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Keywords:

  • Allergy;
  • Th1/Th2 cells;
  • Transcription factors;
  • Transgenic/knockout;
  • Lung

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The transcription factor c-Maf controls IL-4 gene expression in CD4+ T cells, and its expression is up-regulated in human asthmatic airways after allergen challenge. In the present study, we addressed the role of c-Maf in asthma by studying transgenic (Tg) mice overexpressing c-Maf in CD4+ T cells under the control of the CD2 promoter. As shown, lung CD4+ T cells of c-maf-Tg mice produced more IL-5 at the early stage (day 2) of culture in the presence of IL-4 than wild-type control cells. Consistently, c-maf-Tg mice spontaneously showed increased IL-5 expression and eosinophils in the bronchial alveolar lavage fluid (BALF) and activated IL-5 signal transduction via Raf-1 and Ras in lung eosinophils. Finally, IL-13 was suppressed in the BALF of c-maf-Tg mice and in supernatants of Tg lung CD4+ T cells cultured in the presence of IL-2. Consistently, retroviral overexpression of c-Maf suppressed IL-13 production in developing lung Th2 cells. In summary, c-Maf induces IL-5 production in lung CD4+ T cells at an early stage, but along with IL-2 suppresses IL-13 production in differentiating lung Th2 cells, thereby explaining the finding that overexpression of c-Maf does not cause airway hyperresponsiveness, a hallmark feature of asthma.

Abbreviations:
AHR:

Airway hyperresponsiveness

BALF:

Bronchial alveolar lavage fluid

MBP:

Major basic protein

Penh:

Enhanced pause

RV:

Retrovirus

STAT:

Signal transducer and activator of transcription

Tg:

Transgenic

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The development of allergic immune responses and airway hyperresponsiveness (AHR), such as in allergic asthma, is believed to be mediated by CD4+ effector T helper 2 (Th2) cells 18. In contrast to Th1 cells, which produce large amounts of IFN-γ, Th2 cells are known to produce the signature cytokines IL-4, IL-5 and IL-13 5, 910. Increased numbers of CD4+ T cells expressing IL-4 and IL-5 have been found in the airways of asthmatic subjects 2. Whereas IL-4 plays a central role in immunoglobulin E (IgE)-mediated responses, IL-5 is crucial for eosinophil recruitment and activation, which is observed during the late asthmatic reaction 3, 9, 11. Th2 cells develop from naive T cells under the influence of IL-4 produced by T and non-T cells such as IgE-activated mast cells 4, 9. IL-4 activates the transcription factors signal transducer and activator of transcription (STAT)-6 and GATA-3 in T cells, and the latter induces production of Th2 cytokine genes by chromatin remodeling and promoter transactivation 9, 1214. However, the precise molecular pathways causing Th2 cell differentiation in lung T cells in allergic asthma are still poorly understood.

c-Maf is a transcription factor of the maf gene family expressed in Th2 but not Th1 cells 15 that is responsible for tissue-specific expression of the IL-4 gene 1517. The maf gene family includes the large maf genes (c-maf, mafB and Nrl) and the small maf genes (mafK, mafG and mafF), which lack the N-terminal half of the large maf genes containing the transactivation domain 18. Members of both subfamilies possess a well-conserved bZip DNA-binding domain at the C terminus 1820.

Recent studies have indicated an important role for c-Maf in the differentiation of peripheral T cells. In contrast to GATA-3, c-Maf transactivates the IL-4 promoter but not the IL-5 and IL-13 promoters in peripheral lymphatic organs 1517. Consistently, transgenic (Tg) mice overexpressing c-Maf in T cells showed increased production of the Th2 cytokine IL-4 and exhibited elevated serum levels of IgG1 and IgE. The latter findings are IL-4 dependent and could be reversed when the c-maf-Tg animals were backcrossed to an IL-4-deficient background. Furthermore, c-maf–/– mice showed greatly impaired IL-4 production, and their T cells spontaneously polarized towards a Th1 phenotype. However, under Th2-differentiating conditions, c-Maf-deficient Th2 cells produced normal amounts of Th2 cytokines, indicating that c-Maf is not required for chromatin remodeling at the IL-4/IL-13 locus but rather specifically regulates IL-4 gene expression in peripheral T lymphocytes 1517.

