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The Journal of Immunology, 2006, 176: 5788-5796.
Copyright © 2006 by The American Association of Immunologists

ERK1–/– Mice Exhibit Th1 Cell Polarization and Increased Susceptibility to Experimental Autoimmune Encephalomyelitis1

Anshu Agrawal{dagger}, Stephanie Dillon*, Timothy L. Denning* and Bali Pulendran2,*

* Emory Vaccine Center and Department of Pathology, Atlanta, GA 30329; and {dagger} Division of Basic and Clinical Immunology, University of California, Irvine, CA 92697-4096


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation of MAPK ERK1/2 has been shown to play an important role in Th1/Th2 polarization and in regulating cytokine production from APCs. The ERK family consists of two members ERK1 and ERK2, which share ~84% identity at the amino acid level and can compensate for each other for most functions. Despite these features, ERK1 and ERK2 do serve different functions, but there is very little information on the contribution of individual forms of ERK on innate and adaptive immune responses. In this study, we describe that ERK1–/– mice display a bias toward Th1 type immune response. Consistent with this observation, dendritic cells from ERK1–/– mice show enhanced IL-12p70 and reduced IL-10 secretion in response to TLR stimulation. Furthermore, serum from ERK1–/– mice had 100-fold higher total IgG2b and 10-fold higher total IgG2a and IgG1 Ab isotype titers, and enhanced levels of Ag-specific IgG2b Ab titers, compared with wild-type mice. Consistent with this enhanced Th1 bias, ERK1–/– mice showed enhanced susceptibility to myelin oligodendrocyte glycoprotein (MOG)35–55 peptide-induced experimental autoimmune encephalomyelitis (EAE) and developed EAE earlier, and with increased severity, compared with wild-type mice. Importantly, there was a profound skewing toward Th1 responses in ERK1–/– mice, with higher IFN-{gamma} production and lower IL-5 production in MOG35–55-primed T cells, as well as an augmentation in the MOG-specific IgG2a and IgG2b Th1 Ab isotypes. Finally, increased infiltrating cells and myelin destruction was observed in the spinal cord of ERK1–/– mice. Taken together, our data suggest that deficiency of ERK1 biases the immune response toward Th1 resulting in increased susceptibility to EAE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The MAPK signaling cascade is an ancient, evolutionary conserved signal transduction pathway involved in the control of immune responses. It is a key signaling cascade critical for the linking of membrane receptors to cytoplasmic and nuclear effectors. The MAPK cascade is composed of three major groups of kinase, the ERK, stress-activated protein kinase/c-JNK, and p38 (1, 2, 3).

Among these the ERK pathway is activated in response to growth factors and oxidative stress. Phosphorylation of ERK is thought to play a pivotal role in a wide range of cellular activities, including survival, proliferation, and differentiation (1, 2, 3). A number of recent studies have demonstrated a major role for the components of the MAPK pathway in the regulation of innate and adaptive immune responses including Th1/Th2 differentiation (4, 5, 6, 7, 8). For example, naive T cells from JNK1–/– mice preferentially differentiate into a Th2 phenotype on activation, whereas CD4+ T cells from JNK2–/– mice show impaired IFN-{gamma} production and diminished Th1 responses. Similarly, p38 MAPK drives IL-12 production from macrophages inducing a Th1 response. Previous work from our laboratory has also shown that modulation of ERK in dendritic cells (DCs)3 gives rise to distinct Th responses. Selective inhibition of ERK in DCs has been shown to increase IL-12 production associated with concomitant decrease in IL-10 eliciting a Th1 response (5, 6). The inhibitors of the MAPK cascade, PD098059 and U0126, used in previous studies are quite selective in their inhibition of MEK but, unfortunately, do not differentiate between the two ERK isoforms. The two isoforms of ERK, 42 and 44 kDa, share an 84% identity at the amino acid level and can compensate for each other for most functions. However, the individual roles for the two isoforms of ERK have yet to be deciphered. The focus of the present study was to investigate the specific contribution of the ERK1 (p44) isoform of MAPK toward modulation of immune responses with particular emphasis on Th differentiation.

