Evidence for suppressed activity of the transcription factor NFAT1 at its proximal binding element P0 in the IL-4 promoter associated with enhanced IL-4 gene transcription in T cells of atopic patients
Eddy A. Wierenga,
Monika Walchner1,
Gerold Kick1,
Martien L. Kapsenberg,
Elisabeth H. Weiss2 and
Gerald Messer1
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
1 Department of Dermatology and
2 Institute for Anthropology and Human Genetics, Ludwig Maximilians University, 80337 Munich, Germany
Correspondence to:
E. A. Wierenga
 |
Abstract
|
---|
Allergen-specific T cells in atopic patients are polarized IL-4-producing Th2 cells, promoting IgE synthesis by B cells. The molecular basis for increased IL-4 gene expression in atopy is not fully understood. IL-4 gene regulation in general involves the nuclear factor of activated T cells (NFAT) family of transcription factors, of which NFAT1 and NFAT2 are most prominent in peripheral T cells. Recently, a unique inhibitory role of NFAT1 in IL-4 gene control was shown in the mouse. In a series of electrophoretic mobility shift assays with protein extracts of highly polarized Th2 clones from atopics and Th1 clones from controls we compared DNA-binding activities at the two NFAT-binding elements P0 and P1 of the crucial proximal human IL-4 promoter. At the most proximal P0 site, NFAT-containing complexes devoid of NFAT2 were readily inducible in the Th1 clones, but hardly or not in the Th2 clones. In contrast, both in Th1 and Th2 clones NFAT-containing complexes were strongly inducible at the P1 site, consisting of NFAT2 and a P0-competable NFAT activity, without apparent differences between Th1 and Th2 clones. Like in Th2 clones, suppressed NFATP0 complex formation was observed also at the polyclonal level in peripheral blood mononuclear cells (PBMC) of three of five severe atopic dermatitis patients with strongly elevated serum IgE levels, but not in control PBMC. These findings suggest that high-level IL-4 production in atopic Th2 cells is associated with selective reduction of suppressive NFAT1 activity at the IL-4 P0 element and that some patients with this multifactorial disease may have a putative systemic disorder at this level.
Keywords: atopic dermatitis, electrophoretic mobility shift assay, nuclear factor of activated T cells Th1/Th2 cells
 |
Introduction
|
---|
Specific immune responses to exogenous pathogens are largely orchestrated by Th cell-derived cytokines that regulate the functions of the various types of immune cells involved (1). Th cells can be divided into distinct functional subsets based on their cytokine secretion profile (2). Extreme subsets are Th1 cells, which secrete IFN-
but not IL-4, and Th2 cells, which do not secrete IFN-
but high levels of IL-4 (3,4). Animal model studies have indicated that the secretion ratio of IL-4 and IFN-
, in particular, determines the nature of the response and thus may be decisive for protection or progression of infection (5,6). Also non-infectious immune diseases may result from imbalanced T cell cytokine production. In fact, an increasing numbers of human immune diseases are known to result from T cell responses with an inappropriate cytokine secretion profiles (79). Atopic allergy, for example, is associated with excessive, polarized Th2 cell reactivity with selective environmental allergens (1012), in turn responsible for increased IL-4-induced IgE production (13). As for several other immune diseases, selective alteration of the ratio of IL-4 and IFN-
production may be beneficial. Precise knowledge of the mechanisms that regulate the production of these cytokines is a prerequisite to facilitate the development of such therapeutic strategies.
The restricted cytokine profiles of highly polarized Th1 and Th2 cells are evident also at the level of mRNA production, indicating different regulation of transcription. An important question therefore is which mechanisms are responsible for the tissue-specific cytokine gene expression patterns, and how these mechanisms differ in Th1 and Th2 cells.
The nuclear factor of activated T cells (NFAT) family of transcription factors has an essential role in the transcriptional control of several cytokine genes (14), including the IL-4 gene. The mouse and human IL-4 promoter regions share five potential NFAT-binding elements, referred to as P0 to P4 (15,16), with only minimal interspecies variation with respect to their sequence (17), location and orientation (14). Of this growing family (18) of cyclosporin-sensitive (19) transcription factors, NFAT1 (also referred to as NFATp) and NFAT2 (NFATc) are most prominent in peripheral T cells, both in mice and humans (18,2022). Quite unexpectedly, recent reports on NFAT1-deficient mice indicated that NFAT1 has a unique inhibitory, rather than a stimulatory effect on the transcription of various cytokine genes in vitro, including IL-4 (23,24), probably explaining the enhanced immune responses in these mice (21,23).
Although largely involved, it is not likely that NFAT1 levels are decisive for Th2-specific IL-4 gene expression, because this factor is expressed both in Th1 and Th2 cells (25,26). More conceivably, the DNA-binding activity of NFAT1 may be differentially regulated by mechanisms that affect its DNA-binding affinity or the stability of the complexes in which it participates. Indeed, such cooperative binding has been shown for the activator protein (AP)-1 family of transcription factors (14,27) at NFAT sites in the IL-2 and IL-4 promoters that have adjacent AP-1 sites, but this activity was not essentially different in Th1 and Th2 cells. More interesting factors in this regard are probably those that show restricted tissue distribution themselves, such as the recently described Th2-specific transcription factors c-MAF (28) and GATA-3 (29), both promoting IL-4 gene expression when ectopically expressed in Th1 cells or B cells. As for the common transcription factor AP-1, the interference of such Th2- or Th1-specific factors with the IL-4 transcriptional activity of NFAT1 may depend on the promoter sequences flanking the actual NFAT-binding sites and, thus, may affect selective rather than all NFAT sites in the IL-4 promoter.
