Regulation of Interleukin 4 Gene Transcription
Alterations in Atopic Disease?

EDDY A. WIERENGA and GERALD MESSER

Department of Cell Biology and Histology, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands; and Department of Dermatology and Allergology, Ludwig-Maximilians-University, Munich, Germany

	GENERAL REGULATION OF IL-4 GENE TRANSCRIPTION IN HELPER T CELLS

TOP GENERAL REGULATION OF IL-4... Th2-SPECIFIC IL-4 GENE CONTROL ALTERED IL-4 GENE CONTROL... CONCLUSION DISCUSSION REFERENCES

Induction of interleukin 4 (IL-4) transcription in helper T (Th) cells under physiological conditions follows from engagement of the T cell receptor by antigenic peptides presented by antigen-presenting cells in association with MHC class II molecules (1). This trigger will activate two intracellular signaling pathways crucial to IL-4 expression, that is, a protein kinase C (PKC)-dependent pathway leading to the synthesis of activator protein 1 (AP-1) family proteins and a calcium (Ca²⁺)- dependent pathway resulting in calcineurin-mediated dephosphorylation and nuclear translocation of preexisting cytosolic nuclear factor of activated T cells (NFAT). The PKC pathway is potentiated by CD28-mediated costimulation (2), whereas the Ca²⁺ pathway can be fully blocked by cyclosporin A and FK506 (3). Both the mouse and human IL-4 promoters contain five binding elements (designated P0 to P4) for the NFAT family of transcription factors, with only minimal interspecies variation with respect to their sequence, orientation, and location within the proximal 250 base pairs (bp) of the promoter region (4). The P0 element is located just upstream of the transcription start site and P4 is located at about 250 bp in the 5'-flanking region (Figure 1). Although all P elements to some extent contribute to IL-4 gene control, the major positive regulatory P elements seem to be P1 (4, 7) and P4 (reviewed in Reference 1). As are P2 and P3, both P1 and P4 are immediately flanked by sequences with affinity for AP-1 family proteins (8), allowing for cooperative binding with NFAT proteins. The strong reduction of IL-4 promoter activity as observed, for example, after mutation of either the P1-NFAT- or the adjacent AP-1-binding motif (9) emphasizes the importance of collaborative NFAT/AP-1 binding at such composite elements.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Schematic representation of the proximal IL-4 gene promoter, restricted to the transcription factors and promoter elements described in text, which provides further explanation. For reasons of simplicity, data obtained from mouse and human studies are combined.

The proximal part of the IL-4 promoter region, containing the transcription start site (indicated as +1 in Figure 1) and the P0 and P1 elements, can function as a minimal promoter in reporter gene assays, conferring inducible expression and a certain degree of Th2-specific regulation (11, 12). Initial studies gave no indication of Th1- or Th2-specific recruitment of particular NFAT or AP-1 family proteins to these sites (9, 13). Instead, the mechanism underlying the Th2 specificity of IL-4 expression may be based on quantitative differences in the availability of NFAT and/or AP-1 family proteins. Furthermore, because the cooperative DNA binding of NFAT and AP-1 proteins has an auxiliary function for the binding of other transcription factors (5, 14), these additional transcription factors are likely to be involved in tissue-specific IL-4 expression.

	Th2-SPECIFIC IL-4 GENE CONTROL

TOP GENERAL REGULATION OF IL-4... Th2-SPECIFIC IL-4 GENE CONTROL ALTERED IL-4 GENE CONTROL... CONCLUSION DISCUSSION REFERENCES

