (10). We demonstrated that analog peptides with alanine substitution at Val86 and His88 had a unique partial agonistic property and inhibited proliferation of MBP83–99-specific Th0 cell clones, but induced cytokine production profile from Th0->Th1 or Th0->Th2, respectively (10). Th0->Th1 deviation induced by peptide 86V->A correlated with up-regulation of Fyn and ZAP-70 kinase activities. Conversely, Th2 differentiation induced by peptide 88H->A was associated with complete failure to activate Fyn and ZAP-70 kinases (10). The observed T cell phenotype shifts also correlated, to a lesser extent, with Lck kinase activity that is associated with Th1 or Th2 development in MBP-reactive T cell clones. However, it remained unknown whether the observed changes require the activities of MAPKs.

In this study, we report that Th2 deviation of MBP83–99-specific T cells induced by APL-86A is dependent on the activation of p38 and JNK, which is effectively inhibited by specific inhibitors for p38 and JNK and is insensitive to an ERK inhibitor. Conversely, Th1 shift induced by treatment with APL-88A correlated with the activation of ERK. This process was inhibited selectively by an ERK inhibitor, but not by inhibitors for p38 and JNK. The results of the study may have important implications in the understanding of the molecular requirements for the activation of MBP-reactive T cells and for subsequent phenotypic changes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides and kinase inhibitors

MBP83–99 (ENPVVHFFKNIVTPRTP) and a panel of 17 analog peptides substituted at sequential positions with alanine were synthesized by the Merrified solid-phase method and were purified by HPLC (courtesy of S. Boheme, Neurocrine Biosciences, San Diego, CA). The purity of all peptides used in this study was greater than 95%. Specific inhibitors for ERK (PD98059), p38 (SB203580), and JNK (SP600125) were all purchased from Calbiochem (San Diego, CA).

Generation of MBP83–99-reactive T cell clones from patients with MS

To generate specific T cell lines, PBMCs were plated out at 2 x 105 cells/well in U-bottom plates (Costar, Cambridge, MA) in the presence of the 83–99 peptide (10 µg/ml). Seven days later, all cultures were restimulated with irradiated autologous PBMCs as a source of APCs pulsed with the peptide. After another week, each culture was examined for specific proliferation to the 83–99 peptide in [3H]thymidine uptake assays. Briefly, each well was split into four aliquots (~1 x 104 cells per aliquot) and cultured in duplicate in the presence of 1 x 105 APCs pulsed with the 83–99 peptide or a control peptide, respectively. Cells were cultured for 72 h and pulsed with [3H]thymidine (Amersham, Arlington Heights, IL) at 1 µCi/well during the last 16 h of the culture. Cells were then harvested, and [3H]thymidine incorporation was measured in a Betaplate counter (PerkinElmer Wallac, Turku, Finland). A T cell line was considered to be specific for the 83–99 peptide when the cpm were greater than 1500 (in the presence of the peptide) and exceeded the reference cpm (in the absence of the peptide) by at least 3-fold.

To establish stable MBP83–99-reactive T cell clones, the resulting T cell lines were cloned by PHA (Sigma-Aldrich, St. Louis, MO) in the presence of autologous PBMC as accessory cells (17). Briefly, T cells were plated out at 0.3 cell/well under limiting dilution condition and cultured with 105 irradiated autologous PBMC and 2 µg/ml PHA. Cultures were fed with fresh medium containing 50 IU/ml rIL-2 every 3–4 days. After ~10–12 days, growth-positive wells became visible and were tested in proliferation assays for specific responses to the 83–99 peptide.

Proliferation assays with peptides of MBP and analog peptides

To probe the responses of MBP83–99-reactive T cell clones to the wild-type and analog peptides of MBP83–99, DRB1*1501 (DR2b)- and DRB5*0101 (DR2a)-transfected L cells were used as APCs. Irradiated L cells (2 x 104 cells/well) were pulsed with peptides (20 µg/ml) for 3 h, washed, and cocultured with a given T cell clone (5 x 104 cells/well) in the presence or absence of optimal dose (1 µM) of PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor), respectively. Cultures were set up in duplicate for each peptide. In all cases, cell proliferation was measured after 72 h by [3H]thymidine incorporation assays, as described above.

Cytokine quantification

For measurement of IFN-{gamma}, TNF-{alpha}, IL-4, and IL-10, supernatants recovered from duplicate cultures were collected 48 h after stimulation under the identical conditions as for proliferation assay. Culture supernatants were diluted 1/4 with PBS before assays. Cytokines were determined quantitatively using ELISA kits obtained from BD Pharmingen (San Diego, CA). The kits were used according to the manufacturer’s instructions. Briefly, 96-well microtiter plates (Nunc; Maxisorp, Rochester, NY) were coated overnight at 4°C with 2 µg/well respective mouse-capturing mAbs in PBS. Wells were then blocked at 37°C for 2 h with 2% BSA-PBS and washed three times with cold washing solution, containing 0.02% Tween 20. A total of 50 µl of each sample and its control was added to the adjacent wells and incubated for 2 h at ambient temperature simultaneously with 50 µl of a biotinylated detecting Ab (0.25 µg/ml each mAb) in 2% BSA/PBS/Tween 20. Plates were washed and incubated for 30 min with streptavidin-conjugated HRP. A total of 100 µl of 0.0125% tetramethylbenzidine and 0.008% H202 in citrate buffer was used as substrate, and color development was stopped using 100 µl of 1 N HCl. The concentration of each cytokine in a given sample was calculated using a double standard curve of corresponding recombinant cytokine (BD Pharmingen) in each ELISA plate, which also served as a quality control. The detection limits for all cytokine measurements were <35 pg/ml in all assays.