The proximal IL-4 promoter, including the P0 and P1 NFAT-binding elements, confers considerable inducible and tissue-specific expression of the IL-4 gene (15,27,28,30). In the search for differentially regulated NFAT activities at the IL-4 promoter in human Th1 and Th2 cells, we have adopted an experimental approach by conducting a series of comparative gel shift analyses with protein extracts of highly polarized Th1 and Th2 clones of non-atopic and atopic origin, respectively, and oligonucleotides containing the individual P0 or P1 NFAT sites of the proximal human IL-4 promoter. Our data shows that NFAT-containing complex formation at the IL-4 P0 element is devoid of NFAT2 and selectively suppressed in IL-4-producing Th2 clones of atopic patients. Likewise, suppressed NFATP0 complex formation could also be observed at the polyclonal level in peripheral blood mononuclear cells (PBMC) of three of an initial collective of five severe atopic dermatitis patients with strongly elevated serum IgE levels, but not in control PBMC. Given the prominent involvement of NFAT1 and NFAT2 in IL-4 gene regulation in peripheral T cells, the lack of NFAT2 binding at the IL-4 P0 site, and the newly discovered inhibitory role of NFAT1 in IL-4 gene transcription (24), our present results suggest that the mechanisms underlying high IL-4 production in atopic Th2 clones involve selective squelching of NFAT1 activity from the IL-4 P0 element. The data further allows us to speculate that systemic suppression of this inhibitory NFAT1 activity as observed in some severe atopic dermatitis patients may reflect one of the many immunologic phenotypes of this multifactorial disease.
 |
Methods
|
---|
Cells and donors
Housedust mite (Dermatophagoides pteronyssinus)-specific Th1 clones MBE.AA16 and MBE.AA42 were generated as described (31) from the peripheral blood of a nonatopic, healthy individual with a normal serum IgE level (<100 IU/ml), devoid of detectable housedust mite-specific IgE. Cat (Felis domesticus)-specific Th2 clones PBG2 and PBE1 were generated from the peripheral blood of atopic, cat-allergic patients with elevated serum IgE levels, including high titers of cat-specific IgE (32). At the protein level, these clones stably secreted IL-4 without IFN-
(Th2 clones) or IFN-
without IL-4 (Th1 clones). Absence of IL-4 mRNA expression in the activated Th1 clones was confirmed by Northern blot analysis as described (33,34), using a 318 bp human IL-4 cDNA probe. PBMC were isolated from fresh heparinized peripheral blood by density centrifugation. Donors in this study included (i) healthy control individuals with normal serum IgE levels (<100 IU/ml), (ii) severe atopic dermatitis patients, diagnosed according to the criteria described by Hanifin and Rajka (35) and selected for strongly elevated serum IgE levels: patient 1 (35,500 IU/ml), patient 2 (13,000 IU/ml), patient 3 (18,000 IU/ml), and two others with IgE titers of 5000 and 25,000 IU/ml, and (iii) patients suffering from psoriasis vulgaris (PV) or systemic sclerosis (SSc), representing a non-atopic control group with chronic T cell reactivity but normal serum IgE titers. All donors gave informed consent.
Cell culture
Cultures were performed in IMDM (Biowhittaker, Walkersville, MD) with 10% complement-inactivated human serum (Biowhittaker) and gentamycin (80 µg/ml; Duchefa, Haarlem, The Netherlands). T cell clones were maintained by bi-weekly re-stimulation, as described (31). Preceding experiments, cells were rested for 12 days and their exclusive cytokine profiles were confirmed by ELISA, as described (36). PBMC were analyzed immediately after isolation, combining a newly diagnosed severe atopic dermatitis patient with representatives from the control groups.
Antibodies and reagents
T cell clones and PBMC were stimulated with CD3 (CLBT3/4E) plus CD28 (CLB-28/1) mAb (1:1000; Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). CLB-T3/4E is an IgE-switch variant, which efficiently activates T cells without prior cross-linking (37). Antibodies used for supershift experiments were rabbit polyclonal anti-NFAT1 (Upstate Biotechnology, Lake Placid, NY) and mouse monoclonal anti-NFAT2 (MA3-024; Affinity Bio-Reagents, Golden, CO). As evident from the present study, the anti-NFAT1 serum cross-reacts at least with NFAT2 and will further be referred to as anti-NFAT. The specific anti-NFAT2 antibody did not supershift but prevented the formation of NFAT2-DNA complexes.
Protein extraction and electrophoretic mobility shift assay (EMSA)
Nuclear (36) and whole-cell (38) protein extracts were prepared as described before and the protein concentrations were determined by a Bradford microassay (BioRad, Munich, Germany). The samples were aliquoted and stored at 80°C. DNA-binding reactions and EMSA were performed as described (38). Supershift antibodies were added 5 min after (anti-NFAT) or 15 min prior to (anti-NFAT2) the start of the binding reaction. Unlabeled competitor oligonucleotides were added prior to the labeled probe.
All reactions were adjusted to a final volume of 20 µl with distilled water and incubated for 30 min at room temperature. The whole sample was then loaded on a 4% native polyacrylamide gel in 0.5xTrisborateEDTA buffer. ProteinDNA complexes were visualized by autoradiography using Kodak XAR5 films.
