Eosinophils are attracted to sites of allergic inflammation by a number of chemoattractants including eotaxin-1. This chemokine can be secreted from epithelial cells and fibroblasts after IL-4 and TNF-α stimulation in a synergistic fashion. TNF-α activated gene expression at the transcriptional level in a STAT6-dependent manner, because: 1) eotaxin-1 promoter luciferase constructs were TNF-α inducible in STAT6-defective HEK293 cells only on cotransfection of STAT6 expression vector, an effect that was partially mediated by activation-induced binding of NF-κB proteins to a composite STAT6/NF-κB element; 2) reporter constructs defective in STAT6 DNA binding did not respond to TNF-α stimulation; 3) eotaxin-1 protein secretion was detected only in STAT6-transfected HEK293 cell supernatants on TNF-α treatment; and 4) a trans-dominant negative STAT6 protein inhibited TNF-α-induced eotaxin-1 secretion in primary fibroblasts. TNF-α inducibility of the IL-8 and monocyte chemoattractant protein-1 genes was not dependent on STAT6 expression in the same experimental systems. The inducing effect of IL-4 and IL-13 was also mediated by STAT6. The synergistic effect of IL-4 and TNF-α observed at the RNA and the protein level was not seen at the promoter level. The data demonstrate that both IL-4 and TNF-α induce eotaxin-1 expression at the level of transcription via a STAT6-mediated pathway.
Eosinophils serve as major proinflammatory cells during parasitic infections as well as in allergic inflammation. Their products, such as cytotoxic granule proteins, leukotrienes, and cytokines, are involved in pathological changes seen at the sites of inflammation. The selective influx of eosinophils in allergic asthma is a complex, multistep process involving cell recruitment, infiltration, and activation within the target tissue (1). Several members of the C-C branch of chemokines exhibit chemoattractant properties toward eosinophils. These include RANTES; eotaxin-1, -2 and -3; monocyte chemoattractant protein (MCP)-3,2 MCP-4 and monocyte-derived chemokine (2, 3, 4). The specificities of the chemokines are regulated by the presence of chemokine receptors on a given cell type. The eosinophil-specific C-C chemokines differ in receptor usage, target cell specificity, and cellular sources. Although RANTES induces migration of cells expressing the receptors CCR1, CCR3, and CCR5, the three eotaxins specifically bind to CCR3-expressing cells. This receptor is found on eosinophils, T cells, and basophils (5, 6), and a number of studies have demonstrated eotaxin-mediated migration of these cell types (7, 8, 9).
Expression of eotaxin-1 itself is regulated by a number of cytokines. It has been shown that the proinflammatory cytokines TNF-α and IL-1 induce the production of eotaxin-1 (10, 11). More recently, IL-4 and TNF-α were reported to synergize in the secretion of eotaxin-1 in human skin (12) and nasal fibroblasts (13). Similarly, IL-13 which shares many biological properties with IL-4 showed similar effects in human epithelial cells (14, 15). The underlying mechanisms responsible for this effect in epithelial cells have been characterized in more detail (16). In that study, the transcription factor STAT6 was shown to be responsible for the IL-4-mediated induction of eotaxin-1 promoter activity by binding to a specific DNA response element. TNF-α stimulation, in contrast, resulted in binding of NF-κB protein members to a site that overlapped with the STAT6-binding site, and this interaction conferred promoter activation on TNF-α treatment. Incubation with the combination of the cytokines had an additive effect.
The present study describes a similar analysis in human fibroblasts. We confirm and extend the important role of STAT6 in induction of eotaxin-1 promoter activity in response to IL-4 in human fibroblasts. In contrast to the situation in epithelial cells (16), TNF-α stimulation was not additive or synergistic with IL-4. Interestingly, the activating effect of TNF-α on the promoter was dependent on the presence of an intact STAT6-binding site and also on the presence of functional STAT6 protein. In addition, TNF-α-induced eotaxin-1 protein production was detected only in STAT6-expressing cells and could be counteracted by a trans-dominant negative STAT6 protein. Two other known TNF-α-inducible genes, MCP-1 and IL-8, were not affected. The data show that both IL-4 and TNF-α require STAT6 as mediator to activate eotaxin-1 gene expression.