To determine whether ERK1 played a role in modulating Th1/Th2 bias, we examined the nature of cytokine secretion by DCs in ERK1–/– mice (9, 10) and found increased secretion of IL-12p70, a cytokine known to induce Th1 responses. Therefore we hypothesized that ERK1-deficient mice may be prone to autoimmune disease and enhanced Th1 responses. Thus we examined the effect of ERK1 deletion on the development and progression of experimental autoimmune encephalomyelitis (EAE), a prototypic Th1-mediated autoimmune disease of the CNS that is used as an animal model for multiple sclerosis, a human demyelinating disease (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). The Th1 nature of the disease is reinforced by the fact that all encephalitogenic lines and clones express the Th1 phenotype (14). Mice, exhibiting an enhanced Th1 bias due to deletion of a given gene, also show increased susceptibility to EAE (13, 15, 16, 18, 19, 20). We therefore reasoned that EAE would serve as a good model to evaluate whether ERK1–/– mice displayed an enhanced Th1 bias. Furthermore, we used the Ag myelin oligodendrocyte glycoprotein (MOG)35–55 peptide, which is a well-characterized target Ag of encephalitogenic T cells in the mouse model of EAE (10, 12). Our data indicate that deletion of ERK1 isoform alone is sufficient to induce a Th1 bias, which is reflected by an increased IFN-{gamma} production by MOG-specific T cells, and an early onset and increased susceptibility to EAE.


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

ERK1–/– mice of 129S1sv/IMJ background were a gift from Dr. G. Landreth (Case Western Reserve University, Cleveland, OH). Wild-type (WT) 129S1sv/IMJ mice were purchased from The Jackson Laboratory. Mice 6–20 wk of age were maintained under standard pathogen-free housing conditions at the Emory Vaccine Center Vivarium (Atlanta, GA).

Microbial stimuli and MOG peptide

Highly purified Escherichia coli LPS (strain 25922) was a gift from Dr. T. Van Dyke at Boston University (Boston, MA). Pam3Cys was obtained from G. Jung (Eberhard Karls Universität Tübingen, Tübingen, Germany) and reconstituted in endotoxin-free water. The MOG35–55 peptide used corresponds to amino acid residues 35–55 of the mouse sequence (MEVGWYRSPF SRVVHLYRNGK). The peptide was synthesized by Invitrogen Life Technologies and the purity was >97%.

DC purification and stimulation

CD11c+CD11b+ and CD11c+CD11b– DC subsets were purified from spleen cell suspensions from mice treated for 9 days with Flt-3 ligand as described before (5). Briefly, spleens from Flt-3 ligand-treated mice were dissected, cut into small fragments, and digested with collagenase and the resulting cells frozen until used. For DC subset preparation, thawed spleen cells were stained via allophycocyanin-conjugated CD11c (BD Pharmingen) and PE-conjugated CD11b (BD Pharmingen) and sorted into the CD11c+CD11b+ and CD11c+CD11b– DC subsets using a high speed modular flow cytometer (MoFlo; DakoCytomation). Total or sorted DCs from WT or ERK1–/– mice were cultured with E. coli LPS (1 µg/ml) and Pam3Cys (100 µg/ml) for 24 h in the presence of a fibroblast cell line expressing CD40L (5). Supernatant collected was assayed for IL-10 and IL-12p70 by ELISA kits (BD Pharmingen).

DC-naive CD4 T cell coculture

Magnetic bead purified CD11c+ DCs from ERK1–/– and WT mice were loaded with OVA peptide or LPS (1 µg/ml) and were cultured together with purified (flow sorted) naive CD4+CD62L+ T cells from OT-2 mice (transgenic for OVA Ag) for 5 days. At 5 days later, the T cells were restimulated with plate-bound anti-CD3 (10 µg/ml) and anti-CD28 (2 µg/ml) for an additional 3 days. Supernatants were collected before and after CD3 stimulation and assayed for IFN-{gamma}, IL-2, IL-4, IL-5, IL-10, and TNF-{alpha} by ELISA kits (BD Biosciences). IL-17 was assayed with a kit from R&D Systems.

Dinitrophenol-conjugated keyhole limpet hemocyanin (DNP-KLH), DNP-Ficoll, and serum Ab isotyping

ERK1–/– and WT mice were immunized with 10 µg of DNP-KLH in alum. Blood was collected by retro-orbital puncture from ERK1–/– and WT mice on days 7 and 14 and allowed to clot overnight. Serum was separated by centrifugation. Dilutions of serum ranging from 1/100 to 1/100,000,000 were incubated on DNP-OVA-coated plates. Specific Ab detection was done using isotype specific HRP-conjugated anti-mouse Abs (Caltag Laboratories).