Oligonucleotides
NFAT-binding activities at the IL-4 P0 and IL-4 P1 elements were analyzed using the following double-stranded DNA oligonucleotides (the AT/ATTTCCNNT consensus sequences are underlined): P0 (72 to 40) 5'-TTCCAATGTAAACTC-ATTTTCCCTCGGTTTCAG-3' and P1 (88 to 62) 5'-CTGGTGTAACGAAAATTTCCAATGTAA-3', both obtained from the Gene Center (Ludwig Maximilians University, Munich, Germany). Equal protein loading was checked by testing for binding of the constitutive transcription factor SP-1 using the herpes simplex virus SP-1 site containing oligonucleotide 5'-CCGGCCCCGCCCATCCCCGGCCCCGCCCATCC-3' (Gene Center). Probes were 32P-labeled with T4 polynucleotide kinase (Boehringer, Mannheim, Germany).
Activation-induced fragmentation of chromosomal DNA
To monitor activation-induced apoptosis in the Th1 and Th2 clones, 5x106 cells were stimulated for 4 h with CD3 plus CD28 mAb and analyzed for DNA fragmentation as described (39). Fragmented DNA was separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.
 |
Results
|
---|
Lack of IL-4 mRNA induction in Th1 clones
To confirm strongly inducible (Th2 clones) and absence (Th1 clones) of IL-4 mRNA production, Northern blot analysis was performed with total RNA, isolated 6 h after stimulation with CD3 plus CD28 mAb, detecting IL-4 mRNA with a
-32P-labeled IL-4-specific cDNA probe. Shown in Fig. 1
(upper panel) is the strongly inducible IL-4 mRNA accumulation in the activated Th2 clones and the lack of IL-4 transcripts in the stimulated Th1 clones, validating their classification as genuine Th1 and Th2 clones. Activation of all clones was confirmed by strong induction of [3H]thymidine incorporation. Ethidium bromide staining of total RNA (lower panel) indicated the loading of comparable amounts of RNA per lane.

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 1. IL-4 mRNA expression in Th1 and Th2 clones. Rested Th1 (MBE.AA16 and MBE.AA42) and Th2 clones (PBE1 and PBG2) were cultured for 6 h in the absence () or presence (+) of CD3 plus CD28 mAb. IL-4 mRNA was detected by Northern blot analysis using an IL-4-specific cDNA probe. Ethidium bromide staining indicates equal loading of RNA in all lanes.
|
|
Reduced NFAT-binding intensity at the IL-4 P0 site in Th2 clones
The NFAT-binding kinetics at the IL-4 P0 site in these Th1 and Th2 clones were analyzed by EMSA, initially with whole-cell protein extracts obtained at different time points after stimulation with CD3 plus CD28 mAb. Clones were rested for 12 days and all tested in parallel. In rested as well as stimulated cells (Fig. 2A
), a pre-existing (0 h) and in Th1 cells slightly further inducible P0-binding activity (closed arrowhead) was of much higher intensity in the whole-cell Th1 extracts (right panel), as compared to the Th2 extracts (left panel). Both the high-intensity P0-binding complexes in the Th1 extracts and the low-intensity P0-binding complexes in the Th2 extracts were fully recognized by an NFAT1/NFAT2 cross-reactive antiserum (Fig. 2B
), indicating the prominent NFAT participation in this differentially regulated complex. P0-binding intensities were optimal after 15 min, after which they gradually declined to below baseline levels within 4 h (Fig. 2A
). Testing the same whole-cell extracts for the NFAT-binding kinetics at the P1 site gave no indication for a generally reduced NFAT availability in the Th2 clones. The formation of NFAT-containing complexes (Fig. 3A
, arrowhead), which could be supershifted with the common anti-NFAT serum (Fig. 3B
), was similar (PBG2) to slightly stronger (PBE1) in the Th2 extracts as compared to the Th1 extracts, suggesting enhanced rather than reduced NFAT availability in Th2 clones.


View larger version (157K):
[in this window]
[in a new window]
|
Fig. 2. Different intensity of NFAT binding activity at the IL-4 P0 site in Th1 and Th2 clones. (A) Rested Th1 and Th2 clones were stimulated with CD3 plus CD28 mAb for the time periods indicated, after which whole-cell protein extracts were analyzed by EMSA for transcription factor activities at the 32P-labeled double-stranded DNA IL-4 P0 oligonucleotide. The closed arrowhead indicates a reduced P0-binding intensity in Th2 clones (left panel) as compared with Th1 clones (right panel), both in rested (0 h) and stimulated cells. After 2 h of stimulation, P0-binding activities decline. The open arrowhead indicates the free DNA probe. (B) Addition (+) of a common anti-NFAT serum in the P0-binding reaction (1:50 diluted) indicated the crucial involvement of NFAT in the high-intensity complex formed with Th1 extracts, as well as in the low-intensity complex formed with the Th2 extracts.
|
|
Th1 clones are more sensitive to activation-induced apoptosis
Both at the P0 and the P1 elements, the NFAT-containing and other binding activities declined within 2 h after stimulation, especially in the Th1 extracts (Figs 2A and 3A
). Similarly, the binding activity of the constitutive transcription factor SP-1 at an SP-1-consensus element, tested to control for equal protein input in each binding reaction, tended to decline with time in the Th1 extracts, but remained stable in the Th2 extracts (Fig. 4A
). We therefore tested whether this gradual loss of transcription factor activities could be associated with activation-induced apoptosis, to which both mouse (40,41) and human (42) Th1 cells are more sensitive than Th2 cells. Indeed, light microscopic inspection of 4 h-stimulated (CD3 plus CD28 mAb) cultures revealed that the Th1 clones but not the Th2 clones underwent activation-induced cell fragmentation with appearance of apoptotic bodies (not shown) as described (43). At this time point, concomitant activation-induced fragmentation of chromosomal DNA into the characteristic 180 bp ladder pattern (Fig. 4B
) was much more pronounced in the Th1 clones (Fig. 4B
, lanes 3 and 9), as compared to the Th2 clones (Fig. 4B
, lanes 5 and 11). The Th2 clone PBG2 showed some degree of passive, activation-independent apoptosis, which may occur in long-rested cells (44). To avoid possible interference with the activation-induced apoptotic machinery as much as possible, further experiments were performed with 15 min-stimulated cells.