Based on the above data and the recently described overexpression of c-Maf in the airways of asthmatic subjects 21, 22, we thought to delineate the functional role of c-Maf in asthma in the present study by using c-maf-Tg mice. Surprisingly, our results demonstrate that overexpression of c-Maf, in the absence of GATA-3, results in inhibition of IL-13 production in the airways and in AHR. Moreover, we found that overexpression of c-Maf selectively induced IL-5 expression at the beginning of the Th2 differentiation in lung CD4+ T cells, but suppressed IL-13 production in both lung and splenic CD4+ T cells throughout the entire Th2 differentiation period in an IL-2-dependent manner.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Spontaneously increased eosinophilia and IL-5 production in the airways of c-maf-Tg mice

c-Maf is a transcription factor expressed in Th2 cells that induces IL-4 production in peripheral CD4+ T cells. Based on its role in Th2 cytokine gene transcription and its up-regulation in patients with allergic asthma 1719, 21, 22, we analyzed the effect of overexpression of c-Maf in CD4+ cells in the lung. We found that c-maf-Tg mice have lung eosinophilia in the bronchial alveolar lavage fluid (BALF) (Fig. 1A) as compared to wild-type mice. In contrast, after OVA sensitization and challenge, no change in lung eosinophilia was found in the lungs of c-maf-Tg mice as compared to control wild-type mice (Fig. 1B).

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Figure 1. Spontaneous increase of eosinophils in the BALF of c-maf-Tg mice. (A) Eosinophils in the BALF were quantified in DiffQuick-stained cytopreparations. A significant spontaneous increase in the eosinophils could be seen in the airways of c-maf-Tg mice (**p=0.0054) (A). By contrast, no significant changes were observed in the lungs of c-maf-Tg mice after OVA sensitization (B). Per sample, 200 cells were analyzed. Results are reported as means ± SEM; n=8 and n=14 in (A) and in (B), respectively.

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Since IL-5 is a key cytokine responsible for eosinophil differentiation and survival 1, 11, we determined in subsequent studies IL-5 levels in the BALF of c-maf-Tg and control mice. It was found that the level of IL-5 in the BALF of PBS-treated c-maf-Tg mice is significantly increased (16.0±8.5 versus 67.7±23.2 pg/ml in wild-type versus c-maf-Tg mice), while the Th1 cytokine IFN-γ was decreased compared to the wild-type littermates (67.4±22.5 versus 28.1±12.2 pg/ml) (Fig. 2A; left upper and lower panel, respectively). However, after OVA sensitization and challenge, IL-5 levels were significantly decreased in the BALF of Tg mice as compared to control mice, whereas IFN-γ levels were not significantly changed (Fig. 2B; right upper and lower panel, respectively).

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Figure 2. Spontaneous increase in IL-5 expression in the lungs of c-maf-Tg mice. Eosinophil differentiation and survival are supported by the Th2 cytokine IL-5. We thus measured IL-5 expression in BALF and found that IL-5 but not IFN-γ is spontaneously increased in the BALF of c-maf-Tg mice as compared to wild-type littermate controls (A; upper and lower panel, respectively; *p=0.049). By contrast, after OVA sensitization and challenge, IL-5 expression was found to be decreased in the lungs of c-maf-Tg mice as compared to wild-type littermates (B; upper panel; *p=0.029). Instead, IFN-γ expression increased after OVA sensitization in the lungs of c-maf-Tg mice. Results are reported as means ± SEM; n=8 and n=14 in (A) and in (B), respectively.

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2.2 Lung CD4+ T cells from c-maf-Tg mice release increased amounts of IL-5 in the presence of IL-4

The above studies on c-Maf overexpression in lung CD4+ T cells are consistent with a model in which c-Maf induces IL-5 production during early Th2 cell differentiation but partly suppresses Th2 activation in fully differentiated lung Th2 cells. To directly test this concept, we set up a novel model system with isolated lung CD4+ T cells. Accordingly, we purified lung CD4+ T cells from wild-type and c-maf-Tg mice and determined cytokine production in the presence of IL-4, IL-2 or both. Purified lung CD4+ T cells were stimulated with antibodies to CD3 and CD28 in the presence or absence of recombinant IL-4, IL-2 or both for 2 and 6 days. CD4+ T cells from c-maf-Tg mice produced higher amounts of IL-4 and IL-5 early, i.e. 2 days after isolation (Fig. 3A and C, respectively), but not late, i.e. after 6 days under the influence of IL-4, as shown in Fig. 3A for IL-4 and Fig. 3D for IL-5. Furthermore, IL-2, when given in combination with IL-4, demonstrated an inhibitory effect on IL-5 production in lung CD4+ T cells isolated from c-maf-Tg mice (Fig. 3C). By contrast, splenic CD4+ T cells released irrelevant amounts of IL-5 (<30 pg/ml; 106 cells/ml) compared to lung CD4+ T cells. In addition, IL-5 could not be up-regulated in splenic CD4+ T cells (Fig. 3B), indicating different transcriptional regulation of the IL-5 gene in splenic and lung CD4+ T cells.

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Figure 3. Lung CD4+ T cells from c-maf-Tg mice produce increased amounts of IL-5 in the presence of IL-4, which is repressed by IL-2. CD4+ T cells from lungs of c-maf-Tg mice (grey bars) produced significantly higher amounts of IL-4 (A) and IL-5 (C) 2 days after isolation (A, *p=0.048; C, *p=0,047; n=5 and n=12, respectively) but not late after 6 days in the presence of IL-4, as compared to CD4+ T cells isolated from wild-type littermates (white bars); (A) and (D) for IL-4 and IL-5, respectively. This early effect on IL-5 production was inhibited by addition of IL-2 to the cell cultures (C; right bars). No regulation of IL-5 was observed in splenic CD4+ T cells (B). Splenic CD4+ T cells were purified as previously reported 12. Results are reported as means ± SEM.