Materials and Methods
Cell culture and cytokines
Normal human adult dermal fibroblasts and neonatal fibroblasts were cultured in FGM-2 medium (Clonetics, Walkersville, MD). HEK293 cells were carried at 37°C with 5% CO2 in DMEM supplemented with 10% heat-inactivated FCS (Life Technologies, Grand Island, NY), 100 U/ml penicillin, and 100 μg/ml streptomycin. Purified human recombinant IL-4 was obtained from Novartis (Basel, Switzerland) with a specific activity of 0.5 U/ng. Recombinant human TNF-α (Genzyme, Cambridge, MA) and recombinant human IL-13 (PeproTech EC, London, U.K.) were used at a concentration of 100 U/ml. Human eotaxin-1 and MCP-1 proteins were quantitated by commercially available ELISA kits (R&D Systems, Minneapolis, MN).
Total RNA was isolated using the Trizol reagent (Life Technologies) according to the instructions of the manufacturer. Total RNA, 3 μg, was reverse transcribed using avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany) in a total volume of 50 μl. A 260-bp fragment of the eotaxin-1 transcript was amplified using the intron spanning PCR primers 5′-CATGAAGGTCTCCGCAGCACTTCT-3′ and 5′-CCAGATACTTCATGGAATCCTGC-3′ from cDNA corresponding to 20 ng RNA. PCR was performed for 30 cycles at 94°C and 30 s, 56°C and 30 s, and 68°C and 30 s). The PCR primer pair 5′-ATGGATGATGATATCGCCGCG-3′ and 5′-AGTCCATCACGATGCCAGTGG-3′ was used to amplify a 480-bp fragment of the β-actin mRNA using the same reaction conditions .
Cloning of eotaxin-1 reporter constructs
A 1.1-kb eotaxin-1 promoter fragment was amplified from genomic DNA (Roche Molecular Biochemicals) using the PCR primers 5′-CTGACTCGAGCAGGTTTGCAGTACCTCCACACC-3′ and 5′- AGTCAAGCTTGTTGGAGGCTGAAGGTGTGAGC-3′. The PCR fragment was digested with XhoI and HindIII and cloned into pGL3-Basic (Promega, Madison, MA) to give pGL3-EO1. Another promoter fragment was amplified between position −2250 relative to the transcriptional start site (17) and a natural PstI site at position −986 using the primer pair 5′-AGTCACGCGTTTCAGGCGTAGAGTAAATCC-3′ and 5′-AGTCACTGCAGCGGATTACAGC-3′. This fragment was digested with MluI and PstI and inserted into pGL3-EO1 restricted with the same enzymes to give pGL3-EO2 with a total insert size of 2.2 kb. Plasmid pGL3-EO3 was constructed by inserting a 1.4-kb EcoRI/HindIII fragment in which the EcoRI site was made blunt with Klenow polymerase (Roche Molecular Biochemicals) into a SmaI/HindIII-digested pGL3-Basic vector. Site-directed mutations in the composite STAT6/NF-κB site were generated as reported earlier (18) using the following oligonucleotides: M1, 5′-ATGGGCAAAGGCTATCCTGGAATCTCCCACACTGTCTGCT-3′ and 5′-GGGAGCAGACAGTGTGGTCGATTCCAGGGAAGCCTTTGCC-3′; M2, 5′-ATGGGCAAAGGCTTCCCTGCTATCTCCCACACTGTCTGCT-3′ and 5′-GGGAGCAGACAGTGTGGGAGATAGCAGGGAAGCCTTTGCC-3′; M3, 5′-ATGGGCAAAGGCTTCCCTGGAATCGACCACACTGTCTGCT-3′ and 5′-GGGAGCAGACAGTGTGGTCGATTCCAGGGAAGCCTTTGCC-3′. The cloning of the STAT6 expression vector (19) and the IL-8 promoter reporter construct IL-8p (20) has been described. The STAT6-ΔTD expression vector was cloned by insertion of a XhoI/SacI fragment containing the complete human STAT6 cDNA except for the carboxy-terminal trans-activation domain into the pcDNA3.1 vector. Plasmids were analyzed by digestion with restriction endonucleases and DNA sequencing. Constructs used for transient transfections were purified by cesium chloride density gradients.