Further characterization of the reduced P0NFAT activity in nuclear Th2 extracts
The selectively suppressed P0NFAT complex formation in stimulated Th2 clones, as observed in whole-cell extracts, was fully reproducible in nuclear protein extracts, as shown for 15 min-stimulated clone cells (Fig. 5
). Using an NFAT2 (NFATc)-specific mAb, no NFAT2 binding could be identified at the P0 element, neither by titrating the antibody into the P0-binding reactions (not shown), nor by prior pre-incubation of the nuclear extracts with increasing antibody concentrations [up to 10% (v/v) of the binding reaction] (Fig. 6A
), both in Th1 and Th2 extracts. In contrast, the NFAT activity at the P1 site could partially be prevented by pre-incubation of the nuclear extracts with 10% (v/v) of the NFAT2-specific mAb (Fig. 6C
), without obvious quantitative differences between the Th1 and Th2 extracts. Cross-competition experiments with unlabeled oligonucleotides showed that the P0-binding NFAT activity could dose-dependently (not shown) and completely be competed out with 250-fold molar excess of the P1 oligonucleotide (Fig. 6B
, right panel). The inverse experiment showed, however, that the P1-binding NFAT activity could only partially be competed out with 250-fold molar excess of the P0 oligonucleotide (Fig. 6B
, left panel), together suggesting that the P0-binding NFAT activity is part of the P1-binding NFAT activity. In contrast to the full P1-binding NFAT activity, the NFAT2-depleted residual P1-binding activity could completely (Th1) or almost completely (Th2) be competed with excess P0 oligonucleotide (Fig. 6C
), suggesting that the P1-binding NFAT activity consists of NFAT2 and the P0-binding NFAT activity, which therefore most likely is NFAT1.

View larger version (120K):
[in this window]
[in a new window]
|
Fig. 5. Selectively suppressed inducible NFAT binding at the IL-4 P0 site in nuclear Th2 extracts. Rested Th1 and Th2 clones were either (+) or not () stimulated for 15 min with CD3 plus CD28 mAb and nuclear protein extracts were analyzed by EMSA with the IL-4 P0 and P1 probes. Inducible complexes (arrowhead) in Th1 and Th2 extracts are similar at the P1 site but reduced at the P0 site in the Th2 clones, as compared to the Th1 clones.
|
|
Similarly reduced NFATP0 binding in PBMC extracts of atopic patients
Considering the selectively reduced NFAT binding activity at the P0 site in activated Th2 clones and the increased numbers of IL-4-producing T cells in atopic patients (45,46), we extended our studies to the polyclonal level. To this aim we compared the P0NFAT binding activities in nuclear protein extracts of freshly isolated PBMC from atopic eczema patients with strongly elevated serum IgE levels, and, as controls, non-atopic healthy individuals and non-atopic PV or SSc patients with chronic T cell reactivity, but normal serum IgE levels. A specific complex at the P0 probe (Fig. 7A
; closed arrowhead) was up-regulated in control PBMC within 15 min after stimulation with CD3 plus CD28 mAb (Fig. 7A
, lanes 2 and lanes 3), while apparently the same binding activity was already pre-induced in vivo in PBMC of the PV and SSc patients (Fig. 7A
, lanes 6 and lanes 7). Interestingly, this complex was strongly reduced and hardly or not inducible in PBMC of three out of five atopic eczema patients tested (results are shown for patients 1 and 2), while PBMC of the other two atopic eczema patients contained pre-induced activities similar to the psoriasis vulgaris and systemic sclerosis extracts (not shown). Figure 7
(B) shows for nuclear PBMC extracts of atopic eczema patient 3 that this strongly reduced and non-inducible binding activity (arrowhead) is supershifted (doubled arrowhead) by the common NFAT antiserum, suggesting systemically altered regulation of NFAT activity at the IL-4 P0 element in some, but not all atopic dermatitis patients.


View larger version (118K):
[in this window]
[in a new window]
|
Fig. 7. NFAT activity at the IL-4 P0 element in PBMC extracts of atopic eczema patients and non-atopic controls. (A) Nuclear protein extracts were prepared from PBMC of healthy control individuals (controls 1 and 2), atopic eczema patients (AE patients 1 and 2), or patients suffering from PV or SSc, either (+) or not () stimulated with CD3 plus CD28 mAb for 15 min. A NFAT-containing binding activity (closed arrowhead) is inducible in control PBMC, is pre-induced in PV and SSc PBMC, but strongly reduced and hardly inducible in AE PBMC. (B) This reduced activity (closed arrowhead) is supershifted by anti-NFAT (1:50; double arrowhead), as shown for PBMC of AE patient 3.
|
|
 |
Discussion
|
---|
The present comparative study of polarized human Th1 and Th2 clones of non-atopic and atopic origin respectively provides evidence for the involvement of the proximal NFAT site P0 of the IL-4 promoter in the differential transcriptional control of the IL-4 gene. The data suggests that in Th2 clones from atopic patients, the inducible binding activity of an NFAT1-containing transcription factor complex at the IL-4 P0 element is selectively suppressed, despite abundant NFAT1 availability. This reduced NFAT1-containing complex formation at the IL-4 P0 element, at the clonal level associated with strongly inducible IL-4 gene transcription, was also evident at the polyclonal level in PBMC of part of an initial small collective of severe atopic dermatitis patients.