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2.3 Spontaneous activation of IL-5 signaling in the airways of c-maf-Tg mice

We next determined IL-5 signal transduction in c-maf-Tg mice. Since IL-5 receptor signaling results in phosphorylation of Ras, at the transduction domain of the βc chain of the IL-5 receptor (Fig. 4A.1), we performed Ras pull-down assays on CD4-negative cells (CD4) isolated from the lungs of both wild-type and c-maf-Tg mice after saline or OVA sensitization. As shown in Fig. 4A.2, Ras was activated in CD4-negative cells isolated from the lungs of c-maf-Tg mice, as determined by Ras-GTP levels, further suggesting spontaneous activation of IL-5 signal transduction in the lung of c-maf-Tg mice.

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Figure 4. Spontaneously increased activation of IL-5 signal transduction in the lung eosinophils of c-maf-Tg mice. (A.1) The pathway of eosinophil activation by IL-5 signal transduction is shown. Upon binding to its receptor, IL-5 induces Ras activation, which in turn leads to phosphorylation of Raf-1. (A.2) Increased Ras activation and Raf-1 phosphorylation in the lungs of untreated c-maf-Tg mice. Ras activation was measured by Ras activity assay in CD4 lung cells. (A.3) Activation of Raf-1 was determined after immunoprecipitation (I.p.) with phospho-tyrosine antibodies (p-Tyr), followed by Western blot (Wb) analysis with an antibody against Raf-1. (A.5) The same blot was probed with anti-STAT-6 antibodies to detect phospho-STAT-6. The loading control for Raf-1 is shown in (A.4). Double immunofluorescence localization of Raf-1 (B.2) and the eosinophilic MBP (B.1) in the BALF of c-maf-Tg mice indicates that eosinophils express activated Raf-1 in the airways of these mice (see arrows). The lower panels show no staining in cells that were incubated with the corresponding control antiserum or immunoglobulins instead of either anti-Raf-1 (B.4) or anti-MBP (B.3) antibodies.

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Since activated Ras binds to Raf-1 to activate the effector protein ERK-2 (Fig. 4A.1), we then determined Raf-1 expression and activation in c-maf-Tg and wild-type control mice (Fig. 4A.3). Interestingly, we found a spontaneously increased phosphorylation of Raf-1 in the lungs of c-maf-Tg mice as compared to wild-type mice, as shown by immunoprecipitation of total lung proteins and subsequent Western blot analysis for phospho-Raf-1 levels (Fig. 4A.3). In contrast, phospho-STAT-6 levels remained unchanged in the lungs of c-maf-Tg mice as compared to controls (Fig. 4A.5).

2.4 Immuno-localization of Ras activation in eosinophils in the lungs of c-maf-transgenic mice

To understand the cellular source of Ras activation in the lungs of c-maf-Tg mice, we performed double immunostaining of cells obtained from the BALF using antibodies directed against the major basic protein (MBP) of eosinophils and Raf-1. As can be seen, many MBP-positive eosinophils (Fig. 4B.1) in the BALF of c-maf-Tg mice showed colocalization with Raf-1 (Fig. 4B.2), indicating spontaneous activation of IL-5 signaling in eosinophils of c-maf-Tg mice. Negative controls showed no specific staining (Fig. 4B.3, B.4).

2.5 c-maf-Tg mice do not develop AHR

Next, we determined whether selective overexpression of c-Maf in T cells of Tg mice would be sufficient to induce AHR. To address this question, we measured airway reactivity in a series of experiments, using non-invasive plethysmography in unrestrained c-maf-Tg mice and wild-type control mice after different doses of methacholine. Surprisingly, as shown in Fig. 5A and B, OVA-sensitized and challenged c-maf-Tg mice do not develop AHR after methacholine challenge, as compared to wild-type littermate controls (n=5; p=0.0099).

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Figure 5. Lack of AHR in c-maf-Tg mice after OVA sensitization and challenge: different roles of c-Maf and GATA-3. (A) Two representative experiments are summarized after lung plethysmography of c-maf-Tg mice, compared to wild-type mice, performed with the BUXCO non-invasive system. Results are reported as Penh after different doses of methacholine challenge [0, 50, 100 mg/ml after PBS (A) or OVA (B) challenge] (n=5–15 and n=4–5, respectively). A lack of AHR was measured in c-maf-Tg mice after OVA sensitization and challenge as compared to wild-type littermates (p=0.0099). (C) Western blot analysis of c-Maf and GATA-3 expression in the lungs of wild-type mice after PBS or OVA sensitization and challenge in two different genetic strains of mice, BALB/c (upper left panels) and C57BL/6 (lower left panels). Interestingly, c-Maf was not increased both in the lungs of BALB/c and C57BL/6 mice after OVA sensitization and challenge. By contrast, GATA-3 was up-regulated after OVA sensitization in BALB/c mice, but not in the lungs of C57BL/6 mice. On the right hand side, quantification of the Western blot is reported as expression of c-Maf or GATA-3 relative to β-actin. Results are reported as means ± SEM.