Transient transfection of HEK293 cells and primary fibroblasts
The day before transfection, 5 × 104 cells were seeded into 24-well culture plates in fresh medium. Transient transfection of HEK293 cells was achieved using calcium phosphate coprecipitation. Briefly, 1–2 μg plasmid DNA was diluted in 42 μl H2O, mixed with 7 μl 2 M CaCl2, and added dropwise to 50 μl 2× HEPES buffered saline (280 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES (pH 7.05)). After a 2-min incubation period at room temperature, the mixture was added to the cells. Primary fibroblasts were transfected with DNA-containing liposomes using Effectene (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. After 24 h, cells were washed and cultured for 12 h in the presence or absence of 50 ng/ml IL-4, 100 ng/ml IL-13, and/or 100 U/ml TNF-α before luciferase assays were conducted in triplicates according to the instructions of the manufacturer using the Promega Luciferase Assay System (Promega Biotech, Madison, WI). In some experiments, pRL-Tk (Promega) was cotransfected as internal control for normalization of differences in transfection efficiency. Lysates from these cells were quantitated for luciferase using the Dual-Luciferase Reporter Assay System (Promega).
Preparation of nuclear extracts and EMSA
Nuclear extracts from unstimulated adult dermal fibroblasts or cells that had been stimulated for 30 min with IL-4 (50 ng/ml) or TNF-α (100 U/ml) were prepared according to the method described by Andrews and Faller (21). A ds oligonucleotide probe spanning the composite STAT6/NF-κB site from position −82 and −46 was end-labeled using [α32P]dCTP (Amersham, Little Chalfont, U.K.) and Klenow polymerase (Roche Molecular Biochemicals). The nucleoprotein binding reaction was done as described (22) using 5 μg nuclear extracts. For competition and supershift experiments, extracts were preincubated with a 50-fold excess of competitor oligo or 2 μg Ab for 30 min before the radiolabeled probe was added. All Abs used in supershift experiments were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Synergistic induction of eotaxin-1 expression by IL-4 and TNF-α
To determine the kinetics of eotaxin-1 induction in primary fibroblasts, cells derived from two donors (donor 1, adult skin fibroblasts; donor 2, neonatal fibroblasts) were cultured with IL-4, TNF-α, or the combination for different times. The supernatants were analyzed for eotaxin-1 protein by ELISA (Fig. 1,A). Chemokine expression became detectable at 8 h in IL-4 and IL-4/TNF-α-stimulated cells and was maximal at 24 h. TNF-α alone induced eotaxin-1 expression in donor 2 (23) but not in donor 1 cells, whereas both samples were responsive to IL-4 alone. In accordance with published data (12), the cytokine combination had a clear synergistic effect. Essentially the same results were obtained when IL-13 was used as stimulus alone or in combination with TNF-α (Fig. 1,B). TNF-α was able to induce gene expression in donor 1 cells because MCP-1, a chemokine known to be induced by this factor, could be easily detected in the same supernatants (Fig. 1 A).
To confirm the results at the mRNA level, eotaxin-1 transcripts were quantitated by RT-PCR in the same experiment (Fig. 1 C). Eotaxin-1 specific RNA was detectable as early as 2 h in IL-4-induced cells, was maximal at 8 h, and was much less abundant at the later time points in donor 1 cells. In accordance with the protein data, TNF-α stimulation did not induce gene transcription. Donor 2 cells responded more slowly to IL-4 stimulation and produced significant amounts of chemokine transcripts on TNF-α stimulation. In IL-4/TNF-α-stimulated cells, there was significantly more mRNA detectable at 2 h. At the 8 h time point, eotaxin message was maximally induced and remained at that level throughout the observation period. This result suggested that IL-4-induced eotaxin transcripts follow the typical characteristics of an immediate early gene. This also applies for the TNF-α stimulation in donor 2 cells, whereas in donor 1 TNF-α acted on the steady state levels of IL-4-induced transcripts either at the posttranscriptional or at the transcriptional level.
STAT6 mediates eotaxin-1 promoter activation by IL-4 or TNF-α
The kinetics by which IL-4 induced gene expression suggested that this effect may be caused by the transcription factor STAT6. A number of STAT6-regulated genes, such as the IgE germline gene, CD23, or the IL-4 receptor α gene showed similar kinetics of induction (24, 25).