Immune complex kinase assay

T cell clones (5–7 x 106 cells) pretreated with 1 µM inhibitor for 1 h at 37°C were incubated in 24-well flat-bottom culture plates with adherent L cells (1 x 106 cells) prepulsed with 20 µg/ml relevant peptide. After 15 min, T cells were lysed in a buffer (pH 7.4) containing 20 mM HEPES, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, and 1 mM DTT. Cytoplasmic extracts (250 µg) were subjected to immunoprecipitation with 1 µg of anti-ERK, anti-p38, or anti-JNK Abs (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4°C. Immune complexes were collected by incubation with protein A/G-Sepharose beads (Santa Cruz Biotechnology) for 30 min at 4°C. The beads were collected by centrifugation and washed extensively with lysis buffer (4 x 400 µl) and kinase buffer (2 x 200 µl: 20 mM HEPES, pH 7.4, 1 mM DTT, 25 mM NaCl). Kinase assays were performed for 15 min at 37°C in 20 µl of kinase assay buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM DTT, and 10 µCi of [{gamma}-32P]ATP (Amersham)) in the presence of kinase substrates (MBP (sc-3011; Santa Cruz Biotechnology) for ERK and activating transcription factor-2 (sc-4007; Santa Cruz Biotechnology) for JNK and p38). Reactions were stopped with 20 µl of SDS-sample buffer, boiled for 5 min, and subjected to SDS-PAGE. The incorporation of 32P phosphate was quantitated by PhosphorImager analysis (Amersham Pharmacia Biotech, Sunnyvale, CA).

Western blot analysis

The immunoprecipitates were subjected to SDS-PAGE, and the electrophoresed proteins were transferred to a nitrocellulose membrane (Hybond; Amersham) by standard procedures. The membrane was blocked in a solution of 10% nonfat dry milk in PBS for 1 h at room temperature. The membrane was rinsed three times with 100 ml of 0.1% Tween 20 in PBS and incubated with the appropriate dilutions of the specific Abs in PBS-Tween for 1 h at room temperature. The membrane was then washed five times with 100 ml of PBS-Tween and subsequently incubated with a peroxidase-labeled anti-rabbit Ig (Santa Cruz Biotechnology) in PBS-Tween for 1 h. After six washes with 100 ml of PBS-Tween, the membrane was processed by the ECL method, according to the manufacturer’s specifications (Amersham).

Cell viability

T cells were plated at a density of 105 cells/well in U-bottom 96-well plates in medium alone or in medium containing 1 µM PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor) for 48 h. Cytotoxic effect of the inhibitor was determined by the MTT assay. Briefly, the cultures were washed and incubated for 2 h in medium containing MTT (0.42 mg/ml), the medium was removed, and the cells were lysed in acidified isopropanol (0.04 N HCl). Conversion of MTT to formazon by metabolically viable cells was monitored at 570 nm in a plate reader. Percentage of cytotoxicity was calculated using the formula cytotoxicity (%) = (1 – (A/B))/100, where A is the absorbance of treated cells and B is the absorbance of the control cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Involvement of ERK, p38, and JNK in proliferation and cytokine profiles of Th0 MBP83–99-specific T cell clones in response to analog peptides with single alanine substitutions

Four independent MBP83–99-specific T cell clones originally derived from three patients with MS were selected for this study. All four T cell clones were found to express distinct TCR V{alpha} and V{beta} genes with completely unrelated CDR3 sequences. We first examined the involvement of ERK, p38, and JNK in the proliferation and cytokine profiles of the selected MBP83–89-specific T cell clones. To this end, the T cell clones were stimulated with MBP83–89 (= wild type) and, in parallel, with four analog peptides corresponding to 83A, 86A, 88A, and 90A in the absence and presence of specific inhibitors of ERK, p38, and JNK MAPKs. As a negative control, the clones were incubated in medium alone in the absence or presence of inhibitors. DRB1*1501 (DR2b)- and DRB5*0101 (DR2a)-transfected mouse fibroblasts were used as the APCs in all experiments. As shown in Fig. 1, only the wild-type peptide and peptide 83A were able to induce proliferation of the T cell clones contrary to peptides 86A, 88A, and 90A that failed to induce proliferative responses in MBP83–99-specific clones. Increasing concentrations of these peptides at 20–100 µg/ml did not render the T cell clones responsive (data not shown). Wild-type and 83A-induced proliferation was significantly inhibited with an optimal dose (1 µM) of an ERK inhibitor (PD98059), a p38 inhibitor (SB203580), or a JNK inhibitor (SP600125), respectively.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. Effect of ERK, p38, and JNK inhibitors on proliferative responses induced in MBP83–99-specific T cell clones by alanine-substituted peptides. CD4+ MBP83–99-specific T cell clones, MS1-1C no. 7 (A), MS1-2E8 no. 9 (B), MS2-1D9 (C), and MS3-2E4 (D), were stimulated with DR2a- and DR2b-transfected L cells pulsed with 20 µg/ml indicated peptides in the presence or absence of ERK, p38, or JNK inhibitor, respectively, as described in Materials and Methods. Proliferative responses of the T cell clones were determined by [3H]thymidine incorporation, as described in Materials and Methods. Values shown are representative of three independent experiments performed in triplicate. SE in all experiments was <10%. MED, medium alone as a control; WT, wild-type MBP83–99 peptide.