The role of NFAT1 in this P0-mediated differential IL-4 gene control is implicated by different lines of indirect evidence, including the predominant expression of NFAT1 and NFAT2 in peripheral T cells (18), the binding of NFAT1 at the P0 element in the mouse (28), the binding of rNFAT1 but not rNFAT2 at the human P0 element (30), and the lack of detection of NFAT2 binding at the P0 probe in the present study. It should be noted, however, that despite the inability of rNFAT2 to bind the P0 element, some NFAT2 was detected at this site upon stimulation with phorbol myristate acetate and ionomycin (30). The reason for the discrepancy between this and our study is unclear, but may be related to the different mode of stimulation (phorbol myristate acetate/ ionomycin versus anti-CD3/anti-CD28) and the atopic origin of the cells in the present study. The here suggested suppressed NFAT1 binding at the P0 element in Th2 clones would be in line with the recently described inhibitory role of NFAT1 in IL-4 gene transcription (23,24).
The full extent to which the Th1 and Th2 clones in our studies differed in their NFATP0 binding intensity may have been partially masked by the increased susceptibility of the Th1 clones to activation-induced apoptosis, evident especially at later time points after stimulation. Although NFAT family members appeared to be involved in the induction of the apoptotic machinery (47), they may also be sensitive to the more downstream molecular consequences of this process. To limit possible interference we have focused our experiments on an early time point (15 min) after stimulation.
Impaired NFAT1 binding in the Th2 clones seems to selectively occur at the P0 element. Analyses of NFAT binding at the P1 site indicated similar to higher NFAT-binding intensities in the activated Th2 clones, as reported before for mouse Th2 clones (26) and for polyclonal T cells of atopic dermatitis patients (46). Furthermore, in the presence of anti-NFAT2, no apparent differences were noted between Th1 and Th2 clones with respect to the residual NFAT2-depleted and P0-competable NFAT-binding intensities, which as explained above should be NFAT1. The Th2-specific altered regulation of NFAT1 complex formation at the P0 site may, therefore, result from differential expression or activities of other, cooperative transcription factors, specifically interacting with the NFAT1 complexes at the P0 site.
The adjacent downstream sequence from P0 was shown to contain a binding site for c-MAF (28). This proto-oncogene product is selectively expressed in mouse Th2 cells, interacts with NFAT1 and transactivates the IL-4 promoter even when ectopically expressed in B cells or non-lymphoid cells (28). Homologs of c-maf with a high degree of sequence homology occur in the genomes of chicken, mouse, rat and probably humans, although to our knowledge a human equivalent of c-maf has not been reported. We have tested our Th1 and Th2 clones for c-maf expression by semi-quantitative RT-PCR analysis, using primers derived from the mouse c-maf sequence (48), designed in conserved areas with complete sequence homology with rat and chicken c-maf and with v-maf. We repeatedly amplified a fully homologous cDNA fragment to comparable levels in Th1 and Th2 clones (Wierenga et al., unpublished observation), thus not providing evidence for differential c-maf expression in human Th1 and Th2 cells. Further arguing against a decisive role of c-MAF in Th2-specific IL-4 gene regulation is the recent demonstration that mutation of the c-MAF-binding site in the human IL-4 promoter has only limited effects on the promoter function (30).
Another Th2-specific transcription factor promoting type 2 cytokine gene expression is GATA-3 (29), but since this factor has no binding site close to the IL-4 P0 element it is not likely that GATA-3 is directly involved in the presently described differential NFAT1 activity, as detected with the IL-4 P0 probe. Although P0 is not flanked by an AP-1-binding element, recent evidence suggests that AP-1-family proteins have a role in Th2-specific P0-binding activities (30), as have the octamer-binding proteins Oct-1 and Oct-2 (30), involving the ocatmer-like binding site in the 5' P0-flanking sequence (49). Further research will be needed to elucidate the precise constitution of the NFAT1P0 complexes in Th1 and Th2 cells and to identify the factor or condition squelching NFAT1 activity at the P0 element in atopic Th2 cells.
The consequence of differential NFAT1 binding at the P0 element may influence larger areas of the IL-4 promoter, as judged from preliminary experiments in which suppressed NFAT1 complex formation in Th2 cells was observed also at a longer oligonucleotide that, besides the P0 element, also contains the P1 element (Wierenga et al, manuscript in preparation). As mentioned and shown (25) before, when tested as a single element the P1 site allows for strong NFAT1 binding in Th2 cells. The mutual interference of the P0- and P1-bound complexes and the relevance for IL-4 gene expression are currently being studied in more detail.
