Modulation of p38 MAPK Activity in Regulatory T Cells after Tolerance with Anti-DNA Ig Peptide in (NZB × NZW)F1 Lupus Mice

Elaine V. Lourenço, Claudio Procaccini, Francesca Ferrera, Noriko Iikuni, Ram P. Singh, Gilberto Filaci, Giuseppe Matarese, Fu-Dong Shi, Ernest Brahn, Bevra H. Hahn and Antonio La Cava
J Immunol June 15, 2009, 182 (12) 7415-7421; DOI: https://doi.org/10.4049/jimmunol.0804214

Abstract

Treatment of (NZB × NZW)F1 (NZB/W) lupus-prone mice with the anti-DNA Ig-based peptide pConsensus prolongs the survival of treated animals and effectively delays the appearance of autoantibodies and glomerulonephritis. We have previously shown that part of these protective effects associated with the induction of CD4+CD25+Foxp3+ regulatory T cells (Tregs) that suppressed autoantibody responses. Because the effects of pConsensus appeared secondary to qualitative rather than quantitative changes in Tregs, we investigated the molecular events induced by tolerance in Tregs and found that signaling pathways including ZAP70, p27, STAT1, STAT3, STAT6, SAPK, ERK, and JNK were not significantly affected. However, peptide tolerization affected in Tregs the activity of the MAPK p38, whose phosphorylation was reduced by tolerance. The pharmacologic inhibition of p38 with the pyridinyl imidazole inhibitor SB203580 in naive NZB/W mice reproduced in vivo the effects of peptide-induced tolerance and protected mice from lupus-like disease. Transfer experiments confirmed the role of p38 in Tregs on disease activity in the NZB/W mice. These data indicate that the modulation of p38 activity in lupus Tregs can significantly influence the disease activity.

Suppression of effector immune cells by CD4+CD25+Foxp3+ regulatory T cells (Tregs)6 is a major mechanism of peripheral immune tolerance (1, 2). Despite recent progress in understanding key aspects of the biology of the Tregs, it is largely unknown which molecular mechanisms Tregs use in their activity (other than up-regulation of Foxp3), and what biochemical pathways are modulated in relation to the functional changes that occur in these cells. Indeed, little is known about the molecular pathways that promote or inhibit the activity of Tregs in physiologic and pathologic conditions, despite the many advances in the characterization of Treg phenotypes and suppressive functions (3, 4). A better knowledge of these aspects could lead to the development of targeted therapeutic interventions in diseases that are characterized by immune dysregulation and impaired number and/or function of Tregs, such as systemic lupus erythematosus (SLE) (5).

We have previously shown that tolerogenic administration of the anti-DNA peptide pConsensus (pCons) induced functional Tregs in NZB/W lupus-prone mice (6). We extend in this study those findings by showing that phosphorylation of the p38 MAPK (p38) is down-regulated in Tregs of pCons-tolerized mice.

MAPKs are a group of evolutionarily conserved serine/threonine kinases that are activated in response to a variety of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus (7). Four major types of MAPK cascades have been reported in mammalian cells that respond synergistically to different upstream signals. MAPKs are part of a three-tiered phospho-relay cascade consisting of MAPK, a MAPK kinase (MEK) and a MAPK kinase kinase (MEK kinase). Controlled regulation of these cascades is involved in cell proliferation and differentiation, and p38 is activated in response to inflammatory cytokines, endotoxins, heat shock, and osmotic stress (8).

Our herein described finding of a decreased activation of p38 in tolerized Tregs identifies a pathway modulated by immune tolerance that could be targeted in Tregs in SLE.

Materials and Methods

Mice, peptides, and cell preparation

Female (NZB × NWZ)F1 (NZB/W) mice were purchased from The Jackson Laboratory or obtained from our colony at the University of California Los Angeles (UCLA). All animals were treated according to the National Institutes of Health guidelines for the use of experimental animals, with the approval of the UCLA Animal Research Committee for the Use and Care of Animals.