MOG Ab isotyping

ERK1–/– and WT mice were immunized with 200 µg of MOG peptide emulsified in CFA containing 5 µg/ml Mycobacterium tuberculosis. Serum was collected on days 14 and 28 after immunization. MOG-specific Ab titer was determined by ELISA using MOG-coated plates.

B cell proliferation

Spleen cells (5 x 105 cells/well) were stimulated in vitro with CD40, LPS, or IgM for 5 days. A total of 0.5 µCi of [3H]thymidine was added to each well, and plates harvested after an additional 16 h of culture. Supernatants were assayed for isotype-specific Abs using the isotyping kit (BD Biosciences).

T cell type and quantity

The phenotype and percentage of CD4+ and CD8+ T cells were determined by flow cytometry in nonimmunized ERK1–/– and WT mice by staining the total spleen cells with specific Abs (BD Biosciences). A total of 5 x 105 spleen cells were stimulated with CD3 (10 µg/ml) and CD28 (2 µg/ml) or Con A for 48 h, and proliferation determined by uptake of [3H]thymidine. Supernatants collected were assayed for IL-4, IL-5, and IFN-{gamma} by ELISA.

MOG-specific T cell proliferation

Mice were immunized with MOG peptide (200 µg) and emulsified in CFA, containing 5 µg/ml M. tuberculosis, on days 0 and 7 s.c. in the hind flank. Popliteal and inguinal lymph nodes were harvested at days 7 and 14 or day 7 after boosting with MOG peptide. Lymphocytes (5 x 105cells/well) in 200 µl were incubated in 96-well flat-bottom plates with the indicated amounts of Ag in DMEM supplemented with 10% FCS (Mediatech) for 72 h. The 0.5 µCi of [3H]thymidine was added to each well, and plates harvested after an additional 16 h of culture. Cytokine estimation in the supernatant was by ELISA (BD Pharmingen).

EAE induction

EAE was induced by immunization of ERK1–/– and WT mice with 200 µg of MOG35–55 emulsified in CFA, containing 5 µg/ml M. tuberculosis, on days 0 and 7 s.c. in the hind flank. Mice also received 250–500 ng of pertussis toxin i.p. on days 0 and 2. Disease severity was monitored according to the following scale: 0, no disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb weakness; and 5, moribund.

Histology

WT and ERK1–/– mice were immunized with 200 µg of MOG35–55, as discussed earlier. Spinal cords were removed 22–24 days later at the peak of the disease, and fixed in 10% buffered Formalin (Sigma-Aldrich). Paraffin-embedded longitudinal sections (6-µm thick) were stained with either H&E or Luxol fast blue to assess cellular infiltration and demyelination, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DCs from ERK1–/– mice show enhanced IL-12p70 and reduced IL-10 secretion

DCs are the most efficient APCs of the body that can modulate the nature and magnitude of T cell responses. Therefore, we first wanted to determine the effect of ERK1 deletion on the DC function. We did not find significant differences in either the numbers or phenotype of DCs from ERK1–/– and WT mice by flow cytometry (data not shown) but significant functional differences were observed. DCs in mouse can be further divided into CD11c+CD11b– and CD11c+CD11b+ subsets, which are capable of differentially priming Th responses (22, 23). CD11c+CD11b– DCs secrete copious amounts of IL-12p70 on stimulation with TLR ligands and thus induce Th1 responses, whereas CD11c+CD11b+ DCs induce a Th2 bias through secretion of IL-10 (22, 23). To investigate the effect of ERK1–/– deletion on the DC subsets, Flt-3-injected spleen cells from ERK1–/– and WT mice were sorted into CD11c+CD11b– and CD11c+CD11b+ populations. Sorted cells were stimulated with LPS and Pam3Cys, which are TLR ligands that induce Th1 and Th2 response, respectively, as previously described. IL-12p70 and IL-10 levels secreted were determined by ELISA. As shown in Fig. 1, IL-12p70 levels were significantly elevated in ERK1–/– mice in both LPS- and Pam3Cys-stimulated DC subsets. A 6- to 10-fold increase in IL-12 was observed in the CD11c+CD11b– DC subset stimulated with LPS and Pam3Cys. We have earlier reported an increase in IL-12p70 after ERK blockade, but what was remarkable was that this enhancement of IL-12p70 was evident even in the CD11c+CD11b+ DC subset, which is known to be more of a Th2 inducing subset of DCs (22, 23). A concomitant decrease in IL-10 was also observed in both groups compared with WT mice DCs. ERK1–/– thus seems to act as a negative regulator of IL-12 production through secretion of IL-10.