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To start to investigate the reasons for the lack of AHR in c-maf-Tg mice, we analyzed c-Maf and GATA-3 expression in the lung of wild-type mice after PBS or OVA sensitization and challenge in two different genetic strains of mice, BALB/c and C57BL/6. Interestingly, c-Maf was not increased in the lungs of both BALB/c and C57BL/6 mice after OVA sensitization and challenge, suggesting that this transcription factor plays no role on the asthmatic phenotype after OVA sensitization and challenge. By contrast, GATA-3 was up-regulated after OVA sensitization in BALB/c mice, indicating that GATA-3 has a fundamental role in the development of asthmatic features in this experimental model, as previously demonstrated 1214. Interestingly, GATA-3 was not found to be up-regulated by OVA sensitization and challenge in the lungs of C57BL/6 mice (Fig. 5C). We therefore reasoned that this genetic background was an ideal tool to study the effect of c-Maf overexpression on AHR and on Th2 cytokine production in the lung, independently from GATA-3.

2.6 IL-13 levels are decreased in the BALF of c-maf-Tg mice

IL-13 is a cytokine produced by Th2 and other cells that is known to induce airway inflammation and hyperresponsiveness 2325. To evaluate the possibility that the lack of AHR in c-maf-Tg mice might be due to changes in IL-13 levels, we next measured IL-13 in BALF and found that the airways of c-maf-Tg mice contain significantly less IL-13 as compared to wild-type littermates, both spontaneously (Fig. 6A; p=0.048) and after OVA challenge (Fig. 6B; n=4). Taken together, these results suggest that lung T cells from c-maf-Tg mice indeed produce lower levels of IL-13 than T cells from wild-type mice.

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Figure 6. Lack of IL-13 in the airways of c-maf-Tg mice. IL-13 is known to control AHR. We therefore measured IL-13 by ELISA in BALF. As shown, IL-13 is decreased in the BALF of c-maf-Tg mice as compared to wild-type (wt) littermates both after PBS (A; *p=0.048) and after OVA (B) challenge. Results are reported as means ± SEM; n=5 and n=4 in (A) and in (B), respectively.

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2.7 IL-2-dependent decrease of IL-13 cytokine production by lung CD4+ T cells from c-maf-Tg mice in the presence of IL-4 and IL-2

In contrast to IL-4 and IL-5, we found that CD4+ T cells from c-maf-Tg mice failed to produce IL-13 in the presence of IL-4 after 2 days of culture, as compared to lung CD4+ T cells isolated from wild-type mice (Fig. 7C). Taken together, these data suggest a stage-specific effect of c-Maf overexpression on Th2 cytokine production by lung CD4+ T cells. In particular, overexpression of c-Maf drives first the expression of IL-4 (Fig. 3A); then, IL-4 and c-Maf induce IL-5 in lung CD4+ T cells (Fig. 3C). Finally, throughout the entire Th2 development of lung CD4+ T cells, c-Maf together with IL-2 suppresses IL-13 production (Fig. 7C, D). Interestingly, c-Maf could also suppress IL-13 production by splenic CD4+ T cells at an early stage (day 2) in the presence of stimuli inducing IL-2, such as anti-CD28 antibody treatment (Fig. 7B).

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Figure 7. Lung CD4+ T cells from c-maf-Tg mice release decreased amounts of IL-13 in the presence of IL-2. IL-13 production by lung CD4+ T cells from c-maf-Tg mice was significantly inhibited 2 days after isolation (C) in the presence of IL-2 (C; **p=0.011) and IL-2 + IL-4 (*p=0.046) as compared to wild-type mice, as shown by ELISA. Similar inhibition of IL-13 production was observed at day 2 in splenic CD4+ T cells isolated from c-maf-Tg mice after costimulatory signals (anti-CD28 antibody treatment) and in the presence of IL-4 (B). By contrast, the CD4+ T cells isolated from wild-type littermates released increased IL-13 in the presence of IL-2 (C; *p=0.014). The decrease of IL-13 by lung CD4+ cells isolated from c-maf-Tg mice was not due to cell death as shown by a similar apoptosis rate (Annexinhigh, Propidium iodidelow) at day 6 in lung CD4+ cells isolated from both wild-type and c-maf-Tg mice (A). Results are reported as means ± SEM; n=5 and n=4 for wild-type and c-maf-Tg mice, respectively.