Indeed, several transcription factors have been implicated, some of which may explain the Th2 specificity of the proximal IL-4 promoter, while others may act on other genes. The protooncogene product c-Maf has been described as a transcription factor specifically expressed in mouse Th2 clones and not in Th1 clones (15). Similarly, expression of c-Maf was inducible in Th2 development from naive T cells and not in Th1 development. A half c-Maf-binding site referred to as MARE is located just downstream of the P0/NFAT site (Figure 1) and it was postulated that c-Maf and NFAT synergize in trans-activating the minimal IL-4 promoter (15). Interestingly, ectopic expression of c-Maf trans-activated IL-4 expression in Th1 clone cells, B cells, and even in nonlymphoid cells (15). On the basis of these data, it was suggested that c-Maf is responsible for Th2-specific IL-4 gene expression. More recent data from the same group (17) indicate that the regulatory mechanisms involved are more complex. For example, whereas c-Maf-transfected naive T cells developing along the Th1 pathway can still be induced to express IL-4, in addition to typical Th1 cytokines such as interferon  (IFN-), the earlier observation of IL-4 expression in c-Maf-transfected Th1 clone cells could not be reproduced in fully polarized Th1 effector cells. Instead, IFN- production was inhibited in mature Th1 cells, not only suggesting that other genes are targeted by c-Maf, but also that additional transcription factors are required to explain Th2-specific IL-4 expression. The same requirement follows from our own observation in human Th2 cells that c-Maf mRNA levels do not differ in quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of fully polarized Th1 and Th2 clones (E. A. Wierenga and G. Messer, unpublished data, 1999). A likely candidate to fulfill at least part of this requirement is JunB. JunB is one of the AP-1 family transcription factors that was shown to bind the P1/NFAT-flanking AP-1 site (9), originally referred to as OAP40 (18) (Figure 1). Other AP-1 proteins that can bind at this site are JunD, Fra-1, and Fra-2, but not c-Fos, FosB, or c-Jun (9). Although these early studies did not indicate differential availability of AP-1 family members in Th1 and Th2 clones, it later appeared that JunB is selectively induced in developing Th2 cells, but not in Th1 cells, and that JunB-containing AP-1 complexes accumulate in Th2 cells, correlating with increased transcriptional AP-1 activity (16, 19). It was further shown that the binding of JunB to the P1/NFAT-OAP40 composite site synergizes with c-Maf in the trans-activation of the IL-4 gene (Figure 1), suggesting that the increased availability of JunB in Th2 cells provides the Th2 specificity for the activity of c-Maf, which, hence, could be an explanation for the lack of IL-4 expression in c-Maf-transfected Th1 clones (17).

Apart from the P1 site, AP-1 family members may confer Th2 specificity to the P4 region, as well. Fos and c-Jun, in collaboration with NFAT, were shown to significantly enhance the adjacent binding of C/EBP- and C/EBP- (NF-IL6) in human T cells (10, 14) (Figure 1). Fos appears to form a heterodimer with C/EBP- (14). The tissue specificity at this composite site, also referred to as PRE-I (20), is suggested by the finding that c-Jun-containing complexes at PRE-I are formed in Th0-like Jurkat and Th2 cells, but not in Th1 cells (10). The molecular basis for increased c-Jun activity in human T cells remains elusive. Notably, c-Jun levels do not seem to be increased in mouse Th2 cells (16).

Apparently acting independently from c-Maf and AP-1 family proteins is GATA-3. This protein was first described as a T cell-specific transcription factor required for expression of the T cell receptor  and  genes (21, 22). GATA-3 is expressed in naive T cells and this expression increases in T cells developing along the Th2 pathway, but it is suppressed in Th1 lineage cells (23). Inhibition of the activity of GATA-3 by transfection of an antisense GATA-3 construct prevented IL-4 expression, whereas transgenic expression of GATA-3, on the other hand, induced IL-4 expression in developing Th1 cells. It was therefore postulated that GATA-3 is necessary and sufficient for IL-4 gene expression in Th cells (23), at least in the mouse.

The molecular mechanisms underlying the effects of GATA-3, like those of c-Maf, are not restricted to the regulation of IL-4 gene transcription. Also the expression of other Th2 cytokines such as IL-5 are increased by GATA-3 (23), indicating a more general level of activity. The expression of GATA-3 is upregulated by IL-4 and, thus, will be maintained during Th2 cell development (26). An important direct consequence of maintained GATA-3 expression is downregulation of IL-12Rß2-subunit expression (26), explaining IL-12-unresponsiveness of Th2 cells (27). In turn, IL-12 unresponsiveness will block Th1 development and concomitant IFN- production. GATA-3 expression is actively suppressed by IL-12 during Th1 development in a fashion independent of the mere absence of IL-4 (26). These findings strongly implicate GATA-3 as an important and more general instrument in Th2 cell polarization.

Exactly how GATA-3 influences IL-4 gene transcription is not clear. Although the IL-4 gene promoter contains two putative GATA-3-binding sites (9) (Figure 1), it is uncertain whether GATA-3 regulates IL-4 expression via these sites, because retroviral reintroduction of GATA-3 into mature Th1 cells did not induce IL-4 production (26). The apparent contradiction with the previous finding that ectopically expressed GATA-3 in developing Th1 cells does induce IL-4 production (23) suggests an effect of GATA-3 early in Th cell commitment, which seems to be lost on full polarization.