Figure 1
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  		FIGURE 1. DC from ERK1–/– mice show enhanced IL-12p70 and reduced IL-10 secretion in response to TLR stimulation in vitro. Sorted ERK1–/– and WT splenic CD11c+ DCs were cultured for 24 h with different E. coli LPS or Pam3Cys, and cytokines secreted were determined by ELISA. The CD11c+CD11b– DC subset (top panels) and the CD11c+CD11b+ DC subset (bottom panels) are represented. Data are representative of three experiments.

 
Ag-pulsed ERK1–/– DCs induce an enhanced Th1 response in naive Ag-specific CD4 T cells

DCs are capable of modulating the T cell response toward Th1 or Th2 through the cytokines secreted by them. To determine whether indeed the ERK1–/– DCs induce enhanced Th1 differentiation, CD11c+ splenic DCs were isolated from ERK1–/– and WT mice injected with Flt-3 ligand, pulsed with class II-restricted OVA peptide, or OVA peptide plus LPS, and cultured together with purified (FACS sorted) naive CD4+CD62L+ T cells from OT-2 mice (TCR transgenic mice, which express a TCR that is specific for OVA (5)). Five days later, supernatants were assayed for IFN-{gamma}, IL-2, IL-4, IL-5, IL-17, and TNF-{alpha} by ELISA. In addition, the T cells were restimulated with plate-bound anti-CD3 and anti-CD28 for an additional 3 days. Supernatants were collected before and after CD3 stimulation and assayed for IFN-{gamma}, IL-2, IL-4, IL-5, IL-17, and TNF-{alpha} by ELISA. As indicated in Fig. 2A, 5 days of stimulation with OVA-pulsed WT DCs induced significant levels of IL-2, TNF-{alpha}, and IL-17, but did not result in detectable production of IFN-{gamma}, IL-4, or IL-5. In contrast, stimulation with ERK1–/– DCs did result in significant induction of IFN-{gamma}, but no IL-4, IL-5 nor any enhancement of IL-2, TNF-{alpha}, or IL-17. Importantly, the induction of similar levels of IL-2 in T cells by WT DCs, and ERK1–/– DCs, suggests that both types of DCs induce equivalent activation of T cells. After restimulation of the T cells with anti-CD3 and anti-CD28, there is a noticeable enhancement in the induction of IL-2, IFN-{gamma}, TNF-{alpha}, and IL-17 (Fig. 2B), although not IL-4 or IL-5 (data not shown). In particular, ERK1–/– DCs pulsed with OVA, either in the presence of absence of LPS, results in a significant enhancement (p < 0.05) of IFN-{gamma} relative to wild-type DCs (Fig. 2B). There was no significant difference in the levels of IL-17 and TNF-{alpha} secreted between the WT and ERK1–/–. IL-2 levels were similar and IL-4 and IL-5 were undetectable (data not shown). ERK1–/– DCs thus prime the naive T cell responses toward a Th1 type immune response.


Figure 2
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  		FIGURE 2. ERK1–/– DCs induce an enhanced Th1 response in FACS sorted, naive OVA-specific CD4 T cells. CD11c+ splenic DCs from ERK1–/– and WT were pulsed with OVA peptide with or without LPS, and cocultured with naive OT-2 CD4+ T cells. DCs alone plus T cells (DC/T) or DCs pulsed with OVA (DC/T/OVA) peptide cultured with T cells and DCs plus LPS (DC/T/OVA-LPS) cultured with T cells are shown for primary immunization (A) and secondary boost (B). Data are representative of three experiments. Statistical significance (*, p < 0.05), by paired Student’s t test, between WT and ERK1–/– groups.