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2.8 Retroviral overexpression of GATA-3 reverses c-Maf-mediated down-regulation of IL-13 in lung CD4+ cells

We next asked the question whether c-Maf directly down-regulates IL-13 in differentiating Th2 cells and whether this effect could be counteracted by GATA-3. Accordingly, we retrovirally infected primary lung CD4+ T cells isolated from C57BL/6 mice either with a control retrovirus (RV) or with a retrovirus overexpressing c-Maf (c-maf-RV) 26, under Th2-skewing conditions as described above. As shown in Fig. 8A, overexpression of c-Maf in lung CD4+ T cells, in the absence of GATA-3 overexpression, led to down-regulation of IL-13 production both at day 2 and at day 6 (Fig. 8A; left and middle panel, respectively). At day 6, cells were re-challenged overnight only with anti-CD3 antibody, and also under these conditions, c-Maf down-regulated IL-13 in the absence of GATA-3 overexpression (Fig. 8A; right panel). To demonstrate that GATA-3 counteracted c-Maf-induced IL-13 down-regulation, we co-infected lung CD4+ cells with an RV expressing GATA-3. As shown in Fig. 8B, co-infection of lung CD4+ cells with GATA-3 led to an up-regulation of IL-13 production in cells overexpressing c-Maf. These data indicate a direct role of c-Maf in IL-13 production that can be counteracted by GATA-3 overexpression.

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Figure 8. Infection of lung CD4+ T cells with an RV overexpressing GATA-3 inhibits c-Maf-induced down-regulation of IL-13 production. Lung CD4+ T cells were infected either with a control RV or a c-Maf-expressing RV (c-maf-RV) or a GATA-3-expressing RV (GATA-3-RV) or both for 2 or 6 days in the presence of IL-2 and IL-4 (Th2-skewing conditions). As shown in (A), c-Maf overexpression in lung CD4+ T cells led to down-regulation of IL-13 in these cells at days 2, 6 (*p=0.05) and 7. Cotransfection of lung CD4+ T cells with GATA-3-RV inhibited c-Maf-mediated down-regulation of IL-13. Supernatants were collected at the indicated time points, frozen and subsequently analyzed for IL-13 content by ELISA. Results are reported as means ± SEM (two experiments performed in duplicate).

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

c-Maf is a transcription factor expressed in Th2 cells that drives the transcription of IL-4 but not IL-5 and IL-13 in peripheral lymph nodes 1517. In addition, c-Maf is up-regulated in asthmatic airways in humans 21, 22. We therefore used mice overexpressing c-Maf in CD4+ T cells to study the functional role of c-Maf in asthma. In this study, we demonstrate a stage-specific effect of c-Maf overexpression on Th2 cytokine production by lung CD4+ T cells that differed from those previously described for peripheral T cells in lymph nodes 1517. In particular, overexpression of c-Maf resulted in suppression of IL-13 production in lung CD4+ T cells in the presence of IL-2 at days 2 and 6 after isolation, thereby explaining the lack of AHR observed in c-maf-Tg mice after OVA sensitization and challenge.

By contrast, our data indicate that c-Maf is important for eosinophil differentiation and activation in the lung via its effects on IL-5 production by lung CD4+ T cells selectively at the early stage of the Th2 differentiation (day 2). This is consistent with the increased number and activation state of eosinophils through IL-5 receptor signaling in the airways of c-maf-Tg mice. The interactions of eosinophils and bronchial epithelial cells are important for the development of asthma and are mediated by chemokines like cationic granule proteins, MBP, eosinophilic cationic protein (ECP) derived from eosinophils, and eotaxin derived from bronchial epithelial cells after challenge with IL-13 4, 5, 2729. However, upon OVA sensitization, c-maf-Tg mice showed in fact reduced IL-5 production as compared to wild-type mice, suggesting stage-specific effects of c-Maf overexpression on IL-5 and Th2 cytokine production by lung T cells. This concept was confirmed by studies on isolated CD4+ T lymphocytes from the lungs of c-maf-Tg mice, showing that overexpression of c-Maf augments IL-5 production early, but not late, under Th2-skewing conditions.