 
Next, we examined the phenotypic changes in the T cell clones treated with MBP83–99 and the altered peptides. As shown in Fig. 2A, MBP83–99- and peptide 83A-treated cells produced high levels of IFN-{gamma} and TNF-{alpha} and IL-4 and IL-10. Peptide 86A induced a Th1 phenotype in these clones, which produced higher levels of IFN-{gamma} and TNF-{alpha}, but background levels of IL-4 and IL-10. This pattern of cytokine production remained unchanged even at a higher concentration of the peptide used to stimulate the T cells. In contrast, peptide 88A induced a Th2 switch, as characterized by higher IL-4 and IL-10 production in the T cell clones, but failed to induce IFN-{gamma} and TNF-{alpha} production (Fig. 2A). The optimal concentration of the peptide required to induce the observed phenotypic changes was 20 µg (Fig. 2A), which was selected for subsequent characterization of the role of MAPKs in phenotype switch.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 2. Effect of ERK, p38, and JNK inhibitors on the cytokine profile of MBP83–99-specific T cell clones in response to the alanine-substituted peptides. A, The dose-response pattern of the T cell clones induced by the altered peptides. T cells (MS1-1C no. 7) were stimulated for 48 h in triplicates with DR2a- and DR2b-transfected L cells pulsed with the indicated concentrations of MBP83–99 or the altered peptides. Supernatants were tested for the levels of IFN-{gamma}, TNF-{alpha}, IL-4, and IL-10 by ELISA. Data are expressed as mean cytokine concentrations. SE in all experiments was <10%. The results were reproducible with other T cell clones (MS1-2E8 no. 9, MS2-1D9, and MS3-2E4). B, Effect of MAPK inhibitors on T cell clones stimulated with MBP83–99 and APLs. T cell clones, MS1-1C no. 7, MS1-2E8 no. 9, MS2-1D9, and MS3-2E4, were stimulated with DR2a- and DR2b-transfected L cells pulsed with MBP83–99 or indicated peptides in the presence or absence of ERK, p38, or JNK inhibitor, respectively. Supernatants were collected 48 h after stimulation, and the levels of IFN-{gamma}, TNF-{alpha}, IL-4, and IL-10 were determined by ELISA. The data shown are representative of three independent experiments performed in triplicate. SE in all experiments was <10%. Peptide 83–99 = the wild-type MBP83–99 peptide.

 
Next, we examined how the changes in the cytokine profile of the MBP83–89 T cell clones might be associated with different MAPKs. As shown in Fig. 2B, all selected T cell clones exhibited a Th0 phenotype when they were stimulated with MBP83–99 or 83A and produced significantly high levels of IFN-{gamma} and TNF-{alpha} (characteristic of Th1 phenotype) and IL-4 and IL-10 (characteristic of Th2 phenotype). Addition of 1 µM PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor) resulted in significant inhibition of the IFN-{gamma}, TNF-{alpha}, IL-4, and IL-10 cytokine production of the T cell clones treated with the wild-type peptide and peptide 83A, indicating that Th0 phenotype of MBP83–89-specific clones required adequate activity of ERK, p38, and JNK MAPKs. In comparison, treatment of the same MBP83–89 clones with peptide 86A induced the Th0 clones to selectively produce cytokines (IFN-{gamma} and TNF-{alpha}, but not IL-4 and IL-10), characteristic of a Th1 phenotype. Therefore, 86A acted as a partial agonist of MBP83–99 and uncoupled proliferation and induced split cytokine production (Fig. 1 compared with Fig. 2). It was of interest to determine whether phenotypic changes induced by peptide 86A involved selective activation of different MAPKs. Fig. 2B shows that IFN-{gamma} and TNF-{alpha} production of the T cell clones induced by peptide 86A was significantly inhibited by specific inhibitors for p38 and JNK, but was not affected by the ERK inhibitor, thus eliminating ERK MAPKs as a factor. In contrast, analog peptide 88A had the opposite effect upon the cytokine profile. After treatment with peptide 88A, the T cell clones were altered to produce selectively Th2 cytokines (IL-4 and IL-10), but negligible quantities of IFN-{gamma} and TNF-{alpha} (Fig. 2), suggesting that 88A is a partial agonist of MBP83–99 and uncouples proliferation and changes the cytokine profile of MBP83–99-specific T cells (Fig. 1 compared with Fig. 2). The cytokine production of 88A-treated cells was selectively blocked by the ERK inhibitor (PD98059), but not by inhibitors for p38 and JNK. Unlike the differing functions of analog peptides 86A and 88A, alanine substitution at position 90 completely abolished the ability of the 83–99 peptide of MBP to stimulate the proliferation or the production of either Th1 or Th2 cytokines in the same T cells, a process unaffected by the MAPK inhibitors (Fig. 1 compared with Fig. 2). MBP83–99-specific T cells incubated in medium alone in the absence or presence of MAPK inhibitors failed to produce significantly high cytokine levels, and therefore, acted as negative control for the experiments.

Up-regulation of ERK activity is associated with peptide 88A-induced Th0 to Th2 differentiation of MBP83–99-specific T cell clones

The observation that treatment of MBP83–99-specific T cell clones with peptide 88A induced a Th2 phenotype and that the specific inhibitor for ERK blocked the process had raised the possibility that ERK activity is critical to this phenotypic switch. To further characterize the role of ERK in this process, two MBP83–99-specific clones (MS1-1C no. 7 and MS3-2E4) derived from two patients were exposed to DR2-transfected L cells prepulsed with the wide-type peptide 83–99 or analog peptides (86A, 88A, or 90A) in the presence or the absence of ERK, p38, or JNK inhibitors. The ERK activity was measured subsequently in the T cell clones by immune complex kinase assays. As illustrated in Fig. 3, the wild-type peptide and peptide 88A significantly enhanced ERK activity by an increase of 5.6- to 5.9-fold over the control, which was blocked by the ERK inhibitor. However, peptides 86A and 90A did not affect the ERK activity. To examine the possibility that the observed change in the ERK kinase activity may be due to altered ERK protein level, this parameter was determined using Western blotting for various T cell preparations treated with the test peptides. The results revealed that the ERK protein level remained unchanged in T cell clones, regardless of the peptide ligands and inhibitors used (Fig. 3). Thus, the findings have confirmed that the Th0 to Th2 switch induced by alanine substitution at position 88 is associated with ERK kinase activity.