An intriguing finding was that, like in Th2 clones, reduced NFAT complex formation at the IL-4 P0 site could be reproduced polyclonally in PBMC of some but not all severe atopic dermatitis patients. Within the clinical spectrum of atopic allergy, enhanced IL-4-induced IgE production is most pronounced in atopic dermatitis, on which basis the patients in the present study were selected. Conclusive data on the multifactorial pathogenesis of atopic dermatitis is not available yet, but ample evidence suggests a genetic predisposition. Genome-wide searches for genetic linkage in atopic allergy have indicated various associations with markers on different chromosomes, including chromosome 5q31 (5052), where the IL-4 gene is located. Others have reported on IL-4 promoter polymorphisms (5355) or a mutation leading to increased IL-4 receptor function (56), but evidence for genetically determined alterations in the molecular mechanisms underlying IL-4 gene regulation in general or in atopic allergy has not been reported, so far. Although the present sample size of five atopic dermatitis patients is far from sufficient to draw firm conclusions, it is tempting to speculate that the altered NFAT1P0 complex regulation we describe here in three of five severe atopic dermatitis patients reflects a polyclonal immunologic phenotype of enhanced inducibility of the IL-4 promoter, as a result of yet unknown regulatory changes in its transcriptional control. As such, this may be one of the possible mechanisms contributing to the generalized increased IL-4 production in atopic patients, which indeed is not confined to allergen-specific T cells only (45,57). To further address the question to which extent our present observations reflect alterations in atopic disease or are associated with Th2 cells in general, our current studies include the analyses of the IL-4 P0-binding activities in mono- and polyclonal T cells of larger and additional groups of individuals, including patients with IgE-related diseases, such as allergic asthma and hyper-IgE syndrome.
 |
Acknowledgments
|
---|
This work was supported by grants from The Netherlands Asthma Foundation (E. A. W.), The Royal Netherlands Academy of Arts and Sciences (E. A. W.), and the Deutsche Forschungsgemeinschaft (SFB 217) (G. M.). The authors gratefully acknowledge the technical assistance of Ing. J. Wormmeester and F. Stiekema.
 |
Abbreviations
|
---|
AP-1 | activator protein-1 |
EMSA | electrophoretic mobility shift assay |
NFAT | nuclear factor of activated T cells |
PBMC | peripheral blood mononuclear cells |
PV | psoriasis vulgaris |
SSc | systemic sclerosis |
 |
Notes
|
---|
Transmitting editor: J. Borst
Received 27 April 1998,
accepted 26 October 1998.
 |
References
|
---|
-
Arai, K. I., Lee, F., Miyajima, A., Miyatake, S., Arai, N. and Yokota, T. 1990. Cytokines: coordinators of immune and inflammatory responses. Annu. Rev. Biochem. 59:783.[ISI][Medline]
-
Mosmann, T. R. and Coffman, R. L. 1989. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145.[ISI][Medline]
-
Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L. 1986. Two types of murine T cell clone: I.1. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2348.[Abstract/Free Full Text]
-
Wierenga, E. A., Snoek, M., de Groot, C., Chretien, I., Bos, J. D., Jansen, H. M. and Kapsenberg, M. L. 1990. Evidence for compartmentalization of functional subsets of CD4+ T lymphocytes in atopic patients. J. Immunol. 144:4651.[Abstract/Free Full Text]
-
Mosmann, T. R. and Sad, S. 1996. The expanding universe of T cell subsets: Th1, Th2 and more. Immunol. Today 17:138.[ISI][Medline]
-
Abbas, A. K., Murphy, K. M. and Sher, A. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[ISI][Medline]
-
Salgame, P., Abrams, J. S., Clayberger, C., Goldstein, H., Convit, J., Modlin, R. L. and Bloom, B. R. 1991. Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science 254:279.[ISI][Medline]
-
Peltz, G. 1991. A role for CD4+ T cell subsets producing a selective pattern of lymphokines in the pathogenesis of human chronic inflammatory and allergic disease. Immunol. Rev. 123:23.[ISI][Medline]
-
Romagnani, S. 1994. Lymphokine production by human T cells in disease states. Annu. Rev. Immunol. 12:227.[ISI][Medline]
-
Kapsenberg, M. L., Wierenga, E. A., Bos, J. D. and Jansen, H. M. 1991. Functional subsets of allergen-reactive human CD4+ T cells. Immunol. Today 12:392.[ISI][Medline]
-
Van der Heijden, F. L., Wierenga, E. A., Bos, J. D. and Kapsenberg, M. L. 1991. High frequency of IL-4-producing CD4+ allergen-specific T lymphocytes in atopic dermatitis lesional skin. J. Invest. Dermatol. 97:389.[Abstract]
-
Parronchi, P., Macchia, D., Piccinni, M. P., Biswas, P., Simonelli, C., Maggi, E., Ricci, M., Ansari, A. A. and Romagnani, S. 1991. Allergen- and bacterial antigen-specific T cell clones established from atopic donors show a different profile of cytokine production. Proc. Natl Acad. Sci. USA 88:4538.[Abstract]
-
Pene, J., Rousset, F., Briere, F., Chretien, I., Bonnefoy, J.-Y., Spits, H., Yokota, T., Arai, N., Arai, K., Banchereau, J. and de Vries, J. E. 1988. IgE production by normal human lymphocytes is induced by interleukin-4 and suppressed by interferons
and A and prostaglandin E2. Proc. Natl Acad. Sci. USA 85:6880.[Abstract]
-
Rao, A. 1994. NFATp: a transcription factor required for the co-ordinate induction of several cytokine genes. Immunol. Today 15:274.[ISI][Medline]