For tolerance induction, 10- to 12-wk-old NZB/W mice received a single i.v. dose of 1 mg of pCons (which contains T cell determinants from different J558 VH regions of NZB/W anti-dsDNA Ig) dissolved in saline (9). Control mice received an identical volume of saline or equal dose of negative control peptide pNeg i.v. (9). There was no significant difference in the percentage and total numbers of Tregs between mice that received saline and pNeg, as reported before (6). Peptides were synthesized at Chiron Biochemicals, purified to a single peak by HPLC, and analyzed by mass spectroscopy for expected amino acid content before use. One week after treatment, single cell suspensions of splenocytes were prepared by passing cells through a sterile wire mesh. After lysis of RBC with ACK lysing buffer (Sigma-Aldrich), cells were centrifuged, washed, and resuspended in HL-1 medium (BioWhittaker) before experimental use.

Flow cytometry

After cell wash and blockade of Fc-γ receptors, mAb to surface markers or control isotype-matched fluorochrome-labeled Ab in PBS/2% FCS were added for 20 min at 4°C. For surface staining, the following fluorochrome-labeled mAb from eBioscience were used: anti-CD3, anti-CD4, anti-CD25, and anti-CD19. For Foxp3 detection, cells were fixed and permeabilized before incubation with anti-Foxp3-PE (eBioscience). Samples were read on a BD FACSCalibur and analyzed with FCS Express (De Novo Software). For purification of Tregs, sorting was done from splenocytes as CD4+CD25+ T cells by FACSVantage (BD Biosciences) or with the Mouse Regulatory T Cell Isolation kit (Miltenyi Biotec) using an AutoMACS Separator (Miltenyi Biotec). Purity of cells was determined by FACS analysis as >90% Foxp3+ cells among gated CD4+CD25+ T cells. The sorted populations were routinely >95% pure. For signaling proteins, costaining of cells was done after permeabilization and subsequent use of p38 (C-20) and phosphorylated p38 (p-p38) (Tyr182) Abs (Santa Cruz Biotechnology) and fluorochrome-conjugated secondary Ab or matched control Ab (eBioscience) with the Cytofix/Cytoperm Kit (BD Biosciences), following the manufacturer’s instructions.

Western blotting

Western blot analyses were performed as previously described (10). In brief, total cell lysates from sorted cells were obtained in 50 mM HEPES (pH 7.5), 250 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 2 μg/ml pepstatin. Proteins were separated by SDS-PAGE (SDS-PAGE) and then transferred onto PVDF membranes (BioRad Laboratories). Membranes were blocked in 5% nonfat milk/PBS, 0.5% Tween 20 (PBST) at 4°C for 2 h, and then incubated with Ab from Cell Signaling Technology (anti-ZAP-70, anti-p27 Kip1, anti-ERK1/2, anti-STAT1, anti-STAT3, anti-STAT6, anti-stress-activated protein kinase (SAPK), anti-p38) and Santa Cruz Biotechnology (anti-JNK, anti-p38) before being washed in PBST and incubated with peroxidase-conjugated secondary Ab. After an additional wash, peroxidase activity was detected with the ECL system (Amersham) or Femto system (Thermo Scientific). Membranes were stripped and reprobed with anti-phospho Abs from Cell Signaling Technologies (anti-p-ZAP-70Tyr319, anti-p-ERK1/2Thr202/Tyr204, anti-pSTAT1Ser727, anti-STAT3Ser727, anti-STAT6Tyr641, anti-p-SAPKThr183/Tyr185, anti-p- p38Thr180/Tyr182) and Santa Cruz Biotechnology (anti-p-JNKThr183/Tyr185, anti-p-p38Tyr182) and again stripped and reprobed with anti-β-actin Ab (Cell Signaling Technology), to determine equivalency of loading. Exposed films were quantified by densitometric band analysis with the ScionImage program (Scion Corporation).

ELISA

ELISA for IL-1 and TNF-α were performed using commercial ELISA kits (R&D Systems) following the manufacturer’s instructions. IgG and anti-dsDNA Ab were analyzed by standard ELISA, as described elsewhere (6, 11).

p38 inhibition

The p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and negative control molecule 4-ethyl-2(p-methoxyphenyl)-5-(4′-pyridyl)-IH-imidazole (SB202474) were purchased from Calbiochem and dissolved in saline. For in vivo treatment, mice were injected i.p. daily for 2 wk with 2 mg/kg SB203580 or SB202474 or equal volume of saline (12).