The inspection of the published eotaxin-1 promoter sequence (17) revealed the presence of two potential high affinity STAT6-binding sites as defined by the 5′-TTC(N)4GAA-3′ consensus sequence (26). The proximal site between position −74 and position −60 relative to the transcriptional start site overlapped with a putative binding sequence for NF-κB proteins. The distal site is located between positions −2204 and −2195. Reporter constructs were generated in which the firefly luciferase reporter gene is driven by the human eotaxin-1 promoter. The sequence of the 2.2-kB insert in pGL3-EO2 corresponded well with the published sequence. Of interest, one difference was noted in the putative distal STAT6-binding sequence where the cytidine at position 3 in the consensus sequence 5′-TTC(N)4GAA-3′ was changed to a thymidine (Fig. 2). The presence of this substitution was verified by using another PCR primer pair amplifying a small DNA fragment encompassing this site (data not shown). A 5′-TTT(N)4GAA-3′ sequence is generally not recognized by STAT6 with high affinity (26), making the proximal sequence a more likely candidate for a potential regulatory role. Three 5′ deletion promoter constructs (pGL3-EO1, -EO2, and -EO3) (Fig. 2) were transiently transfected into the STAT6-defective HEK293 cell line (27) and tested for cytokine inducibility. In the absence of STAT6 expression, none of the constructs was inducible with IL-4, TNF-α or the combination thereof (Fig. 3, left). The constitutive promoter activity of the shortest EO-1 plasmid was lower than that of the other two constructs, suggesting a positive regulatory element between positions −1036 and −1359. In the presence of cotransfected STAT6 expression vector, all three constructs were inducible on IL-4 stimulation. IL-13 stimulation was as effective as IL-4 and was also dependent on the presence of cotransfected STAT6 (Fig. 3, right). The IL-4 induction index of the plasmids pGL3-EO2 and -EO3 was consistently higher than that of the EO1 construct, suggesting that the sequence between positions −1036 and −1359 contributed to IL-4 inducibility as well as constitutive promoter activity. Because the EO3 plasmid was as IL-4 responsive as the EO2 construct, a major regulatory role of the distal STAT6-like binding site can be ruled out. All three constructs responded to TNF-α treatment albeit at a somewhat lower rate than IL-4. Interestingly, TNF-α responsiveness, like IL-4, was dependent on cotransfected STAT6. The shortest pGL3-EO1 plasmid was less well inducible with TNF-α, suggesting that the same region that contributed to IL-4 up-regulation also was involved in TNF-α regulation. The cytokine combination activated promoter activity to the same degree as IL-4 alone. This demonstrated that the synergistic effect of the stimuli seen at the protein level was not due to synergistic activation of the eotaxin-1 promoter. Overall, these results suggested that the stimulatory potential of these reporter constructs to both IL-4 and TNF-α was dependent on STAT6 coexpression. Because they all contain the proximal STAT6/NF-κB element, an involvement of this sequence motif appeared likely.
Binding of STAT6 to a specific DNA-element in the eotaxin-1 promoter
The interaction of STAT6 with the proximal STAT6/NF-κB binding sequence was assessed by EMSAs. Nuclear extracts prepared from human dermal fibroblasts were incubated with a ds oligonucleotide probe (−82/46), and the nucleoprotein complexes were resolved in native polyacrylamide gels. In uninduced cells, two specific bands were detected (Fig. 4 A). Extracts from IL-4-treated cells produced an additional band which migrated more slowly than the two original complexes. Addition of anti-STAT6 Abs before addition of the labeled probe specifically reduced the large IL-4-induced complex and led to the formation of a supershifted complex. Although an anti-PU.1-specific as well as an Ab directed against the NF-κB family member p50 did not change the banding pattern, preincubation with an anti-p65 Ab led to disappearance of the original two complexes present in uninduced extracts but not the IL-4-induced band. This suggested that small amounts of NF-κB p65 were present in uninduced cells and interacted with the −82/46 ds probe. The addition of an 50-fold excess of unlabeled ds oligonucleotide containing the functional STAT6-binding site of the human IgE germline promoter (IgE104/83) specifically competed with the formation of the IL-4-induced nucleoprotein complex to the radiolabeled probe. These data demonstrated that the IL-4-induced band contains STAT6.
A similar EMSA analysis was conducted with TNF-α-induced extracts (Fig. 4,B). TNF-α induced the formation of two nucleoprotein complexes that migrated at the positions of the weak constitutive complexes and were different from the one induced by IL-4, suggesting that NF-κB p65 was induced. Incubation with Abs directed against STAT6, bcl-6, or NF-κB p50 had no effect on the formation of the complexes. In contrast, NF-κB p65 Abs led to almost complete disappearance of the TNF-α-induced (and constitutive; compare with Fig. 4 A) bands and formation of a supershifted complex. In addition, successful competition with an authentic NF-κB ds oligonucleotide from the murine Ig Cκ enhancer for both TNF-α-induced complexes was observed (data not shown). These data suggested that this cytokine stimulated the interaction of p65 containing κB proteins but not STAT6 to the composite STAT6/NF-κB element.