The data reviewed above indicate that both c-Maf and GATA-3 affect the expression of multiple cytokine genes during a limited period of time after priming of naive T cells and therefore may have to be regarded as T cell polarization factors, rather than mere IL-4- or Th2-specific transcription factors. Similar to the requirement for JunB accumulation to provide Th2 specificity to c-Maf at the IL-4 promoter (16, 19), GATA-3 may act with collaborative factors, as well, either at the putative GATA-3 sites within the IL-4 promoter, or elsewhere.

	ALTERED IL-4 GENE CONTROL IN ATOPIC DISEASE?

TOP GENERAL REGULATION OF IL-4... Th2-SPECIFIC IL-4 GENE CONTROL ALTERED IL-4 GENE CONTROL... CONCLUSION DISCUSSION REFERENCES

Most of the current knowledge of the molecular control of IL-4 gene regulation is derived from mouse studies. Although the underlying mechanisms of human and mouse IL-4 gene control are not identical, for example, with respect to Th2-specific availability of AP-1 family members (10, 16), the obvious strong interspecies similarities may be useful in trying to explain some of the more recently reported alterations in human IL-4 expression, some of which may be associated with atopic disease.

A search for promoter polymorphisms in the so-called IL-4 cytokine gene cluster located in human chromosome 5931.1 identified a C-to-T base substitution at position 590 relative to the transcription start site (30) (Figure 1). The incidence of this polymorphism in an American study was about 40% and the T allele was clearly associated with enhanced total IgE production, skin test positivity, and asthma (30). Japanese studies confirmed the association of this 590 C-to-T substitution both with asthma (31) and with atopic dermatitis (32). Reporter gene transfer experiments in Jurkat cells indicated a threefold higher promoter activity of the T allele and EMSA experiments showed a strong phorbol myristate acetate (PMA)/ ionomycin-inducible transcription factor-binding activity at the T allele, but not at the wild-type C allele (30). The data have not yet been published as such, but the transcription factor complex formed at the T allele at position 590 is claimed to involve NFAT family proteins (33), despite the fact that the C-to-T substitution does not create the characteristic TTTCC core sequence of the NFAT contact site (8), but creates TTCCC instead. Regarding the gradual reduction of IL-4 promoter activity on consecutive deletion of the NFAT sites P0 to P4 (1, 4) and the relatively strong transcriptional activities from artificial promoters consisting of multimer constructs of particular NFAT sites (11, 12), it is not unlikely that the increased IL-4 transcription from the position 590 T allele promoter results from additional NFAT binding, either independently or in collaboration with other transcription factors.

Other investigators have analyzed the proximal human IL-4 promoter sequence in cell lines from different donors and identified no less than 9 different alleles in the 10 cell lines tested (34). Examination of the functional impact of these nucleotide exchanges by transient transfection experiments in Jurkat cells indicated that an A-to-G base substitution at position 81 significantly increased the IL-4 promoter activity. Position 81 is located within the P1/NFAT-flanking OAP40 site (Figure 1) and the A-to-G mutation increases the binding affinity for AP-1 family proteins, as determined by competition experiments. These findings may be explained in terms of enhancement of collaborative AP-1/NFAT binding at this composite site (9).

An intriguing question is whether this increased AP-1 activity influences the binding activity of c-Maf, as does the Th2-specific increased JunB activity at this site in the mouse (16, 19), and whether this base pair substitution could be one of many possible aberrations leading to IL-4 overproduction as part of the multifactorial pathology of atopic disease. Relevant to the latter hypothesis is a comparative study between patients with atopic dermatitis and healthy control individuals (35) in which PMA/ionomycin-stimulated polyclonal T cells from 12 of 12 patients with atopic dermatitis showed a strongly increased inducible transcription factor-binding activity at the OAP40 site, as opposed to only 2 of 12 control donors. These complexes contained both AP-1 and NFAT family proteins and their increased inducibility correlated with increased frequencies of IL-4-producing T cells and polyclonal IL-4 production. Unfortunately, IL-4 promoter sequence data from these patients were not reported.