In vivo cell transfer

Sorted Tregs, CD4+CD25− T cells, or B cells from treated mice or controls were injected into irradiated recipient mice, as described previously (11). In brief, each recipient mouse received 600–800 rads 2 h before an i.v. injection of 10:10:1 ratio of Tregs (1 × 107), CD4+CD25− (effector) T cells (1 × 107), B cells (1 × 106), or isolated sorted lymphocyte subsets or Tregs plus effector T cells suspended in PBS. After transfer, sera of recipient mice were monitored weekly for IgG and anti-dsDNA Ab by ELISA. Early morning urine was monitored at 2 wk intervals for the presence of proteinuria using Albustix strips (Bayer), and animal survival was analyzed using Kaplan-Meier curves.

Statistical analyses

Analyses for statistical significance were performed using Prism 4 software (GraphPad). Parametric testing between two groups was performed by the paired t test or by the Mann-Whitney U test. Nonparametric testing among more than two groups was performed by one-way ANOVA. Values of p < 0.05 were prespecified as significant.

Results

Effects of peptide tolerization on in vivo Treg number and function

We previously reported that NZB/W mice tolerized with the anti-DNA Ig peptide pCons were protected from the development and progression of renal disease (9). The tolerized mice had significantly reduced serum titers of anti-DNA Ab and decreased production of proinflammatory cytokines when compared with control mice (9). Because part of the protective effects could be explained with the suppression of anti-DNA responses by pCons-induced CD4+CD25+Foxp3+ regulatory T cells (6), we studied the quantitative and qualitative characteristics of these regulatory T cells in relation to tolerization with pCons. The number of Tregs in pCons-tolerized and control NZB/W mice did not change significantly after treatment both as percentage (Fig. 1⇓a) and total number of cells (Fig. 1⇓b). This finding could not explain the observation of an increased suppressive efficacy of Tregs after pCons-induced immune tolerance (6). Therefore, we asked whether the lack of quantitative effects on Tregs was concomitant to the presence of qualitative change in these cells. Equal numbers of purified Tregs from tolerized mice or control donor mice were adoptively transferred into syngeneic premorbid NZB/W recipients (16-wk-old) to monitor in vivo the activity of transferred Tregs on development of renal disease. Mice that had been administered Tregs from tolerized mice had delayed development of proteinuria (p < 0.04) and increased survival (p < 0.001) when compared with mice given Tregs from controls (Fig. 2⇓). These data suggest that tolerization of NZB/W mice with pCons can induce qualitative, rather than quantitative changes in Tregs.

FIGURE 1.
FIGURE 1.

Effects of peptide tolerization on in vivo numbers of Tregs. NZB/W mice tolerized with pCons have similar percentage (a) and total (b) numbers of Tregs than age- and weight-matched controls (8 to 12 mice per group). P is not significant between tolerized mice and controls for both panels a and b.

FIGURE 2.
FIGURE 2.

Tregs from tolerized mice confer higher protection than control Tregs after transfer into syngeneic mice. The transfer of equal numbers of purified NZB/W Tregs from pCons-tolerized or control mice into syngeneic 16-wk-old recipient mice associates with a delayed development of renal disease (as manifested by proteinuria, top; ∗, p < 0.04 in the comparison between pCons and control groups), and an increased survival (bottom; ∗, p < 0.001 in the comparison between pCons and control groups) of recipients of pCons-derived Tregs than recipients of control Tregs.

Tolerization with peptide down-regulates p38 activation in Tregs

Because Tregs from tolerized mice were more effective than Tregs from controls to suppress lupus-like disease (Ref. 6 , and Fig. 2⇑) despite a lack of quantitative changes between the two groups of mice (Fig. 1⇑), we investigated whether the differences between the two groups of animals could be explained by a modulation in molecular signaling events in Tregs. Immunoblot analyses indicated no difference in phosphorylation of the ζ-chain-associated tyrosine kinase ZAP70, suggesting that initiation of TCR-proximal T cell signaling events was not significantly affected by tolerance with pCons (Fig. 3⇓). The p27-dependent cell cycle activity in Tregs was also not influenced by tolerance induction, as no changes were found in the expression of the cycline-dependent kinase inhibitor protein p27 (Fig. 3⇓). When STATs were studied for the responsiveness to cytokine signaling and/or cell growth, no differences in activation of STAT1, STAT3, and STAT6 were detected in the Tregs after peptide-induced tolerance (Fig. 3⇓).

FIGURE 3.
FIGURE 3.