Involvement of the STAT6 response element in promoter activation
To determine whether the STAT6/NF-κB-binding site was involved in STAT6-mediated cytokine induction, three different point mutations were introduced into the pGL3-EO2 construct. One mutation (M1) specifically altered the palindromic TTC of the STAT6 site into TAT (Fig. 2). This change has been shown earlier to ablate IL-4-induced STAT6 binding and trans activation in the human IgE germline promoter (28). Mutation M3 affected the polypyrimidine half site of the NF-κB element and mapped outside of the STAT6 core sequence. The M2 mutant affected the overlapping portion of this putative regulatory unit. The effect of these changes on transcription factor interaction was monitored by EMSA. A ds oligonucleotide probe containing the M1 mutation (−82/46 m1) was unable to bind STAT6 in IL-4-induced extracts but still retained the ability to form NF-κB nucleoprotein complexes on TNF-α stimulation (Fig. 5). Conversely, a M3 mutation containing probe was able to bind STAT6 but not NF-κB proteins. STAT6 binding appeared to be much stronger than with the wild-type probe. This may be explained either by a higher specific activity of the M3 mutant ds oligonucleotide or by an increased affinity for the protein. It is known that neighboring nucleotides are involved in fine tuning of the affinity of DNA-binding proteins to its core recognition sequence. The ds oligonucleotide M2 probe did not form any nucleoprotein complexes consistent with the mutation located in the overlapping portion of the composite STAT6/NF-κB element.
Transient transfection of the reporter constructs containing these mutations into HEK293 cells revealed that all three mutants displayed a lower constitutive activity compared with the wild-type plasmid (Fig. 6,A, left). This suggested that the STAT6/NF-κB site acted as a positive regulatory cis element. In STAT6-cotransfected cells, the IL-4 response of the M1 and M2 mutant plasmids was abrogated, demonstrating that cytokine inducibility by STAT6 was mediated via the proximal STAT6-binding site. In contrast, the NF-κB-specific M3 mutation responded to IL-4 in a manner comparable to that of the wild-type plasmid, suggesting that the NF-κB binding site was not involved in IL-4-mediated promoter trans activation. The phenotype of the three mutant reporter constructs was identical when the transfectants were incubated with IL-13 (data not shown). Interestingly, TNF-α inducibility of the STAT6-specific M1 mutation was also completely abrogated. The double defective M2 mutant plasmid showed the same phenotype, whereas in the NF-κB-specific M3 plasmid TNF-α inducibility was partially reduced. These data showed that eotaxin-1 promoter activation by both cytokines required the presence of STAT6 and a functional STAT6-binding site. The overlapping NF-κB element was partially involved in the TNF-α but not in the IL-4 response. To assess the specificity of STAT6 in eotaxin-1 promoter activation after TNF-α stimulation, an IL-8 promoter luciferase reporter construct which has been shown to respond to TNF-α treatment (20) was tested under identical experimental conditions. The inducible phenotype of this plasmid was observed in the absence or presence of cotransfected STAT6 expression vector (Fig. 6 A, right). These results demonstrated that the dependence of eotaxin-1 promoter activation on STAT6 in response to TNF-α was specific.
The same constructs were transiently transfected in donor 2 primary fibroblasts (Fig. 6 B). Similar to the situation in HEK293 cells, the wild-type EO2 construct was inducible with IL-4 or TNF-α alone, whereas the cytokine combination did not lead to a further increase in luciferase expression. Also the phenotype of the three mutation plasmids was essentially identical with the one in HEK293 cells, with the exception that the constitutive promoter activity of the M3 construct was significantly lower compared with the wild-type plasmid. These data demonstrated that in primary human fibroblasts an intact STAT6 site was required for mediating inducibility of the eotaxin-1 promoter against both IL-4 and TNF-α.