IL-13 is a cytokine known to induce airway inflammation, AHR and mucus production 24, 25, 2830. Activation of both IL-13 and IL-4 membrane-bound heterodimeric receptors induces receptor molecule dimerization and activation of the Janus family members of inner membrane-bound tyrosine kinases, which then phosphorylate and initiate the nuclear transport of STAT-6 transcription factors 9. However, studies in murine models of asthma have indicated that IL-13 rather than IL-4 appears to be critical for the development of allergic airway inflammation, hyperresponsiveness and matrix remodeling in asthma 3035. In the present study, we observed that overexpression of c-Maf, in the absence of a coincident overexpression of GATA-3, but in the presence of IL-2, down-regulates IL-13 but not IL-4 production in developing and polarized lung CD4+ T cells (Fig. 7C, D and Fig. 3A, respectively). Interestingly, we found that overexpression of c-Maf also suppressed IL-13 production of developing splenic Th2 cells (day 2), and also in these cells, the suppression of IL-13 was IL-2 dependent. These results are consistent with previous studies indicating a different role of c-Maf in IL-4 and IL-13 production 36. In addition, the dependence on IL-2 for IL-13 suppression is compatible with a model proposed by Shevach et al., in which CD4+CD25+ T cells present in our cell culture system mediate the suppression of CD4+CD25 T cells following production of IL-2 by the responder cells, resulting in both the expansion of the CD4+CD25+ T cells and the induction of their suppressor function 37. Additional experiments are required to verify this possibility in our cell culture. Our data in lung CD4+ T cell culture systems suggest that overexpression of c-Maf causes, in the presence of IL-4 (which is overproduced in peripheral lymph nodes of these mice), IL-5 production during early Th2 T cell differentiation in the lung. However, overexpression of c-Maf during later stages of Th2 T cell differentiation blocks IL-5 and IL-13 production by lung CD4+ T cells. Consistently, IL-13 and IL-5 levels in the BALF of mice overexpressing c-Maf were significantly suppressed as compared to wild-type mice after OVA sensitization and challenge. To start to elucidate the mechanism by which c-Maf induces IL-5 expression at the early stage of lung Th2 cell differentiation and suppresses IL-13 production in lung CD4+ T cells, we analyzed GATA-3 and c-Maf expression in the lungs of wild-type mice with different genetic backgrounds, before and after OVA sensitization. We found that c-Maf is not increased in the lungs after OVA sensitization in mice of both genetic backgrounds. By contrast, GATA-3 is increased exclusively in the lungs of BALB/c mice after OVA sensitization. Therefore, the C57BL/6 genetic background was an ideal experimental system to study the effect of c-Maf in asthma independently from GATA-3 overexpression. In addition, we found that simultaneous overexpression of GATA-3 in lung CD4+ T cells reversed c-Maf-repressed expression of IL-13. These results demonstrate specific regulatory effects of c-Maf on IL-5 and IL-13 production, independently from GATA-3, in lung CD4+ T cells that differ from its known effects in peripheral T cells 1517.

In summary, these results demonstrate that c-Maf regulates IL-5 production in lung CD4+ T cells. Furthermore, we found that c-Maf requires IL-2 to suppress IL-13 production in developing and polarized lung Th2 effector cells, thereby explaining the finding that overexpression of c-Maf in Tg mice does not cause AHR as a hallmark feature of asthma. In contrast to GATA-3, c-Maf with IL-2 can inhibit IL-13 production, thereby counteracting the full development of the Th2 phenotype in lung T cells.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Allergen sensitization and challenge protocol in wild-type and T cell-specific c-maf-Tg mice

C57BL/6 mice (6–8 weeks old) received an intraperitoneal (i.p.) injection of 100 µg OVA (Sigma, Deisenhofen, Germany) complexed with alum on days 0 and 14. On days 25, 26 and 27, mice received OVA aerosol (50 mg OVA/ml PBS). Control animals received i.p. saline complexed with alum on days 0 and 14 and a saline aerosol on days 25, 26 and 27, as described 12, 38.

4.2 Assessment of airway reactivity by body plethysmography

Airway responsiveness in mice was evaluated by using a non-invasive whole-body plethysmograph 24 h after the last OVA aerosol exposure (model PLY 3211; Buxco Electronics, Inc.) as described 12, 38. Briefly, mice were exposed to 0, 50, 100 mg/ml methacholine for 3 min; afterwards, the enhanced pause (Penh) responses of different groups of mice (n=8) in the following 5 min were measured. Ten experiments were performed. Data are reported as the mean value of thirty values of Penh taken every 10 s (for 5 min) after administration of methacholine of two representative experiments. Data are expressed as mean values (Penh) ± SEM.

4.3 Collection and analysis of the BALF

At 24 h after the last intranasal challenge with either OVA or saline, a bronchial alveolar lavage of the right lung was performed four times with 0.75 ml saline. Total BALF was centrifuged, supernatants were collected and stored at –80°C for subsequent cytokine ELISA analysis, and BALF cell pellets were resuspended in 1 ml of PBS. Eosinophils were detected by staining cytospins according to DiffQuick (Dade Behring, Marburg, Germany). Cell differentiation analysis was performed by counting at least 200 cells with an Olympus microscope (Neuss, Germany) at 400× magnification.