Signaling events after tolerization. Immunoblots for phosphorylated (p-) and nonphosphorylated ZAP-70, p27, ERK, JNK, SAPK, STAT1, STAT3, STAT6, and p38 in sorted Tregs from control mice (ctrl) and from mice tolerized (tol) with pCons peptide show a reduced phosphorylation of p38 in Tregs from tolerized mice. A direct comparison of phosphorylated p38 to nonphosphorylated p38 between purified CD4+CD25+ T cells and CD4+CD25− T cells shows that modulated activation of p38 occurs in Tregs after tolerance but not in effector T cells, the latter displaying virtually absent basal activation of p38. Graphs show the densitometric quantitation of protein to the housekeeping gene (p27) and to the nonphosphorylated form (all others). The results were reconfirmed in four to seven independent experiments and using different Ab from two different commercial suppliers (see Materials and Methods). There was no difference in Foxp3 expression by immunoblot in sorted Tregs from control and tolerized mice (data not shown).

Next, the MAPK/ERK pathway was analyzed because in T cells this pathway couples intracellular responses to binding of growth factor, including cytokines, to cell surface receptors. This pathway also promotes T cell differentiation, proliferation, and responsiveness to cytokines and stress stimuli. No significant differences were observed between Tregs from tolerized and control mice in the activation of JNK and the JNK/SAPK, and in the activation of ERK (Fig. 3⇑). The only significant difference between Tregs from tolerized mice and control mice was a decreased phosphorylation of p38 after treatment with pCons (Fig. 3⇑). Interestingly, there was no difference in the phosphorylation of p38 between CD4+CD25− T cells before and after tolerance, indicating that pCons influenced p38 activity in the Tregs but not in CD4+CD25− T cells (Fig. 3⇑). These findings were confirmed by a reduced activation of p38 in Tregs from tolerized mice in flow cytometry experiments (Fig. 4⇓).

FIGURE 4.
FIGURE 4.

Effects of tolerization with peptide on p38 activity in NZB/W Tregs. a, Flow cytometry analysis for the intracellular expression of p38 (left panel) and p-p38 (right panel) in gated Tregs (CD4+CD25+Foxp3+cells) from tolerized (gray line) and control mice (black line). b, Tregs from pCons-tolerized mice have similar mean fluorescence intensity of nonphosphorylated p38 (P ns) and reduced phosphorylation of p38 than controls (n = 8 per group; p < 0.004). c, Cumulative data on the intracellular expression of p38 and p-p38 detected by flow cytometry in CD4+CD25+Foxp3+ T cells from tolerized (n = 8) and control (n = 8) mice; p < 0.01.

Inhibition of p38 delays lupus-like disease in NZB/W mice

To address whether pharmacologically induced down-regulation of p38 (which would mimic pCons-induced down-regulation of p38) could affect disease in NZB/W lupus mice, we treated naive animals with the p38 inhibitor SB203580, the control molecule SB202474, or vehicle, and followed treated animals for survival and for development of anti-DNA Ab. Mice that received SB203580 (that down-regulated p38 activity in Tregs in ex vivo flow cytometry experiments, data not shown) had a significantly reduced titer of anti-DNA Ab (p < 0.04) when compared with control mice treated with SB202474 or vehicle (Fig. 5⇓a). Moreover, inhibition in vivo of p38 also resulted in an increased survival of mice (p < 0.003) as compared with controls treated with SB202474 or vehicle and, interestingly, none of the animals treated with SB203580 had died 6 mo after treatment (Fig. 5⇓b).

FIGURE 5.
FIGURE 5.

Inhibition of p38 delays lupus-like disease in NZB/W mice. Sixteen- to 20-wk-old premorbid mice (n = 6 per group) were treated with p38 inhibitor SB203580, control SB202474, or vehicle as indicated in the Materials and Methods and then monitored for serum anti-DNA (a; ∗, p < 0.04 vs both controls) and survival (b; ∗, p < 0.003 vs both controls).

Considering that phosphorylation of p38 results in proinflammatory activity, we also addressed whether a reduced activation of p38 could associate with a change in the expression of IL-1 and TNF-α, which are cytokines that contribute to chronic inflammatory processes in systemic autoimmunity (13). Both IL-1 and TNF-α were reduced in sera of mice treated with SB203580, as compared with control mice (Fig. 6⇓). These data indicate that the in vivo effect of SB203580 on p38 inhibition associates with a reduced production of inflammatory cytokines and beneficial effects on disease in NZB/W mice.

FIGURE 6.
FIGURE 6.