 
ERK1–/– mice have elevated levels of serum IgG2b and IgG2a and respond efficiently to T cell-dependent Ags

We then determined the influence of ERK1–/– on B cell development and function. Again no significant differences were observed in the percentages, or absolute number of B cells in the spleen, bone marrow, and peritoneal cavity as analyzed by flow cytometry (data not shown). Proliferation of B cells in response to IgM cross-linking, CD40 or LPS was also similar in ERK1–/– and WT mice (Fig. 3A). However, when we determined the Ab in the culture supernatant after stimulation with CD40, we observed significant differences in the isotype of Ab secreted between ERK1–/– and WT mice B cells (Fig. 3B). The level of IgG2b was 3-fold higher in ERK1–/– B cells compared with WT (p < 0.005) (Fig. 3B). IgG3 levels were found to be different but the difference was not statistically significant (p > 0.05). The molecular mechanism resulting in this preferential enhancement of the IgG2b isotype is at present unclear.


Figure 3
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  		FIGURE 3. ERK1 regulates Ab production in B cells. A, B cells from ERK1–/– mice proliferate normally to stimulation with IgM, LPS, or anti-CD40. B, In vitro stimulation of B cells from ERK1–/– mice with anti-CD40 results in enhanced secretion of IgG2b Ab. C, ERK1–/– mice have significantly elevated levels of serum total IgG2b, IgG2a, and IgG1 Ab. D, DNP-specific serum Ab titer was determined by ELISA, day 14 after immunization with DNP-KLH. Data represent mean of five to seven individual mice. Statistical significance (*, p < 0.005), by paired Student’s t test, between WT and ERK1–/– groups.

 
To further confirm the IgG2b bias observed in the culture supernatant, we determined the nature of Ab secretion by these mice. We found that in nonimmunized ERK1–/– mice, the concentrations of serum total IgG2b were profoundly enhanced by >100-fold (p < 0.005) compared with levels in control littermates, whereas levels of IgG2a and IgG1 showed an enhancement of 10-fold (Fig. 3C). In addition, there was also an enhancement of the total IgG1 response, whereas levels of IgM and IgG3 were similar in both groups of mice (Fig. 3C). These data suggest that the absence of ERK1 results in a dramatic enhancement of total IgG2b, in addition to more modest enhancements in total IgG2a and IgG1 isotypes.

We next tested the ability of ERK1–/– to respond to T cell-independent and T cell-dependent Ags (24). For this purpose ERK1–/– and WT mice were immunized with DNP-Ficoll and DNP-KLH in alum, and serum Ab titer was determined at various time points. As shown in Fig. 3D, a 10-fold increase in DNP IgG2b-specific Ab titer was observed in ERK1–/– mice immunized with DNP-KLH over Ab titers in WT mice (p < 0.05). Immunization with T cell-independent Ag DNP-Ficoll did not result in a significant difference in titers of IgM and IgG3 between ERK1–/– mice and controls (data not shown).

Taken together, these data suggest that although the ERK1–/– deficiency is dispensable for B cell development, it affects the nature of T cell-dependent Ab response, resulting particularly in the enhancement of IgG2b secretion (25, 26). Why the deficiency of ERK1–/– results in significant enhancement of IgG2b Ab secretion in the supernatants (Fig. 3B), at the basal level in nonimmunized mice (Fig. 3C), and in the DNP-KLH group (Fig. 3D) is not clear to us at this point. TGF-beta is considered one of the factors responsible for isotype switching to IgG2b; however, we did not observe significant differences in the level of TGF-beta between the serum of ERK1–/– and WT mice (data not shown).

T cells from ERK1–/– mice show reduced proliferation

Next, we determined the effect of ERK1 deletion on peripheral T cells. No significant difference was observed in the number and percentage of CD4+ and CD8+ T cells in spleen and lymph node between ERK1–/– and control mice (Fig. 4A). However, ERK1–/– T cells exhibited significantly lower proliferation when stimulated through TCR or mitogenic stimuli such as Con A (Fig. 4B). This phenomenon was similar to that observed in an earlier study with ERK1–/– mice in which research showed reduced T cell proliferation of the thymic T cells (26). Interestingly, IFN-{gamma}, IL-4, and IL-5 levels were not significantly different between ERK1–/– and WT T cells (Fig. 4C).