Eotaxin-1 protein production is STAT6 dependent
Further confirmation for the involvement of STAT6 in eotaxin-1 regulation was obtained at the protein level. Eotaxin-1 was produced in IL-4-stimulated HEK293 cells that had been transiently transfected with a STAT6 expression vector but not in mock transfected cells (Fig. 7). Interestingly, TNF-α stimulation also led to eotaxin-1 production in STAT6-expressing cells only. Similar to primary fibroblasts, a synergistic effect was measured in the presence of both cytokines. In mock transfected cells, no eotaxin-1 secretion was measured. Importantly, the secretion of the known TNF-α-inducible chemokine MCP-1 measured in the same supernatants was very similar irrespective of the presence of cotransfected STAT6, demonstrating that HEK293 cells are not generally defective in TNF-α signal transduction and that the effect seen with eotaxin-1 was specific. Further evidence for the involvement of STAT6 in eotaxin-1 regulation was obtained in normal fibroblasts transfected with increasing amounts of STAT6-ΔTD DNA. The plasmid expresses a mutant STAT6 protein that lacks the carboxy-terminal trans activation domain. This polypeptide has been shown earlier to act in a trans-dominant negative fashion (29). The transfected cells were induced with cytokines for 48 h before eotaxin-1 protein was monitored in the cell supernatants. Expression of this STAT6 mutant in transiently transfected primary fibroblasts led to strong inhibition of eotaxin-1 protein expression in either IL-4- or TNF-α-induced cells (Fig. 8). Higher amounts of DNA were not well tolerated by the cells. The effect, however, was specific because transfection of empty vector was significantly less inhibitory. In addition, no difference between vector and STAT6-ΔTD plasmid was observed when the same supernatants were assayed for MCP-1 (data not shown). These results supported the conclusion that STAT6 is a mediator of both TNF-α- and IL-4-driven eotaxin-1 secretion in fibroblasts but not of TNF-α-driven MCP-1 synthesis.
This study was aimed at a molecular understanding on the individual roles of IL-4 and TNF-α during their synergistic induction of eotaxin-1 expression in fibroblasts. A similar study was recently conducted in epithelial cells (16). The results obtained in the present effort extend the role of STAT6 as a mediator of IL-4-induced eotaxin-1 production to fibroblasts. In addition, we demonstrate that TNF-α stimulation also is dependent on STAT6; TNF-α-inducible reporter gene expression was dependent on an intact STAT6-binding site both in HEK293 cells and in primary fibroblasts. In addition, eotaxin-1 protein synthesis driven by TNF-α was observed only in STAT6-expressing HEK293 cells and could be inhibited by a trans-dominant negative form of STAT6 in primary fibroblasts. The data demonstrated that HEK293 cells displayed the same phenotype as primary fibroblasts and therefore represent a valid cellular system with which to study these aspects of eotaxin-1 gene regulation. A number of possible scenarios can be envisioned to explain this result. The most straightforward explanation would be the direct activation of STAT6 by TNF-α. Support for this comes from a recent article describing STAT6 phosphorylation in adipocytes upon TNF-α treatment (30). Our efforts including EMSA analysis and immunoblotting with phosphotyrosine-specific anti-STAT6 mAb have thus far failed to detect a similar phenomenon in HEK293 cells or in primary adult fibroblasts (Figs. 4 B and 5 and data not shown). Even in HEK293 cells that ectopically expressed wild-type STAT6, no activation of this factor in response to TNF-α could be measured (data not shown). A more indirect possibility may be that TNF-α induces the expression of IL-4 or IL-13 (31) which then, in an autocrine manner, activates eotaxin-1 promoter activity via the STAT6 pathway. This possibility was addressed by measuring IL-4 and IL-13 protein in supernatants of TNF-α-induced fibroblasts. In addition, we attempted to block a possible autocrine action of IL-4 by adding neutralizing anti-IL-4 Abs to TNF-α-stimulated cultures and to measure IL-4 mRNA (data not shown). All efforts failed to indicate the presence of IL-4, suggesting that a TNF-α-induced IL-4 autocrine mechanism was unlikely. A related possibility may be that TNF-α stimulation induced the expression of other factors able to activate STAT6, such as platelet-derived growth factor (32). Another candidate may be bradykinin, which has been shown recently to induce eotaxin synthesis in fibroblasts (33). Experiments are currently ongoing to address these various possibilities.
The dependence of TNF-α on STAT6 for eotaxin-1 promoter activation appeared to be specific. In the same experimental system, an IL-8 promoter construct was responsive to the cytokine in the absence of functional STAT6, and ectopic expression of the transcription factor did not lead to further activation. Similar results were obtained at the protein level where the TNF-α-induced production of MCP-1 was not dependent on STAT6, whereas eotaxin-1 could be secreted only in STAT6-transfected cells. These results demonstrated that the three different TNF-α-inducible genes could be distinguished by their requirement for functional STAT6 protein to respond to the cytokine stimulus.