In vivo inhibition of p38 in NZB/W mice associates with a reduced serum concentration of inflammatory cytokines. Age matched (12- to 16-wk-old) NZB/W mice (n = 6 per group) were treated with the p38 inhibitor SB203580, SB202474 control, or vehicle, as indicated in the Materials and Methods. Serum concentration of IL-1 (a) and TNF-α (b) were monitored by ELISA; ∗, p < 0.05 for both.

Effects of p38 inhibition on in vivo Treg suppression

Both SB203580 and peptide tolerization inhibit p38 activity. We then explored whether cumulative effects would derive from combining pharmacologic and tolerogenic treatments. The reduced phosphorylation of p38 by pCons peptide in Tregs was decreased further by cotreatment with the p38 inhibitor (p < 0.01, Fig. 7⇓a). The inhibition of p38 in vivo did not affect the number of peripheral Tregs when compared with control-treated animals (Fig. 7⇓b) or Foxp3 expression (data not shown).

FIGURE 7.
FIGURE 7.

Effects of SB203580 and pCons on p38 inhibition in Tregs. a, Ratio of phosphorylated p-p38 to total p38 detected by intracellular flow cytometry in sorted CD4+CD25+Foxp3+ T cells from tolerized (n = 5–8) and control mice (n = 5–8) treated with 2 mg/kg/day i.p. for 2 wk with SB203580, SB202474, or vehicle (∗, p < 0.04; ∗∗, p < 0.01). b, Numbers of Tregs after treatment (p not significant).

Next, we performed in vivo transfer of Tregs with down-regulated p38, following a strategy that allows to monitor anti-DNA Ab production in vivo (11). In brief, mice were irradiated before transfer with Tregs, Th cells, and B cells. The Tregs were derived from tolerized mice treated with either p38 inhibitor or controls, the Th cells were derived from untreated mice, and the B cells were from old mice. The combination of Th and B cells served as positive controls for in vivo production of anti-DNA Ab (11). As shown in Fig. 8⇓, mice receiving Tregs with inhibited p38 displayed reduced IgG and delayed proteinuria than controls.

FIGURE 8.
FIGURE 8.

Effects of in vivo p38 inhibition in Tregs. Irradiated NZB/W mice received 2 × 106 purified Tregs from syngeneic mice tolerized with pCons that had been treated for 2 wk i.p. with 2 mg/kg/day SB203580 (TregsSB203580), SB202474 (TregsSB202474), or vehicle, together with 2 × 106 purified CD4+CD25− Th cells (Th) and 2 × 106 purified B cells (1:1:1 ratio Tregs:Th:B). Control mice received equal numbers of the individual cell subsets (data not shown) or a combination of Th and B cells. Recipient mice (n = 6 per group) were monitored for serum anti-DNA IgG (a), total IgG (b), and development of proteinuria (c). TregsSB203580/Th/B vs controls; ∗, p < 0.05.

Discussion

This study shows that the identification of T cell signaling events in adaptive Tregs induced by tolerance with peptide can identify targets for pharmacologic modulation of murine SLE. We had previously shown that high doses of the anti-DNA Ig-based peptide pCons effectively suppressed autoimmunity in lupus-prone NZB/W mice and that one mechanism responsible for the protection was the induction of functional Tregs, in addition to inhibitory CD8+ T cells (6, 9, 12, 14, 15). However, pCons had little effect on the number of peripheral Tregs (Fig. 1⇑). Because an increased suppressive capacity of Tregs after tolerance (Fig. 2⇑) had to be explained by qualitative rather than quantitative changes, we investigated whether altered intracellular signaling events in Tregs from pCons-treated mice might associate with changes in Treg activity. In conventional T cells, the intracellular signaling events that follow TCR stimulation result in the activation of tyrosine kinases and the assembly of scaffolds of adaptor molecules whose phosphorylation leads to activation of downstream effectors including serine/threonine kinases such as MAPK and the activation of transcription factors including NF-κB (16). The activation of these cascades can engage a variety of T cell functions including the production of cytokines and cell-cycle progression. Although most signaling studies have typically focused on conventional T cells, some studies have also investigated the intracellular signaling in Tregs, and the importance of intracellular signaling in the biology of Tregs as well as its relevance to possible therapeutic applications has been recognized (17). As Tregs express a TCR, they require signaling to NF-κB through IKK2 (18) and, although hyporesponsive to antigenic stimulation (3, 4), Tregs can signal. Tregs could nonetheless have specific signaling that make them respond to stimuli differently than conventional activated T cells (19). For example, Tregs are in an anergic state concomitant with an intact proliferative potential, as indicated by the observation that Tregs unable to flux Ca2+ proliferate in response to lymphopenia after TCR engagement (20). Thus, cells remain anergic but maintain an intact suppressive function. The anergic state of Tregs may not represent a default state but rather an actively maintained gene program (19). Signaling requirements of Tregs might reflect the fact that these cells are activated or are memory T cells that can react to self Ags, in our case pCons (21, 22), and signaling pathways in the Tregs could contribute to their maintenance and function.