Figure 4
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  		FIGURE 4. T cells develop normally in ERK1–/– mice, but proliferate less efficiently than WT T cells upon in vitro stimulation. A, Percentage of CD4 and CD8 cells in the spleen of ERK1–/– and WT mice as determined by flow cytometry. B, Proliferation of ERK1–/– and WT mice splenic lymphocytes. C, Cytokine production after in vitro stimulation is shown, as determined by ELISA. Results are representative of three experiments.

 
ERK1–/– mice exhibit exacerbated EAE compared with WT littermates

Next, we examined the effect of ERK1–/– gene disruption on the development of EAE, which is considered a prototypic Th1-mediated disease (9, 14, 18). EAE was induced in ERK1–/– and WT mice following active immunization with MOG35–55. As shown in Fig. 5, both the onset and severity of disease was enhanced in ERK1–/– mice compared with their WT littermates. The development of severe paralytic symptoms was apparent in ERK1–/– mice by day 20 with the maximal disease severity score of 3.6 being observed by day 21–22. In the WT mice, the mean day of onset of severe paralysis was on day 23, and a maximal severity score never crossed 2.5. Although all mice in both groups succumbed to the disease, there was no mortality associated with the acute attack. It is possible that ERK2 compensated to a certain extent for ERK1 preventing the development of fatal disease. Unfortunately, ERK2 deletion is fatal to mice, thus we were unable to use ERK2–/– mice to determine its effects. Nevertheless these results clearly suggest that the ERK1 deletion alone results in the exacerbation of progression and severity of EAE.


Figure 5
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  		FIGURE 5. ERK1–/– mice exhibit accelerated EAE compared with WT littermates. Mice were immunized with MOG35–55 in CFA and scored for clinical signs of EAE as described in Materials and Methods. Results are plotted as mean clinical scores of all animals in each group (n = 3) vs day of immunization. The data are the mean of three different experiments.

 
ERK1–/– mice develop severe inflammation and demyelination in the spinal cord compared with their WT counterparts

To further confirm that the clinical severity observed in ERK1–/– mice correlated with increased pathological inflammation and demyelination in the CNS, longitudinal spinal cord sections from ERK1–/– mice and their WT littermates were analyzed for myelin loss (demyelination) and infiltration of mononuclear cells (inflammation) after staining with Luxol fast blue and H&E (11, 27). Areas of demyelination and inflammation were more extensively widespread in ERK1–/– compared with WT mice (Fig. 6, A and B). Although the WT mice showed ~42% demyelination (Fig. 6C), the ERK1–/– showed ~73% (1.5-fold increase). Similarly the inflammation was also increased from 33% to 69.2% in ERK1–/– mice. These observations showed a close association between the development of paralysis and the pathological severity of the disease.


Figure 6
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  		FIGURE 6. Pathology of spinal cord sections from ERK1–/– and WT mice induced to develop EAE. Two longitudinal sections per spinal cord were prepared and stained as described. The representative pictures of demyelination (A) and inflammation (B) were taken under microscope (magnification, x20). The spinal cord sections were scored for the presence of demyelination or inflammation in a blinded manner and pathological scores were expressed as percentage of positive spinal cord quadrants over total number of quadrants examined (C). The average number of quadrants examined per section was 20.