The composition of NF-κB proteins induced by TNF-α in fibroblasts contained NF-κB p65 but not p50, whereas both subunits were detected in epithelial cells (16). Perhaps related to this difference was the finding that mutations in the NF-κB-specific pyrimidine half site of the composite STAT6/NF-κB sequence abrogated TNF-α inducibility in an epithelial cell line (16) in contrast to only a partial effect in HEK293 cells.
Our current model of the individual functions of IL-4 and TNF-α to stimulate eotaxin-1 gene promoter in HEK293 and fibroblasts can be summarized as follows. IL-4 activates STAT6 which is able to bind DNA and acts as the predominant player at the level of transcriptional activation of the eotaxin-1 promoter. TNF-α-induced NF-κB may act as transcriptional costimulus in a STAT6-dependent manner using an as yet unknown mechanism.
The synergistic effect of the stimuli on eotaxin-1 protein production is another point worth discussing. The data demonstrate that TNF-α did not synergize with IL-4 in the activation of eotaxin-1 promoter reporter gene constructs both in HEK293 cells and in donor 2 fibroblasts. It is possible, however, that the reporter constructs used did not contain important cis-acting regulatory DNA elements involved in a possible synergism at the level of transcription initiation. Alternatively, synergy may be achieved by a possible role of IL-4 or TNF-α in chromatin opening and gene accessibility in combination with transcription initiation. Such effects would not be seen in transient transfection experiments.
Another explanation for the synergistic effect is suggested by the time course experiments. TNF-α alone was not able to induce eotaxin-1 expression in donor 1 fibroblast cells, yet it synergized with IL-4 to increase the levels of eotaxin-1 mRNA and also secreted chemokine. Its effect on eotaxin-1 transcripts was clearly seen as early as 2 h. This suggested a direct mode of action. Collectively, it is therefore conceivable that TNF-α is the predominant cytokine driving the synergy and that it acts mainly at the posttranscriptional level. This role of TNF-α has been described for a number of other genes, including bradykinin receptor (34), syndecan-4 (35), and FcγRIIb (36).Two AUUUA sequence motifs are located in the 3′-untranslated region of the eotaxin-1 mRNA. These sequences have been implicated in the stabilization of mRNA (37). Therefore, the possibility existed that the synergistic function of TNF-α may be related to stabilization of eotaxin-1 transcripts. We have compared the half-lives of IL-4-induced eotaxin-1 mRNA with the one of IL-4/TNF-α-induced transcripts in cells treated with actinomycin D. The results demonstrated that eotaxin-1 transcripts under both conditions are very stable with a half-life of >18 h (data not shown). The long stability of eotaxin-1 mRNA is in good agreement with previous measurements (38). These data suggested that TNF-α-induced stabilization of eotaxin-1 transcripts is unlikely to be the mode of action of the cytokine to synergize with IL-4.
The architecture of the eotaxin-1 promoter is reminiscent of the regulatory region of the human IgE germline gene (39, 40). In both promoters a STAT6 binding site is flanked by a NF-κB-binding motif. Although binding of κB-factors appear to be dispensable for IL-4-mediated eotaxin-1 promoter activation, NF-κB proteins, together with the transcription factor PU.1, are critically involved in cytokine-driven IgE germline promoter activity (19). This functional difference may be related to the different stimuli that induce DNA binding of NF-κB to their respective binding site. Agonistic anti-CD40 Abs did not synergize with IL-4 to express eotaxin-1 (data not shown), whereas they act as costimulus for IL-4-induced IgE germline gene expression (28).
The dependence of eotaxin-1 synthesis on STAT6 on cytokine stimulation may also explain some aspects of the phenotype observed in STAT6-deficient mice. In mouse models of allergic lung inflammation (41, 42, 43), contact hypersensitivity (44), and allergic diarrhea (45), STAT6 deficiency was accompanied by a lack of eosinophil influx into the sites of allergic inflammation. This effect may be at least partially due to defective eotaxin-1 production in these animals.
In summary, the data strengthen the importance of STAT6 as key player in situations of allergic inflammation and therefore as target for therapeutic intervention.
We thank Ivan Lindley for providing the IL-8p construct and Walter Grimling for skillful artwork.
Abbreviation used in this paper: MCP, monocyte chemoattractant protein.