Because cytokine expression can contribute to the development, maintenance, and differentiation of most immune cells, it would have been possible that the modulated function of Tregs after tolerance could have associated with differences in the expression of STATs. In this context, STAT1 is involved in up-regulating genes related to signaling by either type I or type II IFNs, and STAT3 is activated in response to various cytokines and growth factors including IFNs. Both type I and II IFNs have been linked to the pathogenesis of SLE in humans and in animal models of the disease, and a type I IFN signature is characteristic of ∼50% of SLE patients (23, 24). However, our analysis on phosphorylation of STATs did not find significant differences in the expression of these transcription factors, suggesting that these pathways may not affect significantly the activity of Tregs after pCons-induced tolerance in NZB/W mice. Also, the lack of significant changes in p27 after tolerance with pCons indicated that the effects of pCons on Tregs did not influence cellular expansion, in line with the observations reported in Fig. 1⇑, and the upstream events to the TCR (ZAP70) were unchanged by tolerance with pCons. In contrast, events not affected by mitogenic stimuli and related to cellular stress and modulated by MAPK may suggest that decreased activation of p38 in Tregs of pCons-tolerized mice might represent a sensing mechanism to modulate inflammation in mice poised to develop systemic autoimmunity.

Another consideration relates to the possibility that the modulated activity of p38 in Tregs after peptide-induced tolerance may not exclude the contribution of intermediate immune cell(s) and/or soluble factor(s). We showed that tolerance with pCons in NZB/W mice associated with a quantitative expansion of CD8+ T cells (16) that suppressed anti-DNA Ab (11, 14, 15), and Sharabi and Mozes (25) recently showed that inhibitory/suppressor CD8+ T cells are required for the optimal function/induction of Tregs after anti-DNA peptide-induced tolerance in mice. Other authors found that dendritic cells can influence activity and function of Tregs (26) and convert Foxp3− T cells into Foxp3+ T cells (27). Additionally, the requirement of soluble factors such as IL-10 has been described in the generation (28) and/or activity of Tregs (29), and p38 signaling has been associated with TGF-β-mediated conversion of polyclonal CD4+CD25− T cells into Tregs (30). Investigations are needed to clarify the role of these players in this system to define how pCons can influence the quality (function) and/or quantity (conversion) of Tregs from pre-existing cells. At present, our findings identify p38 as a molecule modulated by peptide tolerance in Tregs and help to explain, at a molecular level, some of the beneficial effects caused by p38 inhibition in animal models of arthritis (31, 32) and SLE (33) through a novel link between modulation of p38 and Tregs.

To conclude, we have identified through a model of induced tolerance a molecular signaling pathway that allows targeted manipulation of Tregs through pharmacologic intervention. This system could represent a new tool to modulate Treg-mediated suppression in SLE.

Disclosures

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 the National Institutes of Health Grants AR53239 (to A.L.C.), AI065645 and AR054034 (to R.P.S.), AR42200 (to E.B.), AI46776 (to B.H.H.), the Arthritis Foundation Southern California Chapter (to A.L.C.), and the Arthritis National Research Foundation (to E.V.L.). G.M. is supported by the ERC-Starting Grant 202579 and by JDRF-Telethon Grant GJT08004.

  • ↵2 E.V.L., C.P., and F.F. contributed equally to this study.

  • ↵3 Current address: Department of Biology and Cellular and Molecular Pathology, Federico II University of Naples, Italy.

  • ↵4 Current address: Center of Excellence for Biomedical Research, University of Genoa, Italy.