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 3. ERK activity is up-regulated in T cell clones treated with MBP83–99 and analog peptide 88A. T cell clones, MS1-1C no. 7 and MS3-2E4, were stimulated for 15 min at 37°C with L cells pulsed with various peptides in the presence or absence of ERK, p38, and JNK inhibitors, respectively. Immune complex kinase assays were performed to determine the ERK kinase activity (upper panel). ERK protein level in the T cell clones was determined by immunoblotting. Blots shown are representative of three independent experiments. Med, medium alone as a control; 83–99 = the wild-type MBP83–99 peptide.

 
p38 and JNK MAPK activity in MBP83–99-specific T cell clones is associated with a Th0 to Th1 switch induced by analog peptide 86A

As described above, the observed inhibition of IFN-{gamma} and TNF-{alpha} production by specific inhibitors for p38 and JNK in MBP83–99-specific T cells treated with peptide 86A is suggestive of a potential association with the activity levels of both the p38 and JNK MAPK pathways. We therefore examined the possibility that the two partial agonist peptides might exert their modulatory effect through regulation of the activity of p38 and JNK kinases. The results presented in Fig. 4 confirmed that stimulation of the T cell clones with either the wild-type peptide or peptide 86A substantially augmented the activities of both p38 and JNK. In contrast, exposure of these clones to peptide 88A or peptide 90A failed to activate or only slightly activated (JNK activity in clone MS3-2E4) the kinases (Figs. 4 and 5). Parallel experiments with the inhibitors for p38 and JNK further established that the molecular events induced by peptide 86A could be blocked only by the inhibitors for p38 or JNK, but not by the ERK inhibitor, confirming that the kinase activity of p38 and JNK is associated with the influence of analog peptide 86A. As shown in the lower panel of Fig. 5, these changes in the JNK activities were not due to the changes in the protein level of JNK, which remained unchanged after various treatments during the time of analysis. The observation that the ERK inhibitor further increased JNK activity in the peptide-treated T cell clones suggested that a basal level of ERK activity may play a regulatory role in JNK activity. Moreover, inhibition of the basal level of ERK activity may remove this regulation, leading to further augmentation of JNK activity.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 4. Modulation of p38 kinase activity in MBP83–99-specific T cell clones by analog peptides. T cell clones, MS1-1C no. 7 and MS3-2E4, were stimulated for 15 min at 37°C with L cells pulsed with the indicated peptides in the presence or absence of ERK, p38, and JNK inhibitors, respectively. Immune complex kinase assays were performed to determine the p38 kinase activity (upper panel). p38 protein level in the T cell clones was determined by immunoblotting. Blots shown are representative of three independent experiments. Med, medium alone as a control; 83–99 = the wild-type MBP83–99 peptide.

 


View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 5. JNK activity in MBP83–99-specific T cell clones treated with analog peptides. T cell clones, MS1-1C no. 7 and MS3-2E4, were stimulated for 15 min at 37°C with L cells pulsed with various peptides in the absence or presence of ERK, p38, and JNK inhibitor. Immune complex kinase assays were performed to determine the JNK activity (upper panel). JNK protein level in the T cell clones was determined by immunoblotting. Blots shown are representative of three independent experiments. Med, medium alone as a control; 83–99 = MBP83–99.

 
It should be noted that the suppression of cytokine production as well as the observed phenotypic switch in MBP83–99 T cell clones were not due to potential nonspecific cytotoxicity of cellular inhibitors. In parallel control experiments, viability of the T cell clones incubated for 48 h with different inhibitors was measured. None of the inhibitors at the concentrations used affected the viability of the T cell clones as measured by the MTT assay (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MS is an inflammatory disease of the CNS white matter characterized by demyelination and some axonal damage in areas of focal inflammation. Autoreactive T cell responses to candidate myelin Ags, including MBP, are thought to play an important role in the disease process in MS (18). T cells recognizing immunodominant regions of MBP (residues 83–99) may have particular relevance to MS, as they are found at high frequency in the blood of MS patients (17, 19) and are implicated in the brain lesions of MS (20). The T cell responses to the 83–99 immunodominant epitope in MS are also associated with DRB1*1501, an MS-related HLA-DR that has high binding affinity to the 83–99 peptide of MBP (21). DRB5*0101 molecule (HLA-DR2a) also binds the 83–99 peptide of MBP with high affinity. However, it has been suggested that DRB1*1501 (HLA-DR2b)-peptide complex is more immunogenic (22). There is increasing evidence indicating that cytokines play an important role in the pathogenesis of MS (13). In particular, the cytokine profile (Th1 or Th2 phenotype) is critical to the proinflammatory nature of MBP-reactive T cells that are capable of migrating to the site of inflammation in the CNS. MBP-reactive T cells capable of producing proinflammatory Th1 cytokines (TNF-{alpha} and IFN-{gamma}) are known to facilitate migration of inflammatory cells into the CNS and are thought to exacerbate myelin-destructive inflammatory processes in MS (13). Th1 cytokines administered directly or released after therapeutic modulation are associated with the worsening of MS (23). In contrast, Th2 cytokines (e.g., IL-4 and IL-10) have anti-inflammatory properties and are known to down-regulate Th1 response (13). The beneficial effect of IFN-{beta} and glatiramer acetate in the treatment of MS is at least partially attributable to their ability to regulate cytokines (18, 24, 25, 26, 27, 28).