-
Szabo, S. J., Gold, J. S., Murphy, T. L. and Murphy, K. M. 1993. Identification of a cis-acting regulatory element controlling interleukin-4 gene expression in T cells: roles for NF-Y and NFATc. Mol. Cell. Biol. 13:4793.[Abstract]
-
Li-Weber, M., Salgame, P., Hu, C. and Krammer, P. H. 1997. Characterization of constitutive and inducible transcription factors binding to the P2 NF-AT site in the human interleukin-4 promoter. Gene 188:253.[ISI][Medline]
-
Yokota, T., Arai, N., de Vries, J. E., Spits, H., Banchereau, J., Zlotnik, A., Rennick, D., Howard, M., Takebe, Y., Miyatake, S., Lee, F. and Arai, K. 1988. Molecular biology of interleukin-4 and interleukin-5 genes and biology of their products that stimulate B cells, T cells and hematopoietic cells. Immunol. Rev. 102:137.[ISI][Medline]
-
Rao, A., Luo, C. and Hogan, P. G. 1997. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15:707.[ISI][Medline]
-
Schreiber, S. L. and Crabtree, G. R. 1992. The mechanism of action of cyclosporin A and FK506. Immunol. Today 13:136.[ISI][Medline]
-
Loh, C., Carew, J. A., Kim, J., Hogan, P. G. and Rao, A. 1996. T cell receptor stimulation elicits an early phase of activation and a later phase of deactivation of the transcription factor NFAT1. Mol. Cell. Biol. 16:3945.[Abstract]
-
Xanthoudakis, S., Viola, J. P. B., Shaw, K. T. Y., Luo, C., Wallace, L. D., Bozza, P. T., Curran, T. and Rao, A. 1996. An enhanced immune response in mice lacking the transcription factor NFAT1. Science 272:892.[Abstract]
-
Hoey, T., Sun, Y. L., Williamson, K. and Xu, X. 1995. Isolation of two new members of the NFAT gene family and functional characterization of the NFAT proteins. Immunity 2:461.[ISI][Medline]
-
Hodge, M. R., Ranger, A. M., de la Brousse, F. C., Hoey, T., Grusby, M. J. and Glimcher, L. H. 1996. Hyperproliferation and dysregulation of IL-4 expression in NF-ATp-deficient mice. Immunity 4:397.[ISI][Medline]
-
Kiani, A., Viola, J. P. B, Lichtman, A. H. and Rao, A. 1997. Down-regulation of IL-4 gene transcription and control of Th2 cell differentiation by a mechanism involving NFAT1. Immunity 7:849.[ISI][Medline]
-
Rooney, J. W., Hodge, M. R., McCaffrey, P. G., Rao, A. and Glimcher, L. H. 1994. A common factor regulates both Th1- and Th2-specific cytokine gene expression. EMBO J. 13:625.[Abstract]
-
Rincon, M. and Flavell, R. A. 1997. Transcription mediated by NFAT is highly inducible in effector CD4+ T helper 2 (Th2) cells but not in Th1 cells. Mol. Cell. Biol. 17:1522.[Abstract]
-
Rooney, J. W., Hoey, T. and Glimcher, L. H. 1995. Coordinate and cooperative roles for NFAT and AP-1 in the regulation of the murine IL-4 gene. Immunity 2:473.[ISI][Medline]
-
Ho, I. C., Hodge, H. R., Rooney, J. W. and Glimcher, L. H. 1996. The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 85:973.[ISI][Medline]
-
Zheng, W. and Flavell, R. A. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587.[ISI][Medline]
-
Li-Weber, M., Salgame, P., Hu, C., Davydov, I. V., Laur, O., Klevenz, S. and Krammer P. H. 1998. Th2-specific Protein/DNA interactions at the proximal nuclear factor-AT site contribute to the functional activity of the human IL-4 promoter. J. Immunol. 161:1380.[Abstract/Free Full Text]
-
Wierenga, E. A., Snoek, M., Bos, J. D., Jansen, H. M. and Kapsenberg, M. L. 1990. Comparison of diversity and function of housedust mite-specific T lymphocyte clones from atopic and non-atopic donors. Eur. J. Immunol. 20:1519.[ISI][Medline]
-
Van Neerven, R. J. J., van der Pol, M. M., van Milligen, F. J, Jansen, H. M., Aalberse, R. C. and Kapsenberg, M. L. 1994. Characterization of cat dander-specific T lymphocytes from atopic patients. J. Immunol. 152:4203.[Abstract/Free Full Text]
-
Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanatephenolchloroform extraction. Anal. Biochem. 162:156.[ISI][Medline]
-
Messer, G., Spengler, U., Jung, M. C., Honold, G., Blömer, K., Pape, G. R., Riethmüller, G. and Weiss, E. H. 1991. Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-ß gene correlates with a variant amino acid in position 26 and a reduced level of TNF-ß production. J. Exp. Med. 173:209.[Abstract]
-
Hanifin, J. and Rajka, G. 1980. Diagnostic features of atopic dermatitis. Acta Dermatol. Venerol. 92:44.
-
Hilkens, C. M. U., Messer, G., Tesselaar, K., van Rietschoten, A. G. I., Kapsenberg, M. L. and Wierenga, E. A. 1996. Lack of IL-12 signaling in human allergen-specific Th2 cells. J. Immunol. 157:4316.[Abstract]
-
Van Lier, R. A. W., Boot, J. A. H., Verhoeven, A. J., de Groot, E. R., Brouwer, M. and Aarden, L. A. 1987. Functional studies with anti-CD3 heavy chain isotype switch-variant monoclonal antibodies: accessory cell-independent induction of interleukin responsiveness in T cells by
-anti-CD3. J. Immunol. 139:2873.[Abstract/Free Full Text]
-
Kick, G., Messer. G., Goetz, A., Plewig, G. and Kind, P. 1995. Photodynamic therapy induces expression of interleukin 6 by activation of AP-1 but not NF-
B DNA binding. Cancer Res. 55:2373.[Abstract]
-
Lindhout, E., Lakeman, A. and de Groot, C. 1995. Follicular dendritic cells inhibit apoptosis in human B lymphocytes by rapid and irreversible blockade of preexisting endonuclease. J. Exp. Med. 181:1985.[Abstract]
-
Varadhachary. A. S., Perdow, S. N., Hu, C., Ramamarayanan, M. and Salgame, P. 1997. Differential ability of T cell subsets to undergo activation-induced cell death. Proc. Natl Acad. Sci. USA 95:5778.