  • ↵5 Address correspondence and reprint requests to: Prof. Antonio La Cava, University of California, Los Angeles, 1000 Veteran Avenue 32–59, Los Angeles, CA 90095-1670. E-mail address: alacava{at}mednet.ucla.edu

  • ↵6 Abbreviations used in this paper: Treg, CD4+CD25+Foxp3+ regulatory T cell; SLE, systemic lupus erythematosus; p-p38, phosphorylated p38; SAPK, stress-activated protein kinase; pCons, pConsensus.

  • Received December 17, 2008.
  • Accepted April 14, 2009.

References

  1. ↵
    Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Asano, M., M. Toda, N. Sakaguchi, S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184: 387-396.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Sakaguchi, S.. 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22: 531-562.
    OpenUrlCrossRefPubMed
  4. ↵
    Shevach, E. M.. 2002. CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2: 389-400.
    OpenUrlPubMed
  5. ↵
    La Cava, A.. 2008. T-regulatory cells in systemic lupus erythematosus. Lupus 17: 421-425.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    La Cava, A., F. M. Ebling, B. H. Hahn. 2004. Ig-reactive CD4+CD25+ T cells from tolerized (New Zealand Black × New Zealand White)F1 mice suppress in vitro production of antibodies to DNA. J. Immunol. 173: 3542-3548.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Chang, L., M. Karin. 2001. Mammalian MAP kinase signalling cascades. Nature 410: 37-40.
    OpenUrlCrossRefPubMed
  8. ↵
    Cook, R., C. C. Wu, Y. J. Kang, J. Han. 2007. The role of the p38 pathway in adaptive immunity. Cell. Mol. Immunol. 4: 253-259.
    OpenUrlPubMed
  9. ↵
    Hahn, B. H., R. R. Singh, W. K. Wong, B. P. Tsao, K. Bulpitt, F. M. Ebling. 2001. Treatment with a consensus peptide based on amino acid sequences in autoantibodies prevents T cell activation by autoantigens and delays disease onset in murine lupus. Arthritis Rheum. 44: 432-441.
    OpenUrlPubMed
  10. ↵
    De Rosa, V., C. Procaccini, G. Calì, G. Pirozzi, S. Fontana, S. Zappacosta, A. La Cava, G. Matarese. 2007. A key role of leptin in the control of regulatory T cell proliferation. Immunity 26: 241-255.
    OpenUrlCrossRefPubMed
  11. ↵
    Hahn, B. H., R. P. Singh, A. La Cava, F. Ebling. 2005. Tolerogenic treatment of lupus mice with consensus peptide induces Foxp3-expressing, apoptosis-resistant, TGF-β-secreting CD8+ T cell suppressors. J. Immunol. 175: 7728-7737.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Peng, T., X. Lu, M. Lei, G. W. Moe, Q. Feng. 2003. Inhibition of p38 MAPK decreases TNF-α expression and improves myocardial function and survival in endotoxemia. Cardiovasc. Res. 59: 893-900.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Lourenço, E. V., A. La Cava. 2009. Cytokines in systemic lupus erythematosus. Curr. Mol. Med. 9: 242-254.
    OpenUrlCrossRefPubMed
  14. ↵
    Singh, R. P., A. La Cava, M. Wong, F. Ebling, B. H. Hahn. 2007. CD8+ T cell-mediated suppression of autoimmunity in a murine lupus model of peptide-induced immune tolerance depends on Foxp3 expression. J. Immunol. 178: 7649-7657.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Singh, R. P., A. La Cava, B. H. Hahn. 2008. pConsensus peptide induces tolerogenic CD8+ T cells in lupus-prone (NZB × NZW)F1 mice by differentially regulating Foxp3 and PD1 molecules. J. Immunol. 180: 2069-2080.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Ashwell, J. D.. 2006. The many paths to p38 mitogen-activated protein kinase activation in the immune system. Nat. Rev. Immunol. 6: 532-540.
    OpenUrlCrossRefPubMed
  17. ↵
    Turka, L. A., P. T. Walsh. 2008. IL-2 signaling and CD4+CD25+Foxp3+ regulatory T cells. Front. Biosci. 13: 1440-1446.
    OpenUrlCrossRefPubMed
  18. ↵