Another example of altered IL-4 gene control in atopic patients has been described by our own laboratories (36). On stimulation with CD3 and CD28 monoclonal antibody (MAb), allergen-specific Th2 cells clones and peripheral blood mononuclear cells (PBMCs) of some but not all patients with atopic dermatitis showed a selectively reduced binding of NFAT1 at the IL-4 P0 element, associated with high IL-4 production. NFAT1 binding at the IL-4 P1 element was not reduced in these patients. A functional explanation for this finding is not at hand, but it may be related to the unique inhibitory effect of this particular NFAT family member on the transcription of the IL-4 gene, as it has on several other cytokine genes (37, 38). Analyses of in vitro Th1- and Th2-polarized naive T cells from atopic patients and control subjects showed that this phenomenon is subject dependent rather than Th2 related (E. A. Wierenga and G. Messer, unpublished observations, 1999). These data suggest a novel systemic and possibly genetically determined dysregulation of the IL-4 gene, which may be predisposed by alterations of the NFAT1 gene or due to the regulation of its activity by other factors.

	CONCLUSION

TOP GENERAL REGULATION OF IL-4... Th2-SPECIFIC IL-4 GENE CONTROL ALTERED IL-4 GENE CONTROL... CONCLUSION DISCUSSION REFERENCES

It is to be expected that more polymorphisms and mechanisms of altered IL-4 gene control will be identified in the near future. Combined with the increasing knowledge obtained from genetic linkage studies and murine and human IL-4 promoter studies, these findings may help to develop and refine therapeutic strategies for atopic disorders in individual patients, based on the exact origin of IL-4 overproduction.

	DISCUSSION

TOP GENERAL REGULATION OF IL-4... Th2-SPECIFIC IL-4 GENE CONTROL ALTERED IL-4 GENE CONTROL... CONCLUSION DISCUSSION REFERENCES

Holgate: When we challenge bronchial mucosal explants from asthmatic subjects with dust mite allergen, for 24 hr in vitro, high levels of IL-5 and IL-13 are released, but IL-4 is almost undetectable. The IL-5 and IL-13 release was inhibitable by CTLA4-Ig and antibodies to either B7.1 or B7.2, indicating a key role for antigen presentation and costimulation. Do you have any data on polymorphism of IL-13?

Aarden: Dr. Van der Pouw Kraan has some data on IL-13 polymorphism.

Van der Pouw Kraan: The regulation of IL-13 turns out to be different from that of IL-4. A signal through the T cell receptor provides a positive as well as a negative signal for IL-13 production. The positive signal is probably protein kinase C activation. The negative signal is due to an elevation of calcium. We can mimic this negative signal by taking a calcium ionophore. The calcium-dependent route is inhibitory for IL-13 production, message as well as protein. We can reverse the inhibition by the addition of cyclosporin A. This suggests that NFAT inhibits IL-13 production, because NFAT is calcium dependent and inhibited by cyclosporin A. In allergic asthma patients this negative signaling through a calcium-dependent signal was reduced. Because other NFAT-dependent cytokines, such as IL-4, were not downregulated we thought that nothing was wrong with NFAT in these T cells. This led us to hypothesize that in the promoter of IL-13 something is wrong in an NFAT site. We sequenced the IL-13 promoter and indeed found one nucleotide exchange immediately adjacent to an NFAT site. Patients that were homozygous for the low-frequency genotype showed a diminished negative signal through the T cell receptor. We observed in band shift assays an increased binding of nuclear proteins to this region. We still have to find out what NFAT members are present there. This polymorphism is associated with allergic asthma with an odds ratio of 11.

Wierenga: Which isoform of NFAT could be involved here?

Van der Pouw Kraan: In mice NFAT-1 and NFAT-4 negatively regulate Th2 development and Th2 cytokines, but NFATc is expressed at a higher level and this could be the reason why Th2 responses are going up. So, perhaps it is NFATc that is positively regulating IL-13 production.

Wierenga: It will be interesting to investigate IL-13 regulation in patients that are deficient in NFAT-1 binding at P0 in IL-4.

Van der Pouw Kraan: Yes, we will have to exchange materials.

Wierenga: One additional remark with respect to IL-13 is that we have seen IL-13 transcription in T cells that were completely devoid of any IL-4 transcripts, so it is probably quite different.

Aarden: It is clear that, for example, CD8⁺ cells make a lot of IL-13 upon stimulation, so there must be a difference between IL-4 and IL-13 there.

Wierenga: And naive T cells make it.

Van der Pouw Kraan: If you give cyclosporin A to T cells, IL-4 production is inhibited and IL-13 is enhanced, so that is completely different.

Wierenga: A different dependence on NFAT.

Kauffman: When you look at naive T cells most of the cytokine genes are silent, but become activated during immune activation. By what mechanisms are these genes kept in a silent state and what are the activation mechanisms?