-
Zhang, X., Brunner, T., Carter, L., Dutton, R. W., Rogers, P., Bradley, L., Sato, T., Reed, J. C., Green, D. and Swain, S. L. 1997. Unequal death in T helper cell (Th) 1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185:1837.[Abstract/Free Full Text]
-
Ramsdell, F., Seaman, M. S., Miller, R. E., Picha, K. S., Kennedy, M. K. and Lynch, D. H. 1994. Differential ability of Th1 and Th2 cells to express Fas ligand and to undergo activation-induced cell death. Int. Immunol. 6:1545.[Abstract]
-
Cohen, J. J. 1993. Apoptosis. Immunol. Today 14:126.[ISI][Medline]
-
Lenardo, M. J. 1996. Fas and the art of lymphocyte maintenance. J. Exp. Med. 183:721.[ISI][Medline]
-
Parronchi, P., De Carli, M., Manetti, R., Simonelli, C., Piccinni, M. P., Macchia, D., Maggi, E., Del Prete, G. F., Ricci, M. and Romagnani, S. 1992. Aberrant interleukin (IL)-4 and IL-5 production in vitro by CD4+ helper T cells from atopic subjects. Eur. J. Immunol. 22:1615.[ISI][Medline]
-
Chan, S. C., Brown, M. A., Willcox, T. M., Li, S. H., Stevens, S. R., Tara, D. and Hanifin, J. M. 1996. Abnormal IL-4 gene expression by atopic dermatitis T lymphocytes is reflected in altered nuclear protein interactions with IL-4 transcriptional regulatory element. J. Invest. Dermatol. 106:1131.[Abstract]
-
Shi, Y., Sahai, B. M. and Green, D. R. 1989. Cyclosporin A inhibits activation-induced cell death in T cell hybridomas and thymocytes. Nature 339:625.[ISI][Medline]
-
Kurschner, C. and Morgan, J. I. 1995. The maf proto-oncogene stimulates transcription from multiple sites in a promoter that directs Purkinje neuron-specific gene expression. Mol. Cell. Biol. 15:246.[Abstract]
-
Chuvpilo, S., Schomberg, C., Gerwig, R., Heinfling, A., Reeves, R., Grummt, F. and Serfling, E. 1993. Multiple closely-linked NFAT/octamer and HMG (I)Y binding sites are part of the interleukin-4 promoter. Nucleic Acids Res. 21:5694.[Abstract]
-
Marsh, D. G., Neely, J. D., Breazeale, D. R., Ghosh, B., Freidhoff, L. R., Ehrlich-Kautzky, E., Schou, C., Krishnaswamy, G. and Beaty, T. H. 1994. Linkage analysis of IL-4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations. Science 264:1152.[ISI][Medline]
-
Postma, D. S., Bleecker, E. R., Amelung, P. J., Holroyd, K. J., Xu, J., Panhuysen, C. I., Meyers, D. A. and Levitt, R. C. 1995. Genetic susceptibility to asthma-bronchial hyperresponsiveness coinherited with a major gene for atopy. New Engl. J. Med. 333:894.[Abstract/Free Full Text]
-
Ulbrecht, M., Eisenhut, T., Bonisch, J., Kruse, R., Wjst, M., Heinrich, J., Wichmann, H. E., Weiss, E. H. and Albert, E. D. 1997. High serum IgE concentrations: association with HLA-DR and markers on chromosome 5q31 and chromosome 11q13. J. Allergy Clin. Immunol. 99:828.[ISI][Medline]
-
Rosenwasser, L. J., Klemm, D. J., Dresback, J. K., Inamura, H., Mascali, J. J., Klinnert, M. and Borish, L. 1995. Promoter polymorphisms in the chromosome 5 gene cluster in asthma and atopy. Clin. Exp. Allergy 25 (Suppl. 2):74.[ISI][Medline]
-
Song, Z., Casolaro, V., Chen, R., Georas, S. N., Monos, D. and Ono, S. J. 1996 Polymorphic nucleotides within the human IL-4 promoter that mediate overexpression of the gene. J. Immunol. 156: 424.
-
Walley, A. J. and Cookson, W. O. 1996. Investigation of an interleukin-4 promoter polymorphism for associations with asthma and atopy. J. Med. Genet. 33:689.[Abstract]
-
Khurana Hershey, G. K., Friedrich, M. F., Esswein, L. A., Thomas, M. L. and Chatila, T. A. 1997. The association of atopy with a gain-of-function mutation in the
subunit of the interleukin-4 receptor. N. Engl. J. Med. 337:1720.[Abstract/Free Full Text]
-
Rousset, F., Robert, J., Andary, M., Bonnin, J. P., Souillet, G., Chretien, I., Briere, F., Pene, J. and de Vries, J. E. 1991. Shifts in interleukin-4 and interferon gamma production by T cells of patients with elevated serum IgE levels and the modulatory effects of these lymphokines on spontaneous IgE synthesis. J. Allergy Clin. Immunol. 87:58.[ISI][Medline]