    Schmidt-Supprian, M., J. Tian, E. P. Grant, M. Pasparakis, R. Maehr, H. Ovaa, H. L. Ploegh, A. J. Coyle, K. Rajewsky. 2004. Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-κB activation. Proc. Natl. Acad. Sci. USA 101: 4566-4571.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Matarese, G., V. De Rosa, A. La Cava. 2008. Regulatory CD4 T cells: sensing the environment. Trends Immunol. 29: 12-17.
    OpenUrlCrossRefPubMed
  20. ↵
    Gavin, M. A., S. R. Clarke, E. Negrou, A. Gallegos, A. Rudensky. 2002. Homeostasis and anergy of CD4+CD25+ suppressor T cells in vivo. Nat. Immunol. 3: 33-41.
    OpenUrlCrossRefPubMed
  21. ↵
    Su, L., R. J. Creusot, E. M. Gallo, S. M. Chan, P. J. Utz, C. G. Fathman, J. Ermann. 2004. Murine CD4+CD25+ regulatory T cells fail to undergo chromatin remodeling across the proximal promoter region of the IL-2 gene. J. Immunol. 173: 4994-5001.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Chan, S. M., J. Ermann, L. Su, C. G. Fathman, P. J. Utz. 2004. Protein microarrays for multiplex analysis of signal transduction pathways. Nat. Med. 10: 1390-1396.
    OpenUrlCrossRefPubMed
  23. ↵
    Crow, M. K., K. A. Kirou, J. Wohlgemuth. 2003. Microarray analysis of interferon-regulated genes in SLE. Autoimmunity 36: 481-490.
    OpenUrlCrossRefPubMed
  24. ↵
    Kirou, K. A., C. Lee, S. George, K. Louca, I. G. Papagiannis, M. G. Peterson, N. Ly, R. N. Woodward, K. E. Fry, A. Y. Lau, et al 2004. Coordinate overexpression of interferon-α-induced genes in systemic lupus erythematosus. Arthritis Rheum. 50: 3958-3967.
    OpenUrlCrossRefPubMed
  25. ↵
    Sharabi, A., E. Mozes. 2008. The suppression of murine lupus by a tolerogenic peptide involves Foxp3-expressing CD8 cells that are required for the optimal induction and function of Foxp3-expressing CD4 cells. J. Immunol. 181: 3243-3251.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Pasare, C., R. Medzhitov. 2003. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299: 1033-1036.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Yamazaki, S., D. Dudziak, G. F. Heidkamp, C. Fiorese, A. J. Bonito, K. Inaba, M. C. Nussenzweig, R. M. Steinman. 2008. CD8+CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells. J. Immunol. 181: 6923-6933.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Adler, H. S., S. Kubsch, E. Graulich, S. Ludwig, J. Knop, K. Steinbrink. 2007. Activation of MAP kinase p38 is critical for the cell-cycle-controlled suppressor function of regulatory T cells. Blood 109: 4351-4359.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Wilczynski, J. R., M. Radwan, J. Kalinka. 2008. The characterization and role of regulatory T cells in immune reactions. Front. Biosci. 13: 2266-2274.
    OpenUrlCrossRefPubMed
  30. ↵
    Huber, S., J. Schrader, G. Fritz, K. Presser, S. Schmitt, A. Waisman, S. Lüth, M. Blessing, J. Herkel, C. Schramm. 2008. p38 MAP kinase signaling is required for the conversion of CD4+CD25 −T cells into iTreg. PLoS ONE 3: e3302
    OpenUrlCrossRefPubMed
  31. ↵
    Mihara, K., C. Almansa, R. L. Smeets, E. E. Loomans, J. Dulos, P. M. Vink, M. Rooseboom, H. Kreutzer, F. Cavalcanti, A. M. Boots, R. L. Nelissen. 2008. A potent and selective p38 inhibitor protects against bone damage in murine collagen-induced arthritis: a comparison with neutralization of mouse TNFα. Br. J. Pharmacol. 154: 153-164.
    OpenUrlCrossRefPubMed
  32. ↵
    Badger, A. M., D. E. Griswold, R. Kapadia, S. Blake, B. A. Swift, S. J. Hoffman, G. B. Stroup, E. Webb, D. J. Rieman, M. Gowen, et al 2000. Disease-modifying activity of SB242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis. Arthritis Rheum. 43: 175-183.
    OpenUrlCrossRefPubMed
  33. ↵
    Iwata, Y., T. Wada, K. Furuichi, N. Sakai, K. Matsushima, K.-I. Kobayashi. 2003. p38 mitogen-activated protein kinase contributes to autoimmune renal injury in MRL-Faslpr mice. J. Am. Soc. Nephrol. 14: 57-67.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section