 
MOG-primed T lymphocytes from ERK1–/– mice show increased Th1 cytokine secretion

To further understand the mechanistic basis of exacerbated EAE observed in ERK1–/– mice, we examined whether MOG-specific T cells generated in mice deficient in ERK1 respond to MOG peptide in a manner similar to T cells generated in WT. First, a kinetic study was done in which WT or ERK1–/– mice were immunized s.c. with MOG peptide, and lymph node cells were collected at 7 or 14 days later. In addition, a separate group of mice was immunized with MOG peptide and given a secondary boost at day 28, and draining lymph node cells were harvested 7 days after the boost. The lymph node cells were cultured in vitro with MOG peptide for 72 h, then [3H]thymidine uptake assayed, and the culture supernatants assayed for cytokines by ELISA. The results indicated that ERK1–/– T cells proliferated to a moderately lower extent than their WT counterparts (Fig. 7A), consistent with what was observed after mitogenic stimulation with Con A (Fig. 4B). However, increased secretion of IFN-{gamma} was observed in the ERK-deficient MOG-primed T cells at day 14, and after boosting compared with WT cells. IFN-{gamma} secretion increased with time and this difference became significant (p < 0.05) after boosting. There was an increase of nearly 4-fold in the ERK1–/– mice compared with WT mice at higher concentrations. Similar to Nekrasova et al. (27), we did not observe a difference at earlier time points such as day 7. It is possible that an amplification of the response in the form of boosting was required for the difference to be visible. We also determined the level of IL-17 and TNF-{alpha}. We were unable to detect IL-17 or IL-4 reproducibly, and IL-10 and TNF-{alpha} levels were similar, although 7 days after primary immunization there was slightly higher levels of IL-10 in ERK1–/– mice (data not shown). These results suggest that ERK1–/– mice developed augmented Th1 response to neuronal Ag MOG, contributing to the increased susceptibility of these mice to EAE. T cells are thought to play a key role in initiating and perpetuating the disruptive inflammatory process associated with EAE, which is regarded as a Th1 MHC class II-restricted CD4+ T cell-mediated disease of the CNS.


Figure 7
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  		FIGURE 7. MOG-primed T lymphocytes from ERK1–/– mice show increased Th1 cytokine secretion despite reduced proliferation. A, Proliferation of Ag-specific cells from MOG35–55 primed WT and ERK1–/– mice. Results are mean of counts per minute ± SEM. B, Cytokine production as determined by ELISA, by Ag-specific cells from MOG35–55 primed WT and ERK1–/– mice after in vitro restimulation with varying concentrations of MOG peptide. The results are plotted as mean ± SEM.

 
ERK1–/– mice have higher IgG2a and IgG2b MOG-specific serum Ab titers

Multiple studies have documented the importance of anti-MOG Abs in the pathogenesis of EAE. Increased levels of Abs directed to proteolipid protein and MOG correlate with relapse and demyelination (28, 29, 30, 31, 32). Injection of anti-MOG mAb at the onset of acute EAE exacerbated clinical neurological disease and induced demyelination in the CNS (31). Therefore we investigated the effect of ERK1 deletion on the MOG-specific Ab response. The results were similar to what we observed with DNP-KLH immunization. As shown in Fig. 8, a 4-fold increase in MOG-specific IgG2a and IgG2b Ab titers was observed at day 14, which increased to 16-fold at day 28 in ERK1–/– mice immunized with MOG over the increase in WT mice (p < 0.05). Total MOG IgG Ab titer was also significantly higher in the ERK1–/– mice compared with WT mice.


Figure 8
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  		FIGURE 8. ERK1–/– mice have significantly elevated levels of MOG-specific IgG2a and Ig2b. Serum Ab titers at days 14 and 28 after immunization with MOG35–55 in CFA are shown. ERK1–/– mice ({blacksquare}) and WT mice ({square}) are indicated. At day 28, significant difference (**, p < 0.005; *, p < 0.05) in Ab titers in ERK1–/– mice immunized with MOG over the increase in WT mice were present. Data represent mean of five to seven individual mice. Total IgG is represented as T-IgG.

 
In summary, ERK1–/– mice display a higher IgG2 to IgG1 ratio for all Ags tested, suggesting a Th1 type immune response. Furthermore, elevated levels of MOG Ab are observed in ERK1–/– mice compared with WT mice. Abs to MOG have been implicated in increasing demyelination and severity of the EAE.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present data suggest that ERK1 does not appear to be involved in the development of T cells, B cells, or DCs in vivo. However, the data point to a critical role for ERK1 as a negative regulator of autoimmune Th1 immune responses, and are consistent with previous findings that suggest a role for ERK1 and ERK2 in controlling the Th1/Th2 balance in T cells (1, 2, 3, 4, 5, 9, 33), and in APCs (5, 6, 8, 34). Our results further indicate that ERK1 regulates the generation of autoimmune T cells and inflammation of CNS associated with EAE. These effects are likely to be mediated by the effects of ERK1 on multiple cell types. First, ERK1 may regulate the Th1/Th2 balance in T cells, as has been shown previously for both ERK1 and ERK2 (1, 2, 3, 4, 5, 9). Second, ERK1 may regulate cytokine production in APCs, particularly in DCs, suppressing the induction of IL-12 and favoring IL-10 production (5, 6, 8, 34). Finally, ERK1 may also play a direct role in B cells, as suggested by the enhanced secretion of IgG2b Ab by B cells activated in vitro (Fig. 4B). Although the precise contribution of each of these mechanisms to the development of Th1 autoimmune responses, and the subsequent immunopathology, remains to determined, it is likely all of these mechanisms act in concert.