To elicit an immune response, a given peptide has to correctly bind to the MHC molecule, and this MHC-peptide complex is then presented to and recognized by the TCR. The crystal structure of the DRB1*1501 molecule complexed with MBP83–99 (ENPVVHFFKNIVTPRTP) has been elucidated (29). The anchor residues of MBP83–99 that bind to the MHC molecule and the TCR contact residues of MBP83–99 have also been identified (29). APLs of a given immunogenic epitope carry amino acid substitution at defined positions, which may affect their binding to either MHC molecule and/or TCR. As a result, APLs can modulate the signals transduced by the TCR complex, which potentially result in the activation of different set(s) of transcription factors. This alteration in the activation pattern of transcription factors may have specific consequences such that the T cells may acquire an entirely distinct phenotype with characteristic Th1 or Th2 cytokine profile. The findings described in this work demonstrate that alanine substitutions at Val86 and His88 within the MBP83–99 epitope conferred upon these peptides unique partial agonist properties capable of inducing Th1 and Th2 deviations in well-characterized Th0 MBP83–99-specific T cell clones. Both Val86 and His88 are important anchor residues for DRB1*1501 and bind to the P1 and the P2 pockets of DRB1*1501 (29). The mechanism by which subtle changes in exogenous binding of an antigenic peptide to the MHC molecule and/or {alpha}{beta} recognition unit of a TCR lead to different signaling events and altered functional responses is unclear. Possible explanations may relate either to a failure to induce a required conformational change in the TCR or failure to assemble the essential molecules in the T cell contact cap (10). Consistent with this possibility is the notion that, upon ligand binding, both receptor cross-linking and subsequent conformational change must occur for successful activation (21).

There is evidence suggesting that members of the Src family of nonreceptor protein-tyrosine kinases, such as Lck and Fyn, are intimately associated with the TCR-CD3 complex and play an important role in T cell activation (10, 30). In addition, the overall phosphorylation within the TCR-CD3 complex is controlled by either of the two tyrosine kinases of the Syk family, namely Syk and ZAP-70 (31). We have previously demonstrated that engagement of the TCR complexes with analog peptides (86A and 88A) presented on DRB1*1501 (DR2b)- and DRB5*0101 (DR2a)-transfected L cells differentially activates Lck, Fyn, and ZAP-70 kinase activities in MBP83–99-specific T cells, leading to Th1 and Th2 deviations (10). The present study has identified, for the first time, three members of the MAPK family that are differentially regulated by the wild-type 83–99 peptide, and analog peptides 86A and 88A. The wild-type MBP83–99 peptide and peptide 83A induced Th0 phenotype, characterized by high levels of both Th1 (IFN-{gamma} and TNF-{alpha}) and Th2 (IL-4, IL-10) cytokines, a process that was associated with high levels of ERK, p38, and JNK activities. As demonstrated in this study, coincubation of these T cell clones with MBP83–99 or peptide 83A and an inhibitor (for ERK, p38, or JNK) resulted in the inhibition of both Th1 (IFN-{gamma} and TNF-{alpha}) and Th2 (IL-4 and IL-10) cytokines. However, the effects were not reflected in the kinase activities in which the ERK inhibitor suppressed only the ERK kinase level without a significant effect on MAPK activities induced by MBP83–99 or peptide 83A. Similarly, the p38 inhibitor only suppressed MBP83–99- or 83A-induced p38 kinase activity without altering ERK or JNK kinase activity. Additionally, the JNK inhibitor suppressed MBP83–99- or 83A-induced JNK kinase level, but did not alter ERK or p38 kinase level. These findings are significant because various MAPK cascades (e.g., ERK, p38, and JNK) are often considered in the literature as linear cascades, and an indication of cross talk between various cascades is limited (for a better understating of cross talk and feedback regulations, please see Refs.32, 33, 34, 35, 36, 37). Most of the reports link activation of ERK with growth factors and activation of p38 and JNK with stress stimuli. However, our findings suggest that activation of all three MAPKs is required for a TCR-regulated Th0 response (high production of IL-4, IL-10, IFN-{gamma}, and TNF-{alpha}), and that equilibrium in the activation of these kinases and possibly substrates generated as a result are critical for determining either Th1 or Th2 polarization of an ensuing response. The findings described in this work further suggest that when the balance of ERK, p38, and JNK is tilted in favor of p38 and JNK by peptide 86A, the resulting Th1 phenotype can be characterized by a high level of IFN-{gamma} and TNF-{alpha} production and a decreased production of IL-4 and IL-10. These conclusions are supported by the results of the parallel experiments using specific inhibitors for the MAPKs. In contrast, application of peptide 88A leaves basal activity of p38 and JNK unenhanced, but significantly activates ERK activity, reducing to background levels the production of IFN-{gamma} and TNF, while keeping Th2 cytokine production consistently high. The ERK inhibitor effectively inhibited the modulatory effects of peptide 88A on the cytokine profile of the T cell clones, thus confirming that Th2 deviation of MBP83–99 T cells requires enhanced activation of the ERK kinase.