4.4 Immunohistochemistry

For immunocytochemical identification of double-positive cells in the BALF, cytospins were fixed in ice-cold acetone, washed twice in PBS, permeabilized for 4 min in permeabilization buffer (0.2% Triton X-100 in PBS) and washed twice in TBS buffer (0.05 M Tris hydrochloride). After 1 h of incubation in blocking buffer (3% BSA, 0.05% Tween-20 in TBS), the rabbit polyclonal anti-Raf-1 antibody (Santa Cruz Biotechnology, Heidelberg, Germany) was applied (1:100) in blocking buffer and incubated overnight at 4°C. Detection of positive cells was performed as previously described by incubation with a corresponding biotinylated secondary antibody, followed by exposure to a Cy2-streptavidine (1:500 in PBS) cytochrome solution 12. Next, mouse monoclonal anti-eosinophilic MBP antibody (Chemicon, Hofheim/Ts., Germany) was applied in antibody diluent (1:100; DAKO, Carpinteria, CA) and incubated overnight at 4°C. Detection of positive cells was performed as described above by using a Cy3-streptavidine (1:500 in PBS) solution as cytochrome solution detection system 12. Negative control cytospins were incubated with either rabbit normal serum (R&D, Wiesbaden, Germany) as negative control for Raf-1 or mouse IgG (Santa Cruz, Heidelberg, Germany) antibodies as control for MBP. Sections were analyzed with a Zeiss microscope (Axioscope 2 mot plus, Jena, Germany).

4.5 Protein extraction and Western blot analysis

Total lung proteins were extracted and 50 µg of this protein was separated by 15% SDS-PAGE, as described 12. Specific proteins were detected with polyclonal rabbit anti-STAT-6 or anti-Raf-1 antibodies or with mouse monoclonal anti-GATA-3 antibody (Santa Cruz, Heidelberg, Germany). c-Maf protein was detected by using a polyclonal rabbit anti-c-Maf antibody (1 mg/ml; kindly provided by Prof. I-Cheng Ho). Specific binding was visualized with the ECL Western blotting analysis system after 1 h of incubation with the correspondent secondary horseradish peroxidase-conjugated antibody (Amersham, Eschwege, Germany). Quantification was performed on developed biomax light films (Kodak, Germany) by using the BioDOC Analyze software program (Biometra, Göttingen, Germany).

4.6 Immunoprecipitation

For immunoprecipitation, 250 µg of total lung proteins were precleared with 1 µg of appropriate IgG, according to the primary antibody, and 20 µl A/G plus agarose (Santa Cruz, Heidelberg, Germany) for 30 min at 4°C. After centrifugation at 2,500 rpm for 5 min, the supernatant was collected and incubated with 2 µg primary monoclonal antibody against phospho-tyrosine (Santa Cruz, Heidelberg, Germany) for 1 h at 4°C, followed by addition of 20 µl A/G plus agarose. Immunoprecipitation was completed by incubation at 4°C overnight. Western blot was performed as described above with 12 µl of phospho-tyrosine-immunoprecipitated proteins and detected with specific rabbit polyclonal antibodies against STAT-6 or Raf-1 (Santa Cruz, Heidelberg, Germany). Specific binding was visualized with the ECL Western blotting analysis system as described above.

4.7 Isolation and culture of lung CD4+ and CD4 T cells

Lungs were removed and transported on ice in RPMI medium (Biochrom, Berlin, Germany). Tissue pieces were suspended in Dulbecco's PBS containing 300 U/ml collagenase type II (Worthington, Lakewood, NJ) and 0.001% DNase (Roche, Basel, Switzerland). The lung digest was filtered, centrifuged, and erythrocytes were removed from cell suspensions by hypotonic lysis in ammonium chloride and potassium chloride (ACK) buffer. Lung CD4+ T cells were purified by using anti-CD4 beads from Dynal [DYNABEADS Mouse, CD4 (L3T4); Dynal, Hamburg, Germany]. The resulting CD4 T cells were saved and used for the Ras activity assay. Lung CD4+ T cells were cultured in RPMI complete medium in anti-CD3 (5 µg/ml, Santa Cruz) antibody-coated wells in the presence of anti-CD28 antibody (2 µg/ml), IL-2 (10 ng/ml) or IL-4 (10 ng/ml) or both for 2 days. At this time point, supernatants were frozen and cells were incubated for four additional days in the presence of IL-4 or IL-2 or both as indicated (day 6).

4.8 Measurements of lung CD4+ T cell apoptosis

Lung CD4+ T cells were cultured in RPMI medium in anti-CD3 (5 µg/ml, Santa Cruz) antibody-coated wells in the presence of anti-CD28 antibody (2 µg/ml) for 2 days. At this time point and at day 6 after lung CD4+ T cell isolation, apoptotic cells were detected by FACS analysis after staining with annexin V and propidium iodide, according to the manufacturer's protocol (Annexin V-FITC apoptosis detection Kit II; PharMingen, San Diego, CA).