Interestingly, one difference between our data and those of Nekrasova et al. (27) concerns the role of ERK1 in the effector functions of peripheral T cells. Although our data suggest that immunization of ERK1–/– mice with MOG35–55 in CFA results in a significant enhancement of IFN-{gamma}, and diminution of IL-5 (Fig. 7), this study by Nekrasova et al. (27) suggests that there is no difference in the Th1/Th2 balance. The reason for this discrepancy is not apparent, but may reside in the effects caused by environmental differences in the housing conditions of the two mouse colonies.

With respect to the relative roles played by IFN-{gamma}-secreting prototypic "Th1" cells vs IL-17-secreting "Th17" cells in the pathogenesis of EAE, there is presently some debate. Numerous previous studies have addressed the role of so-called "Th1" vs "Th2" in EAE and multiple sclerosis, with IL-12 and Th1 responses shown to play critical roles in mediating the pathogenesis of disease (e.g., 16, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47). However, some recent studies have demonstrated that IL-23, rather than IL-12, is the critical cytokine for the establishment and persistence of inflammatory lesions in EAE (48, 49, 50, 51). IL-17 is a crucial effector cytokine that is associated with many inflammatory diseases, including EAE, and is specifically induced by IL-23 (52, 53, 54, 55). Given that current thinking favors a dominant role for IL-23-induced "Th17" cells in the pathogenesis of EAE, we investigated the possibility that DCs from ERK1–/– mice produce greater levels of IL-23, which might result in enhanced IL-17. However, we failed to detect any significant difference in the secretion of IL-23 protein DCs from WT and ERK1–/– mice, stimulated in vitro, with TLR4 or TLR2 ligands (data not shown). Furthermore, as observed in Fig. 2, we could not detect any enhanced secretion of IL-17 by OT-2 cells stimulated in vitro by ERK1–/– DCs vs WT DCs. Thus, our data suggests that although ERK1 plays a critical role in regulating the Th1/Th2 balance by modulating IL-12 and IL-10 in DCs, it does not exert any significant effect on IL-23 production by DCs. These data might explain the disconnect between the significant effects of ERK1 deficiency on the induction of IL-12 and IL-10 in DCs (Fig. 1), and the consequences of this result for T cell responses (Figs. 2 and 7) and the more modest effects on EAE pathogenesis (Fig. 5).

Finally, regarding the role of MAPKs in EAE, there is little information (56, 57, 58). The recent study by Nekrasova et al. (27), consistent with our study, suggests that ERK1-deficient mice show somewhat enhanced susceptibility to EAE. Our present studies, however, do not allow us to clearly delineate the functions of the two isoforms of ERK. ERK2 might be acting in a synergistic fashion along with ERK1 in the regulation of the Th1 immune response because both become activated through a similar signaling pathway. To what extent it compensates or differs from ERK1 is to be determined in future studies. Nevertheless our data clearly show the role ERK1 in modulating the balance of IL-12 and IL-10 in DCs, and the consequences of this role for the Th1/Th2 balance and the regulation of autoimmune disorders.


   Acknowledgments
 
We thank Mandy Ford and Brian Evavold for helping with the EAE experiments and members of Emory University Hospital, Pathology Section, for help with histochemistry. We also thank Denyse Levesque and staff for animal husbandry.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 by National Institutes of Health Grants DK0 57665, AI48638, AI056499, U19 AI05726601, and Regional Centers of Excellence Grants U54 AI057157 and AI056947. Back

2 Address correspondence and reprint requests to Dr. Bali Pulendran, Emory Vaccine Center, 954 Gatewood Road, Atlanta, GA 30329. E-mail address: bpulend{at}rmy.emory.edu Back

3 Abbreviations used in the paper: DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; WT, wild type; MOG, myelin oligodendrocyte glycoprotein; DNP-KLH, dinitrophenol-conjugated keyhole limpet hemocyanin. Back

Received for publication November 18, 2005. Accepted for publication February 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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