In this study, DR2-transfected L cells were used as APCs (16) to avoid potential influence of the costimulatory signals that can be generated through interactions of T cell surface molecules, such as CD28, CD2, LFA-1, VLA-4, or heat stable Ags. Although costimulation clearly plays an important role in T cell signaling and activation, there is mounting evidence that certain conditions may render T cells less dependent on the costimulatory molecules. Recently, Lovett-Racke et al. (38) and Scholz et al. (39) have shown that activation and expansion of MBP-reactive T cells derived from patients with MS are independent of costimulatory signals delivered by B7-CD28 interaction.

Several studies have examined the role of ERK in cytokine gene expression and cytokine production with conflicting results. Some reported that the ERK activity has no effect on IL-4 production (40), while others found changes in the production of IL-4 (41, 42). One explanation of the contradictory results may be related to the cell types used in the different experimental systems and the affinity of the peptide to the TCR. In the present study, we used memory Th0 cells specific for the MBP83–99 peptide, which acquired Th1 or Th2 phenotype after treatment with APLs 86A and 88A, respectively. Our findings that Th0 clones acquired a Th2 phenotype after treatment with 88A, which was inhibited in the presence of ERK inhibitor, seem to be in disagreement with the findings reported by Badou et al. (40), in which the inhibition of ERK activity by its specific inhibitor had no effect on IL-4 production in Th2 clones. Our findings are consistent with those reported by Rincon et al. (43), in which p38 activity was found to correlate with Th1, but not Th2 phenotype in systems using p38 inhibitors and transgenic mice lacking p38 MAPK. Furthermore, our study has demonstrated that the molecular/signaling process of p38 activation pairs with the activation of JNK, all of which is associated with the Th0->Th1 deviation induced by peptide 86A. To the best of our knowledge, no study has been done to date to address the association of ERK, p38, and JNK MAPK activity with phenotypic switch of Th0 cells through induction by APL. The results described in this work indicate that the engagement of the TCR-CD3 complex with APL in the absence of costimulatory signals can lead to selective intracellular signaling pathways, depending upon the nature of the peptide ligands. The alteration of the initial signals results in modulation of MAPK activity and subsequent T cell function, highlighting the exquisite sensitivity of the TCR to subtle changes in its recognition unit.

The finding that single alanine substitutions at given positions within the immunodominant peptide of MBP, leading to the production of Th2 cytokines and the inhibition of the Th1 cytokines, has important therapeutic implications for MS. Because of their potential role in the disease process, MBP83–99-specific T cells have been one of the primary targets in the ongoing efforts to develop a peptide-based immunotherapy for MS. For example, an analog peptide with alanine substitution at the P89L and K91A was tested in clinical trials for MS. The clinical studies were suspended because of unexpected problems related to allergic reactions and other abnormalities induced by the peptide (44, 45). Better understanding of the association of TCR signaling and the kinase activity induced by APL in the context of Th1 or Th2 deviation of MBP83–99-reactive T cells may lead to an improved design for APL-based therapeutics.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by research grants from the National Institutes of Health (NS41289 and NS48860 to J.Z.Z.) and the Richardson Foundation (to J.Z.Z.). Back

2 Address correspondence and reprint requests to Dr. Jingwu Z. Zhang, Department of Neurology, Baylor College of Medicine, 6501 Fannin Street, NB302, Houston, TX 77030. Back