4.9 Ras activity assay

Lung CD4 cells were then analyzed for GTP binding activity of Ras by using the Ras Activation Assay Kit according to the manufacturer's instructions (Upstate Biotechnology, Lake Placid, NY). Briefly, the cell pellet of 5×105 CD4 lung cells was lysed for 10 min by 1× lysis/wash buffer (MLB) containing protease inhibitors. As positive control, 5×105 Jurkat cells that had been stimulated for 18 h with 20 ng/ml PMA and 1 µg/ml Ca-ionophore A23187 in RPMI complete medium were incubated for 15 min at 30°C with γ-GTPS as substrate. The reaction was stopped with 5 µM EDTA before the cells were lysed with 200 µl 1× MLB. Cell lysates were incubated with 0.1 M MgCl2. The cell homogenate was then treated with 4 µl glutathione-agarose (50% w/v suspension; BD PharMingen, München, Germany) for 2 min for preclearing and centrifuged for 5 min at 2,000 rpm at 4°C. The supernatant was collected, 10 µl Raf-1 Ras-binding domain (Raf-1-RBD) was added, and incubation was performed for 30 min at 4°C. After centrifugation, the antibody-protein-complex was washed with 1× MLB buffer and then resuspended in 30 µl sterile water and 5 µl SDS loading buffer. After denaturation for 5 min, the probes were loaded onto a 15% polyacrylamide gel. Western blot analysis was then performed with 1 µg/ml of a monoclonal anti-Ras primary antibody (anti-ras clone 10, 1 µg/µl). Positive Ras activity was detected with a chemiluminescence system (ECL, Amersham, Eschwege, Germany).

4.10 ELISA

Mouse IL-5, IL-4 and IFN-γ were detected using specific sandwich ELISA systems (OptEIA; PharMingen, Becton Dickinson, Heidelberg, Germany). IL-13 was detected by using a mouse-specific ELISA kit (Duo set-IL-13; R&D Systems, Wiesbaden, Germany).

4.11 Transfection of CD4+ T cells with retroviral expression vectors for c-Maf or GATA-3

For retroviral transfer experiments, lung CD4+ T cells were purified using mouse anti-CD4+ bead-conjugated monoclonal antibodies (Miltenyi, Bergisch Gladbach, Germany) in a multiparameter magnetic sorter system (MACS; Miltenyi, Bergisch Gladbach, Germany). The resulting CD4+ cells had 97% purity. The Phoenix-Eco packaging cell line, obtained from G. Nolan 26, was used to produce high amounts of RV to infect lung CD4+ T cells. Briefly, Phoenix-Eco cells were transfected with the green fluorescent protein (GFP)-expressing vector (GFP-RV) and its derivates. The GFP-RV bistronic vector was constructed by inserting the encephalomoycarditits virus ribosomal entry sequence (IRES) and the GFP allele into the MSCV2.2 retroviral vector. The cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing 10% FCS. For transfection experiments, semi-confluent Phoenix-Eco cells were then incubated for 15 min with 50 µM chloroquine (Sigma, Deisenhofen, Germany) in the same medium as used for the target primary lung T cells before transfection. For transfection, 1 ml transfection buffer (50 mM HEPES pH 7.5, 10 mM KCl, 12 mM dextrose, 250 mM NaCl, 1.5 mM Na2HPO4), 0.14 mM CaCl2 and 10 µg retroviral DNA were used. This suspension was then added to the cell supernatant overnight at 37°C and 5% CO2. The uptake of the retroviral DNA was indicated by GFP expression in the target Phoenix-Eco cells. After removal of the transfection medium, cells were incubated for two additional days in RPMI 1640, used later on for the target lung CD4+ T cells. The supernatants containing the retrovirus overexpressing c-Maf or GATA-3 (kindly provided by Prof. I.-Cheng Ho and Prof. K. Murphy, respectively) or the corresponding control virus were collected and used for transfection of lung CD4+ T cells.

4.12 Infection of lung CD4+ T cells with GATA-3 and c-Maf RV

Lung CD4+ T cells from wild-type C57BL/6 mice were isolated and challenged with anti-CD3/anti-CD28 antibodies as described above. The day after, they were infected with either an RV overexpressing c-Maf or GATA-3 or both in the presence of polybrene (20 µg/ml; Sigma Aldrich, Germany). Control cell cultures were incubated with an empty RV instead. After 48 h (day 0), the cell supernatant was washed away and substituted with fresh medium containing IL-2 and IL-4, as described above. At day 2, supernatants were removed, frozen and later analyzed for IL-13 production by ELISA. Lung CD4+ T cells were further incubated for 4 days in the presence of IL-2 and IL-4 (day 6). At day 6, supernatants were analyzed for IL-13 production, and cells were washed and incubated overnight in the presence of soluble anti-CD3/anti-CD28 antibodies (both at 1 µg/ml). The day after, supernatants were analyzed for IL-13 content.

4.13 Statistical analysis

Differences were evaluated for significance (*p⩽0.05; **p⩽0.01) by the Student's two-tailed t-test for independent events (Excel, PC). Data are given as mean values ± SEM.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The authors thank Profs. J. M. Drazen and M. F. Neurath for critical reading of the manuscript. In addition, the authors would like to thank K. Sternemann for her technical help. This work has been supported by a DFG (Deutsche Forschungsgemeinschaft) grant to S. Finotto (FI817/2–1) and the SFB548 of the DFG.

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