Wierenga: At the Lake Tahoe meeting (January 1999) it was claimed that a certain number of cell divisions are needed before IL-4 transcription becomes evident. It could reflect the detection limits of the various assays that IFN- is seen already after one division and IL-4 after three or four, but if this is really the case, it suggests relevant changes during the first rounds of proliferation.

Aarden: In all cell lines activation of PKC by PMA is a positive signal, but in primary human T cells it is a very strong negative signal for IL-4 production. If your culture these cells for 2 days, then they change phenotype and are positively regulated by PMA. Perhaps T cells need a few days before they become responsive to PMA.

Wierenga: There is also another explanation for the negative effect of PMA. It has been reported by Casolaro that stimulation of Jurkat cells with PMA activates NF-B, which then competes with NFAT for the P1 site and inhibits IL-4 production.

Aarden: The strange thing remains that after 2 days all the cell lines lose that sensitivity.

Savelkoul: It is my understanding that GATA-3 is necessary to push T cell development into the Th2 direction, but once you have them there it does not matter what you do to the promoter.

Wierenga: That is also the way I think about it, at least for the mouse. It may also hold true for c-MAF and STAT-6 and maybe even for JunB. It seems that these factors are indeed mainly important for driving naive T cells into the Th2 development pathway. To date, little GATA-3 data are available from human Th1/Th2 studies. Our own quantitative RT-PCR analyses of human fully polarized Th1 and Th2 cells indicate that increased GATA-3 mRNA levels in Th2 cells are sustained after repeated polarizing restimulations. Whether or not this contributes to maintaining the Th2 phenotype remains to be established.

Weiss: IL-4 receptor polymorphism has been linked to various asthma phenotypes in inbred populations with a very high linkage disequilibrium, which could mean, simply, that his is also just due to linkage disequilibrium and that it is not the IL-4 receptor polymorphism per se. So, even though there seems to be a relationship between the biology here and what is going on, we need to integrate this with human genetics, if we really want to understand what is going on.

Wierenga: I fully agree. I don't think that any of these polymorphisms will be decisive, but once you get a Th2 response, it will just increase it a little bit more and may add to the severity rather than to the phenomenon itself.

Aarden: Once we get that far, once we can really link these polymorphisms to asthma, what then do we get out of that for the clinic, for the patient? We learn a little bit about mechanisms, certainly, but would it also offer a diagnostic tool to make subdivisions of patients that maybe require different treatments? Would you expect that a patient, who has a tendency to become allergic or to get asthma because of an overproduction of IL-13, would have to be treated differently from a patient with, say, a deficiency of NFAT-1?

Wierenga: If I worked for a pharmaceutical company, I would focus on things that are different, but that act at a point where all these patients somehow come together, and try to block that stage, so that you would tackle all the patients instead of different ones. In fact, such medication exists but is rather unspecific. Classification of patients according to the various factors involved may enable adjustment and improvement of the treatment for individual patients. However, to achieve this there is still a long way to go.

Aalberse: IL-4 and particularly IL-13 might act on asthma expression independent of IgE. Do you agree that the IL-4 (as well as IL-13) story is too complicated to take it in one step directly from gene to asthma? One approach would be to look for association between patients without asthma, but with or without IgE to inhalants, and see whether that is correlated, and then take the next step, i.e., look at subjects with specific IgE with or without asthma and see how that is correlated.

Wierenga: Yes, I agree. Moreover, the number of patients that have been tested so far is too small. So, I don't want to draw any conclusions about whether the different NFAT activity we observed has any relation to the occurrence or development of asthma.

Holgate: We have become almost dominated by the influence of IL-4 and IL-13 on B cells, without asking the question which you just asked: Which other cells are the IL-4 and IL-13 receptors expressed on and which other cells contain the STAT-6 signaling system? When we stain asthmatic tissue as we have done for STAT-6, of course a few B cells stain up, but the major staining area is the bronchial epithelium, which is absolutely filled with STAT-6. Because nobody really studied in detail the effects of IL-4 or IL-13 on epithelial regulation. These are important questions, because, as you correctly said, the biological readout may be wrong.

	Footnotes

Correspondence and requests for reprints should be addressed to E. A. Wierenga, M.D., Academic Medical Centre of the University of Amsterdam, Laboratory of Cell Biology and Histology, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. E-mail: e.a.wierenga{at}amc.uva.nl

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