3 Abbreviations used in this paper: APL, altered peptide ligand; MBP, myelin basic protein; MS, multiple sclerosis. Back

Received for publication May 7, 2004. Accepted for publication October 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zamoyska, R., A. Basson, A. Filby, G. Legname, M. Lovatt, B. Seddon. 2003. The influence of the src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation. Immunol. Rev. 191:107.[Medline]
  2. Rincon, M.. 2001. MAP-kinase signaling pathways in T cells. Curr. Opin. Immunol. 13:339.[Medline]
  3. Kyriakis, J. M., P. Banerjee, E. Nikolakaki, T. Dai, E. A. Rubie, M. F. Ahmad, J. Avruch, J. R. Woodgett. 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156.[Medline]
  4. Alberola-Ila, J., G. Hernandez-Hoyos. 2003. The Ras/MAPK cascade and the control of positive selection. Immunol. Rev. 191:79.[Medline]
  5. Platanias, L. C.. 2003. MAP kinase signaling pathways and hematologic malignancies. Blood 101:4667.[Abstract/Free Full Text]
  6. Wilkinson, M. G., J. B. Millar. 2000. Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J. 14:2147.[Abstract/Free Full Text]
  7. Sette, A., J. Alexander, J. Ruppert, K. Snoke, A. Franco, G. Ishioka, H. M. Grey. 1994. Antigen analogs/MHC complexes as specific T cell receptor antagonists. Annu. Rev. Immunol. 12:413.[Medline]
  8. Jameson, S. C., M. J. Bevan. 1995. T cell receptor antagonists and partial agonists. Immunity 2:1.[Medline]
  9. Jameson, S. C.. 1998. T cell receptor antagonism in vivo, at last. Proc. Natl. Acad. Sci. USA 95:14001.[Free Full Text]
  10. Singh, R. A., Y. C. Zang, A. Shrivastava, J. Hong, G. T. Wang, S. Li, M. V. Tejada-Simon, M. Kozovska, V. M. Rivera, J. Z. Zhang. 1999. Th1 and Th2 deviation of myelin-autoreactive T cells by altered peptide ligands is associated with reciprocal regulation of Lck, Fyn, and ZAP-70. J. Immunol. 163:6393.[Abstract/Free Full Text]
  11. Swanborg, R. H.. 2001. Experimental autoimmune encephalomyelitis in the rat: lessons in T-cell immunology and autoreactivity. Immunol. Rev. 184:129.[Medline]
  12. Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, H. L. Weiner. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265:1237.[Abstract/Free Full Text]
  13. Willenborg, D. O., M. A. Staykova. 2003. Cytokines in the pathogenesis and therapy of autoimmune encephalomyelitis and multiple sclerosis. Adv. Exp. Med. Biol. 520:96.[Medline]
  14. Chitnis, T., S. J. Khoury. 2003. Cytokine shifts and tolerance in experimental autoimmune encephalomyelitis. Immunol. Res. 28:223.[Medline]
  15. Hermans, G., P. Stinissen, L. Hauben, E. Van den Berg-Loonen, J. Raus, J. Zhang. 1997. Cytokine profile of myelin basic protein-reactive T cells in multiple sclerosis and healthy individuals. Ann. Neurol. 42:18.[Medline]
  16. Kozovska, M., Y. C. Zang, I. Aebischer, S. Lnu, V. M. Rivera, P. D. Crowe, S. A. Boehme, J. Z. Zhang. 1998. T cell recognition motifs of an immunodominant peptide of myelin basic protein in patients with multiple sclerosis: structural requirements and clinical implications. Eur. J. Immunol. 28:1894.[Medline]
  17. Zhang, J., S. Markovic-Plese, B. Lacet, J. Raus, H. L. Weiner, D. A. Hafler. 1994. Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J. Exp. Med. 179:973.[Abstract/Free Full Text]
  18. Zhang, J., G. Hutton, Y. Zang. 2002. A comparison of the mechanisms of action of interferon {beta} and glatiramer acetate in the treatment of multiple sclerosis. Clin. Ther. 24:1998.[Medline]
  19. Bieganowska, K. D., L. J. Ausubel, Y. Modabber, E. Slovik, W. Messersmith, D. A. Hafler. 1997. Direct ex vivo analysis of activated, Fas-sensitive autoreactive T cells in human autoimmune disease. J. Exp. Med. 185:1585.[Abstract/Free Full Text]
  20. Krogsgaard, M., K. W. Wucherpfennig, B. Cannella, B. E. Hansen, A. Svejgaard, J. Pyrdol, H. Ditzel, C. Raine, J. Engberg, L. Fugger, B. Canella. 2000. Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody specific for the human histocompatibility leukocyte antigen (HLA)-DR2-MBP 85–99 complex. J. Exp. Med. 191:1395.[Abstract/Free Full Text]
  21. Valli, A., A. Sette, L. Kappos, C. Oseroff, J. Sidney, G. Miescher, M. Hochberger, E. D. Albert, L. Adorini. 1993. Binding of myelin basic protein peptides to human histocompatibility leukocyte antigen class II molecules and their recognition by T cells from multiple sclerosis patients. J. Clin. Invest. 91:616.
  22. Wucherpfennig, K. W., A. Sette, S. Southwood, C. Oseroff, M. Matsui, J. L. Strominger, D. A. Hafler. 1994. Structural requirements for binding of an immunodominant myelin basic protein peptide to DR2 isotypes and for its recognition by human T cell clones. J. Exp. Med. 179:279.[Abstract/Free Full Text]
  23. Moreau, T., A. Coles, M. Wing, J. Isaacs, G. Hale, H. Waldmann, A. Compston. 1996. Transient increase in symptoms associated with cytokine release in patients with multiple sclerosis. Brain 119:225.[Abstract/Free Full Text]
  24. Rep, M. H., R. Q. Hintzen, C. H. Polman, R. A. van Lier. 1996. Recombinant interferon-{beta} blocks proliferation but enhances interleukin-10 secretion by activated human T-cells. J. Neuroimmunol. 67:111.[Medline]
  25. Hussien, Y., A. Sanna, M. Soderstrom, H. Link, Y. M. Huang. 2004. Multiple sclerosis: expression of CD1a and production of IL-12p70 and IFN-{gamma} by blood mononuclear cells in patients on combination therapy with IFN-{beta} and glatiramer acetate compared to monotherapy with IFN-{beta}. Mult. Scler. 10:16.[Abstract/Free Full Text]
  26. Vieira, P. L., H. C. Heystek, J. Wormmeester, E. A. Wierenga, M. L. Kapsenberg. 2003. Glatiramer acetate (copolymer-1, copaxone) promotes Th2 cell development and increased IL-10 production through modulation of dendritic cells. J. Immunol. 170:4483.[Abstract/Free Full Text]
  27. Putheti, P., M. Soderstrom, H. Link, Y. M. Huang. 2003. Effect of glatiramer acetate (copaxone) on CD4+CD25high T regulatory cells and their IL-10 production in multiple sclerosis. J. Neuroimmunol. 144:125.[Medline]
  28. Franciotta, D., E. Zardini, R. Bergamaschi, L. Andreoni, V. Cosi. 2003. Interferon {gamma} and interleukin 4 producing T cells in peripheral blood of multiple sclerosis patients undergoing immunomodulatory treatment. J. Neurol. Neurosurg. Psychiatry 74:123.[Abstract/Free Full Text]
  29. Smith, K. J., J. Pyrdol, L. Gauthier, D. C. Wiley, K. W. Wucherpfennig. 1998. Crystal structure of HLA-DR2 (DRA*0101, DRB1*1501) complexed with a peptide from human myelin basic protein. J. Exp. Med. 188:1511.[Abstract/Free Full Text]
  30. Wucherpfennig, K. W., J. Zhang, C. Witek, M. Matsui, Y. Modabber, K. Ota, D. A. Hafler. 1994. Clonal expansion and persistence of human T cells specific for an immunodominant myelin basic protein peptide. J. Immunol. 152:5581.[Abstract]
  31. Sloan-Lancaster, J., P. M. Allen. 1996. Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology. Annu. Rev. Immunol. 14:1.[Medline]
  32. Reth, M., T. Brummer. 2004. Feedback regulation of lymphocyte signalling. Nat. Rev. Immunol. 4:269.[Medline]
  33. Roux, P. P., J. Blenis. 2004. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol. Mol. Biol. Rev. 68:320.[Abstract/Free Full Text]
  34. Shen, Y. H., J. Godlewski, J. Zhu, P. Sathyanarayana, V. Leaner, M. J. Birrer, A. Rana, G. Tzivion. 2003. Cross-talk between JNK/SAPK and ERK/MAPK pathways: sustained activation of JNK blocks ERK activation by mitogenic factors. J. Biol. Chem. 278:26715.[Abstract/Free Full Text]
  35. Xiao, Y. Q., K. Malcolm, G. S. Worthen, S. Gardai, W. P. Schiemann, V. A. Fadok, D. L. Bratton, P. M. Henson. 2002. Cross-talk between ERK and p38 MAPK mediates selective suppression of pro-inflammatory cytokines by transforming growth factor-{beta}. J. Biol. Chem. 277:14884.[Abstract/Free Full Text]
  36. Gallo, K. A., G. L. Johnson. 2002. Mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat. Rev. Mol. Cell Biol. 3:663.[Medline]
  37. Garrington, T. P., G. L. Johnson. 1999. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11:211.[Medline]
  38. Lovett-Racke, A. E., J. L. Trotter, J. Lauber, P. J. Perrin, C. H. June, M. K. Racke. 1998. Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients: a marker of activated/memory T cells. J. Clin. Invest. 101:725.[Medline]
  39. Scholz, C., K. T. Patton, D. E. Anderson, G. J. Freeman, D. A. Hafler. 1998. Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation. J. Immunol. 160:1532.[Abstract/Free Full Text]
  40. Badou, A., M. Savignac, M. Moreau, C. Leclerc, G. Foucras, G. Cassar, P. Paulet, D. Lagrange, P. Druet, J. C. Guery, L. Pelletier. 2001. Weak TCR stimulation induces a calcium signal that triggers IL-4 synthesis, stronger TCR stimulation induces MAP kinases that control IFN-{gamma} production. Eur. J. Immunol. 31:2487.[Medline]
  41. Yamashita, M., M. Kimura, M. Kubo, C. Shimizu, T. Tada, R. M. Perlmutter, T. Nakayama. 1999. T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation. Proc. Natl. Acad. Sci. USA 96:1024.[Abstract/Free Full Text]
  42. Egerton, M., D. R. Fitzpatrick, A. Kelso. 1998. Activation of the extracellular signal-regulated kinase pathway is differentially required for TCR-stimulated production of six cytokines in primary T lymphocytes. Int. Immunol. 10:223.[Abstract/Free Full Text]
  43. Rincon, M., H. Enslen, J. Raingeaud, M. Recht, T. Zapton, M. S. Su, L. A. Penix, R. J. Davis, R. A. Flavell. 1998. Interferon-{gamma} expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J. 17:2817.[Medline]
  44. Bielekova, B., B. Goodwin, N. Richert, I. Cortese, T. Kondo, G. Afshar, B. Gran, J. Eaton, J. Antel, J. A. Frank, et al 2000. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83–99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat. Med. 6:1167.[Medline]
  45. Kappos, L., G. Comi, H. Panitch, J. Oger, J. Antel, P. Conlon, L. Steinman. 2000. Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial: The Altered Peptide Ligand in Relapsing MS Study Group. Nat. Med. 6:1176.[Medline]



This article has been cited by other articles:


Home page
 Mult SclerHome page
M. Han and L Steinman
Systems biology for identification of molecular networks in multiple sclerosis
Multiple Sclerosis, May 1, 2009; 15(5): 529 - 530.
[PDF]

Home page
 J. Leukoc. Biol.Home page
S. M. Shin, Y. H. Kim, B. K. Choi, P. M. Kwon, H.-W. Lee, and B. S. Kwon
4-1BB triggers IL-13 production from T cells to limit the polarized, Th1-mediated inflammation
J. Leukoc. Biol., June 1, 2007; 81(6): 1455 - 1465.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. Viardot, S. T. Grey, F. Mackay, and D. Chisholm
Potential Antiinflammatory Role of Insulin via the Preferential Polarization of Effector T Cells toward a T Helper 2 Phenotype
Endocrinology, January 1, 2007; 148(1): 346 - 353.
[Abstract] [Full Text] [PDF]

Home page
 Ann Rheum DisHome page
L Xia, L Ru, and L Zhanguo
Altered influenza virus haemagglutinin peptides inhibit T cell responses to type II collagen in rheumatoid arthritis
Ann Rheum Dis, December 1, 2005; 64(12): 1790 - 1791.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
P. G. Thomas, M. R. Carter, A. A. Da'dara, T. M. DeSimone, and D. A. Harn
A Helminth Glycan Induces APC Maturation via Alternative NF-{kappa}B Activation Independent of I{kappa}B{alpha} Degradation
J. Immunol., August 15, 2005; 175(4): 2082 - 2090.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Singh, R. A. K.
Right arrow Articles by Zhang, J. Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Singh, R. A. K.
Right arrow Articles by Zhang, J. Z.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS