Jun 2, 1997 - Yvonne Samstag2, Hans-Martin Jáck3, Andre Gessner, Martin Ro¨llinghoff and. Michael Lohoff. Institute of Clinical Microbiology, University of ...
International Immunology, Vol. 9, No. 9, pp. 1347–1353
© 1997 Oxford University Press
A dominant mechanism coordinately suppresses the expression of Th2 lymphokines Angela Weiss*, Johannes Waiser, Edgar Serfling1, Gabriele Nebl2, Yvonne Samstag2, Hans-Martin Ja´ck3, Andre Gessner, Martin Ro¨llinghoff and Michael Lohoff Institute of Clinical Microbiology, University of Erlangen/Nu¨rnberg, Wasserturmstrasse 3, 91054 Erlangen, Germany 1Institute of Pathology, University of Wurzburg, 97080 Wurzburg, Germany ¨ ¨ 2Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany 3Med Klinik III, University of Erlangen/Nurnberg, 91054 Erlangen, Germany ¨ Keywords: IL-4, lymphokine regulation, Th1/Th2
Abstract this study, we present evidence for a negatively acting control mechanism that coordinately suppresses the synthesis of the Th2 lymphokines IL-4, IL-5 and IL-6. This control mechanism operates in the murine thymoma cell line BW 5147. When cells of this line were fused to four independently established, well-defined Th2 cell clones, all resulting 74 lymphokine-secreting hybridomas secreted IL-2 which was not secreted by any of the parental Th2 cell clones. Most interestingly, however, none of the 74 hybridomas retained the capacity of the parental Th2 cells to express IL-4. Likewise, the secretion of IL-5 and IL-6 was also suppressed. Obviously, BW 5147 cells dominated the pattern of lymphokines produced, although the lymphokine pattern of Th2 cells was previously considered to be irreversibly fixed due to terminal differentiation of these cells. Suppression of IL-4 production was also observed at the mRNA level, as tested in Northern blot assays. Putative DNA target sequences for suppression of IL-4 gene transcription were not part of the proximal IL-4 promotor regions. Remote DNA control sequences may exist which coordinately regulate the proper, stage-specific expression of the Th2 lymphokines IL-4, IL-5 and IL-6.
Introduction T helper cells have been subdivided into two major subsets, designated Th1 and Th2 cells, according to the lymphokine pattern they secrete. Th1 cells secrete IL-2, lymphotoxin and IFN-γ, whereas Th2 cells produce IL-4, IL-5, IL-6, IL10 and IL-13 (1). Recent studies have suggested that both subsets differentiate from a common precursor cell, termed Thp, that predominantly expresses IL-2, but not IFN-γ (2,3). IL-12 and IL-4 respectively have been shown to drive the development of Thp cells into either the Th1 or the Th2 subset (4,5). Despite our knowledge on the development of these cells, there is little information on the putative molecular mechanisms leading to the selective lymphokine
production in Th cells. The majority of studies conducted so far have addressed this issue by analyzing the proximal segments of the IL-2 and IL-4 promoters that are required for the optimal expression of these lymphokines in tumor cell lines and, in a few cases, in Th2-like cells (6–11). No extensive studies have been performed to analyze why T cells belonging to the subsets of Th1 and Th2 cells coordinately express one whole Th-specific set of lymphokines. In the present study, we approach this question using T cell hybridomas made by fusion of BW 5147 thymoma cells (secreting lymphokines of Thp type) with T cells from four different, well-defined long-term cultured Th2
*In partial fulfilment of the requirements of the doctoral thesis Correspondence to: M. Lohoff Transmitting editor: K. Eichmann
Received 23 February 1997, accepted 2 June 1997
1348 Coordinate regulation of Th2 lymphokine production cell clones. We show that the thymoma cells dominantly suppress the production of the Th2-type lymphokines IL-4, IL-5 and IL-6 upon fusion with Th2 cells. At the same time, the expression of a Thp-type lymphokine pattern becomes manifest in these T cell hybridomas. Methods Cell lines and fusion The characterization of the four Th2 cell clones, L1/1 (from a BALB/c mouse; specific for a Leishmania major antigen), D10G4.1 (AKR/J mouse; specific for conalbumin), LNC-4 (BALB/c mouse; specific for tuberculin) and BEL-16 (BALB/c mouse; specific for a L. major antigen), and their in vitro propagation have been described (12–15). All T cells were cultured in Clicks-RPMI medium (16). T cells to be fused had been re-stimulated with syngeneic spleen cells and antigen at least 2 weeks earlier. Before fusion, a Ficoll (Pharmacia, Freiburg, Germany) density gradient was run to remove residual cell debris. Fusion to BW 5147 thymoma cells (17) and to rat C58 thymoma cells (18) was performed as published (17); however, using a fusion medium consisting of 50% (w/v) PEG 4000, 10% (v/v) DMSO and 40% medium. Successful fusion was verified by immunofluorescence testing for cell surface markers of parental and hybridoma cells: Thy-1.1 expressed by BW 5147 cells, but not by L1/1, LNC-4 or BEL16 Th2 cells; CD3 and CD4 expressed by all Th2 cells, but none of the thymoma cells; Ox 18 expressed by C58 thymoma cells, but none of the Th2 cells. After fusion, all growing cells that responded to anti-CD3 stimulation simultaneously expressed the markers of both parental cell lines and therefore were considered to be hybridomas. In vitro stimulation and lymphokine assays For the analysis of lymphokine secretion, Th2 cells and T cell hybridomas were stimulated at a density of 23104/well with either ionomycin (500 ng/ml) and phorbol myristate acetate (PMA; 50 ng/ml; both from Sigma, Deisenhofen, Germany) or immobilized anti-CD3 antibodies (19), coated on plastic wells (2 µg/ml) (20). All cultures were performed in 96-well culture plates (Nunc, Roskilde, Denmark) in a final volume of 200 µl. Supernatants were removed after culture for 24 h and tested for the presence of lymphokines. For screening, IL-2 and IL4 were tested by bioassay on HT-2 cells (21), as described (22). For confirmation, IL-2 and IL-4, as well as IL-5 and IFNγ were measured in commercial ELISAs (PharMingen, San Diego, CA). IL-6 was measured by bioassay using 7TD1 cells (23), confirmed by blocking anti-IL-6 antibodies [hybridoma 6B4 (24)], as described (22). The detection limits of these assays are provided in Table 1. For analysis of lymphokine mRNA, T cells were stimulated in 24-well microtiter plates (Nunc; total volume: 1.2 ml; 13106 cells/well) using either immobilized anti-CD3 or PMA plus ionomycin. RNA analysis Total RNA was isolated using the RNeasy Total RNA kit (Quiagen, Hilden, Germany), fractionated on 1.0% agarose gels (20 µg/lane) and blotted onto GeneScreen Plus nylon membranes (NEN, Dreieich, Germany). The Northern blots
were hybridized (25) with the following [α-32P]dCTP-labeled DNA probes: murine IL-2: 40–377 (PstI–HindIII fragment), murine IL-4: 1–356 (BamHI–PstI fragment). Murine β-actin was detected using a PCR-amplified fragment of 574 bp length (26). The Northern blots were scanned using the Epson GT 8000 and ScanPack software (both from Biometra, Go¨ttingen, Germany). For reverse transcription, cDNAs were synthesized at 42°C for 90 min in 20 µl reaction mixtures (27) containing 1 µg RNA, 50 mM Tris–HCl, pH 8.3, 5 mM MgCl2, 1 mM of each dNTP, 2.5 mM oligo(dT), 32 U RNAguard and 17 U AMV reverse transcriptase (all from Pharmacia). For PCR, cDNA was amplified in a 40 µl reaction volume containing 50 mM Tris–HCl, pH 8.3, 2.5 mM MgCl2, 10 mM of each dNTP, 1 U Taq DNA polymerase (Pharmacia) and 100 nM primers during 35 cycles (1 min denaturation 94°C, 1 min annealing 58°C, 1 min extension 72°C). Samples were analyzed on a 1.5 % agarose gel, followed by scanning on the Epson GT 8000. Primers used were as follows: β-actin sense primer 59-CACCCGCCACCAGTTCGCCA-39, β-actin antisense primer 59-CAGGTCCCGGCCAGCCAGGT-39 (amplified fragment 574 bp); IL-4 sense primer 59-TCTCAACCCCCAGCTAGTTGTCAT-39, IL-4 antisense primer 59-CCAGGCATCGAAAAGCCCG-39 (amplified fragment 320 bp); IL-2 sense primer 59-AACAGCGCACCCACTTCAA-39, IL-2 antisense primer 59-TTGAGATGATGCTTTGACA-39 (amplified fragment 441 bp) (28). All primers were located exon-spanning and no PCR products were obtained with genomic DNA under the PCR conditions used. Cell transfections and chloramphenicol-acetyl-transferase (CAT) assays Cells were washed twice and resuspended (53106) in 235 µl CG-medium (Vitromex, Vilshofen, Germany). Then, 25 µg of DNA per sample in 40 µl water was added and electroporated using the gene pulser (BioRad, Mu¨nchen, Germany) set at 960 mF capacitance and 280 V. The following CAT constructs were used: pmIL-4 270 CAT 5/21, pmIL-4 307 CAT 5/21, pmIL-4 797 CAT 5/21 and pmIL-4 6300 CAT 5/21 containing the base pairs –270 to –12, –307 to –12, –797 to –12 or –6300 to –12 of the murine IL-4 promoter; pBL CAT 5/3xPubA IL-4 containing three copies of the Pub A element of the IL-4 promoter and pIL CAT 2/11 containing the base pairs –293 to –7 of the murine IL-2 promoter (11,29). After transfection, cells were allowed to rest (53106/5 ml of culture medium) for 24 h, and then were stimulated for a further 18 h by adding ionomycin and PMA. Then, the cells were harvested, washed, lyzed and used in CAT assays (11,29). Autoradiographies were scanned using the Epson GT 8000 and ScanPack software. Results A total of 302 T cell hybridomas was generated by fusion of four different murine Th2 cell clones to the murine thymoma cell line BW 5147. The hybridomas were stimulated via their TCR complex using anti-CD3 and screened for their lymphokine secretion by testing the supernatant on HT-2 cells, the growth of which is stimulated both by IL-2 and IL-4. A total of 74 of the original 302 hybridomas secreted HT-2growth-promoting activity. The remaining 228 hybridomas
Coordinate regulation of Th2 lymphokine production 1349 Table 1. Summary of lymphokine-production after anti-CD3 stimulation by 74 hybridomas generated by fusion of the indicated Th2 cell lines and BW 5147 thymoma cells Assay
IL-2 ELISA IL-4 ELISA IL-5 ELISA IL-6 bioassay IFN-γ ELISA
Amount of cytokinea
20 , x ,100 pg/ml .100 pg/ml . 10 pg/ml 50 , x ,150 pg/ml .150 pg/ml .0.07 U/ml .100 pg/ml
No. of hybridomas positive (parental Th2 cell clone) L1/1 (n 5 58b)
LNC-4 (n 5 7)
D10G4.1 (n 5 5)
BEL-16 (n 5 4)
21 37 0 0 0 2 0
2 5 0 2 0 ND 0
3 2 0 0 0 ND 0
2 2 0 1 0 ND 0
All T cell hybridomas were stimulated (23104 cells/well) with immobilized anti-CD3 antibodies (2 µg/ml). The 24 h supernatants were harvested and tested for the indicated lymphokines, as described in Methods. aThe smallest numbers refer to the detection limits of the respective assay. bTotal number of hybridomas tested.
were non-reactive to anti-CD3-stimulation (as also found by microscopic examination, unpublished results). Post-fusion events, e.g. lack of CD3 expression, may explain this nonreactivity. For hybridomas scoring positive, the HT-2 growthpromoting activity was identified in ELISAs specific for IL-2 or IL-4. The results of the ELISAs (Table 1) document a very surprising observation: all of the 74 reactive hybridomas produced IL-2, but not IL-4. Secretion of IL-4, but not IL-2 would have been anticipated, because the parental Th cell clones were of Th2 phenotype and secreted 3200 (L1/1), 1200 (LNC-4), 2000 (D10G4.1) and 200 (BEL-16) pg/ml respectively of IL-4, and no detectable IL-2 upon stimulation with anti-CD3. The other parental partner, BW 5147 cells, secreted no detectable IL-2 or IL-4 after stimulation with antiCD3, as anticipated by the lack of surface CD3 in these cells. They did secrete trace amounts of IL-2 (50 pg/ml), but still no IL-4, after stimulation with the phorbol ester PMA and the Ca21 ionophore ionomycin. Similar to IL-4, the synthesis of the Th2-type lymphokines IL-5 and IL-6 was distinctly suppressed in the hybridomas. Only three out of 74 hybridomas secreted trace amounts of IL-5 ,150 pg/ml (Table 1). In control experiments, all parental Th2 cells secreted between 1 and 6 ng/ml of IL-5. Similarly, only two out of 58 hybridomas tested secreted measurable IL-6 bioactivity (0.5 and 700 U/ml respectively), whereas parental L1/1 Th2 cells produced .900 U/ml IL-6. Cell fusion did not create a Th1-like phenotype, because none of the hybridomas expressed measurable amounts of IFN-γ. In summary, these results demonstrate that, after fusion, the phenotype of the Th2 cells changed to a precursor Thp phenotype secreting IL-2, but no IL-4, IL-5, IL-6 and IFN-γ. In order to investigate whether such changes in Th phenotype are possibly created by the fusion of Th2 cells to any tumor cell, we fused one of the Th2 cell clones to a different fusion partner, the rat thymoma cell line C58. Nine hybridomas were generated and two of them produced HT-2 growthpromoting activity. The supernatant of these hybridomas contained both murine IL-2 and IL-4, as tested by a speciesspecific ELISA (exemplified by data for hybridoma CL-8-20 in Table 2). Therefore, fusion to a rat thymoma cell did not suppress the production of IL-4 by the parental murine
Table 2. Lymphokine production by a hybridoma derived from murine Th2/rat thymoma cells T cell
Stimulus
Murine IL-2 (pg/ml)
Murine IL-4 (pg/ml)
C58 C58 C58 L1/1 L1/1 CL-8-20 CL-8-20 Rat spleen cells
none anti-murine CD3 PMA/ionomycin none anti-murine CD3 none anti-murine CD3 concanavalin A
,10 ,10 ,10 ,10 ,10 ,10 550 ,10
,10 ,10 ,10 ,10 2000 ,10 400 ,10
Parental C58 rat thymoma cells, murine L1/1 Th2 cells and hybridoma cells derived thereof (CL-8-20) were cultured in the presence or absence of anti-murine CD3 or PMA and ionomycin, as indicated. After 24 h, supernatants were removed and tested for murine IL-2 and murine IL-4 by ELISA. To control for the species specificity of the ELISA, supernatant derived from rat spleen cells activated by concanavalin A (2.5 µg/ml) were also tested. Recombinant rat IL-4 also scored negative (,10 pg/ml) in the IL-4 ELISA.
Th2 clone. This finding indicates that the phenotype-switch described above for the BW 5147-derived hybridomas is no trivial fusion-mediated event, but rather is due to a suppressive mechanism specific for the murine BW 5147 thymoma cells. In the following experiments, we tested whether suppression of Th2-type lymphokine secretion was due to lack of mRNA. To this end, mRNA of the two hybridomas B10.2 and NR12 originating from fusions of BW 5147 cells with the Th2 clones L1/1 and LNC-4 respectively was analyzed in Northern blot assays after stimulation of the cells with anti-CD3. As shown in Fig. 1 for one out of two experiments with equal results, the RNA from the two anti-CD3-activated hybridomas reacted strongly with an IL-2 probe, but not an IL-4 probe. As expected, mRNA of the parental Th2 cells reacted with the IL-4, but not the IL-2 probe. In all cells, lymphokine mRNA was only detectable upon stimulation. Neither IL-2 mRNA nor IL-4 mRNA was found in the parental BW 5147 cells upon stimulation with anti-CD3 (unpublished results). The hybridoma cells yielded identical results when stimulated for
1350 Coordinate regulation of Th2 lymphokine production
Fig. 1. Northern blot analysis. Cells of the two Th2 clones L1/1 and LNC-4 (A) and of the two hybridomas B10.2 and NR.12 derived from these Th2 clones (B) were stimulated with immobilized anti-CD3 in 24-well microtiter plates (total volume 1.2 ml; 13106 cells/well) for 10 h. The cells were harvested, total RNA was isolated and Northern blot analysis was performed, as described in Methods.
Fig. 2. RT-PCR analysis of BW 5147 cells, B10.2 hybridoma cells and D10G4.1 Th2 cells. Cells were activated either with PMA (50 ng/ml) and ionomycin (500 ng/ml) (BW 5147) or with 2 µg/ml solid-phase coupled anti-CD3 (B10.2 and D10G4.1) for 4 h, RNA was isolated and RT-PCR was performed as described in Methods. Lane 1, marker; lanes 2, 6 and 10, BW 5147; lanes 3, 7 and 11, B10.2; lanes 4, 8 and 12, D10G4.1; lanes 5, 9 and 13, H2O control. Lanes 2–5, IL-2specific RT-PCR; lanes 6–9, IL-4-specific RT-PCR; lanes 10–13, actinspecific RT-PCR.
4, 8 or 12 h with PMA and ionomycin instead of anti-CD3 (not shown). The RNA preparations obtained from B10.2 hybridoma cells were also analyzed using the RT-PCR technique for the presence of IL-2 or IL-4 mRNA (Fig. 2). As anticipated, a strong signal for IL-2 mRNA was detected in B10.2 hybridoma
cells, while no such signal was found in control D10G4.1 Th2 cells which showed an expectedly strong signal for IL-4 mRNA. In addition, a strong IL-4 mRNA signal, but no IL-2 mRNA signal, was detected in parental L1/1 Th2 cells in two different experiments (not shown). Parental BW 5147 cells (stimulated with PMA and ionomycin) displayed a strong signal for IL-2, but no signal for IL-4, confirming the above reported data on their lymphokine secretion in response to these stimuli. Importantly, however, with this very sensitive technique IL-4 mRNA could clearly be identified (Fig. 2, lane 7) in B10.2 hybridoma cells (three experiments), while it was undetectable in control Th1 cells (data not shown). Restriction fragment analysis confirmed that the signal was indeed attributable to IL-4-mRNA (data not shown). This finding indicates that IL-4 transcription was not entirely suppressed in the hybridoma cells. It also shows that the lack of IL-4 synthesis in the hybridoma cells was not due to a fusionrelated trivial event, e.g. an incidental loss or damage of chromosomes. To test for putative cis-acting elements that might be responsible for a suppression of IL-4 gene transcription, we performed transient transfection experiments in hybridoma cells. For comparison, we also transfected control D10G4.1 Th2 cells to be able to refer to already existing transfection data (9) for this Th2 cell clone. We used plasmids containing various segments of the IL-4 promoter linked to a CAT reporter gene. As a control, plasmids containing the murine IL-2 promoter/enhancer were transfected in parallel. The PMA/ ionomycin-induced promoter activities were then analyzed in CAT assays. Results of one out of two CAT experiments with similar results are given in Fig. 3. They show that CAT reporter genes under the control of the IL-4 promoter segments up to a length of 6.3 kb were transcribed in the stimulated hybridoma cells to the same extent as a CAT reporter gene under the control of the IL-2 promoter. In contrast to the results obtained for the hybridoma cells, in Th2 cells only the IL-4 promoter, but not the IL-2 promoter, was active, as reported (9). These data indicate that if DNA sequences are responsible for a putative suppression of IL-4 gene transcription, they are localized outside of the proximal part of the IL-4 promoter. In addition, the data show that the hybridoma cells habor the relevant intracellular signalling pathways and transcription factors necessary for the activation of the IL-4-promoter. Discussion In this study, we have shown that upon fusion of murine Th2 cells to BW 5147 thymoma cells, the phenotype has been changed to a Thp type. This conclusion is derived from the observation that the generated T cell hybridomas lost their capacity to produce the Th2-type lymphokines IL-4, IL-5 and IL-6. Instead, they gained the ability to synthesize the Thpand Th1-product IL-2, but not IFN-γ, another Th1 lymphokine. The switch-off of IL-4 synthesis occurred without exception in 74 individual hybridomas obtained after fusion of four Th2 clones established from different mice and specific for different antigens. Only one hybridoma secreted normal amounts of IL-6, while trace amounts of either IL-5 or IL-6 were secreted by three other hybridomas. The reason for normal IL-6 secretion in one single hybridoma is unclear, but
Coordinate regulation of Th2 lymphokine production 1351
Fig. 3. (A) B10.2 hybridoma cells were transfected with the indicated plasmids, cultured and processed for the CAT assay, as described in Methods. During culture, the cells were either unstimulated (–) or triggered with a combination of PMA and ionomycin (1). Scans of the autoradiographs obtained for the indicated transfections are shown. (B) Integrals of the scans depicted in (A) and of similar scans from D10G4.1 Th2 cells (not shown) were obtained, and used to calculate the induction of CAT activity in stimulated cells. All cells were transfected in duplicates. Bars denote the SD.
may reside in a mutation of the IL-6 promoter in that individual hybridoma. From the uniformity of the results, we conclude that the suppression of Th2 lymphokine synthesis is due to a dominant suppressive control mechanism that coordinately affects Th2type lymphokines. The mechanism was introduced into the hybridomas by the parental BW 5147 thymoma cells which secreted small amounts of IL-2, but not IL-4, upon stimulation with PMA and ionomycin. Thus, according to their lymphokine synthesis, the hybridoma cells may be considered as BW 5147 cells expressing TCR. The suppressive control mechanism does not operate in any thymoma cell, because no comparable suppression of Th2-type lymphokine secretion was observed after fusion of Th2 cells with rat thymoma cells. The mechanism appears to be very potent, because it even dominates a functional transcription machinery for Th2-type lymphokines, a phenotype previously considered to be irreversibly fixed (30,31). A different finding of this study also suggests that, at least in principle, the dormant IL-2 gene of a Th2 cell can be reactivated: when murine Th2 cells were fused to rat thymoma cells, the introduced rat genes in the hybridomas clearly helped to reactivate the murine IL-2 gene (Table 2). In accordance, regained IL-2 synthesis of Th2 cells has also been demonstrated after treatment with cycloheximide (32).
A BW 5147-like suppressive mechanism may explain why Thp cells secrete a lymphokine pattern (2) similar to BW 5147 cells in vivo. However, the fact that Thp cells are CD41CD8–, while BW 5147 cells are CD4–CD8– (data not shown), suggests that the state of differentiation of these cells is not totally comparable. Possibly, BW 5147 cells represent a cell type less mature than Thp cells and contain a transcriptional machinery which normally is present only after differentiation of naive Th cells into Thp cells. The suppressive control mechanism for Th2 lymphokine secretion appears to operate at the level of mRNA, because we were unable to detect IL-4 mRNA in activated hybridoma cells by Northern blotting. However, IL-4 mRNA was detected with the very sensitive RT-PCR technology. This finding indicates that, unlike in Th1 cells, IL-4 transcription was not shut off in the hybridoma cells. In agreement with this result, we have occasionally detected trace amounts of IL-4 in the supernatant of one hybridoma (B10.2) that was extensively studied throughout this analysis and was mostly negative for IL-4 protein. Two mechanisms are conceivable for the suppression of IL-4 secretion at the level of mRNA. IL-4 mRNA could be synthesized at normal levels, but could be altered by post-transcriptional modifications. It has been shown that such modifications may alter the half-life of mRNA for a lymphokine considerably (33): the half-life of IL-2 mRNA in concanavalin A-stimulated human T lymphocytes was increased 5 times by co-stimulation with phorbol esters. Maximal levels of IL-2 mRNA were noted after combined stimulation with ionomycin plus phorbol esters (33). However, even this maximal stimulus yielded only small amounts of IL-4 mRNA (detectable by PCR, but not Northern blotting) in our hybridoma cells. This suggests that the mechanism which modified the amount of cytokine mRNA in the cited study (33) may not be accessible in our hybridoma cells. Recently, another type of post-transcriptional modification has been reported: the level of mature mRNA for a lymphokine can be reduced by influencing splicing pathways which lead to accumulation of more immature RNA species (34,35). However, during our studies we have not obtained any evidence for a significant accumulation of precursor IL-4 RNA in our hybridomas. Also, the uniform suppression of secretion of three different Th2 lymphokines seems to suggest that posttranscriptional modifications are not decisive for the phenotype of the hybridomas. However, experiments are in progress to formally rule out this possibility. A second explanation for the lack of IL-4 mRNA may be suppression of transcription of the IL-4 gene. Such suppression could be caused by selective methylation and subsequent inactivation of all Th2 lymphokine genes in the hybridoma cells. However, the finding that the hybridoma cells are in principle able to synthesize IL-4 mRNA (as tested by RT-PCR) argues against this notion. Alternatively, the reason for reduced Th2 cytokine production in the hybridoma cells may reside in DNA-binding factors that bind to control sequences in the promoter of the respective lymphokine genes and suppress their transcription. If such factors exist, they do not mediate suppression of transcription through proximal IL-4 promoter sequences, because even up to a length of 6.3 kb, the proximal IL-4 promotor region mediated
1352 Coordinate regulation of Th2 lymphokine production the expression of a reporter gene in the hybridoma cells. Thus, the sequences reported (11,36–38) to exert a suppressive effect on IL-4 transcription (including sequences that down-regulate IL-4 transcription in Th1 cells) are not responsible for the suppression of IL-4 synthesis in the hybridoma cells. Instead, remote DNA sequences located in front of or behind the IL-4 gene, or intragenic IL-4 sequences, may be the target of such putative IL-4 suppressors. The existence of such remote target sequences would offer an explanation on why the production of the Th2-type lymphokines IL-5 and IL-6 is also down-regulated in the hybridoma cells: these cytokine genes might have similar target sequences. A possible coordinate control of Th2-type lymphokine synthesis is facilitated by a closely associated location of the IL-4 and IL-5 (and IL-13) genes on chromosome 11 in the mouse and 5 in the human. Common regulatory sequence motifs for the IL-4 and IL-5 genes are also suggested by the observation that IL-4-deficient mice have a disturbed capacity to produce IL-5 (39). A transcriptional enhancer that coordinately regulates the expression of IL-3 and granulocyte macrophage colony stimulating factor in hematopoetic cells has already been described, and is located between these closely linked cytokine genes (40). Such regulator sequences could lead to a change in the chromatin structure of the promotor, as previously described for the proximal IL-2 promotor (41). A change in the chromatin structure would also explain why the production of Th2-type lymphokines was dominantly suppressed in the hybridoma cells, although all transcription factors necessary for successful Th2-type lymphokine synthesis were already present and operative in the parental Th2 cells. After fusion, these factors may no longer have gained access to their target sequences. In conclusion, we have demonstrated a mechanism that coordinately suppresses the secretion of the Th2 lymphokines IL-4, IL-5 and IL-6. Closer analysis of this mechanism may open possibilities for a common exogenous manipulation of the Th2-type immune response. Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 263). The authors thank Ms Susi Bischof for technical assistance.
Abbreviations CAT PMA
chloramphenicol-acetyl-transferase phorbol myristate acetate
References 1 Mosmann, T. R., Cherwinsky, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L. 1986. Two types of murine helper T-cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2348. 2 Swain, S. L., Bradley, L. M., Croft, M., Tonkonogy, S., Atkins, G., Weinberg, A. D., Duncan, D. D., Dedrick, S. M., Durron, R. W and Huston, W. 1991. Helper T-cell subsets: phenotype, function and the role of lymphokines in regulating their development. Immunol. Rev. 123:115. 3 Street, N. E and Mosmann, T. R. 1991. Functional diversity of T lymphocytes due to secretion of different cytokine patterns. FASEB J. 5:171.
4 Hsieh, C. S., Macatonia, S. E., Wolf, S. F., O’Garra, A. and Murphy, K. M. 1993. Development of TH1 cells through IL-12 produced by Listeria-induced macrophages. Science 260:547. 5 Seder, R. A., Paul, W. E., Davis, M. M. and de St Groth, B. F. 1992. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD41 T cells from T cell receptor transgenic mice. J. Exp. Med. 176:1091. 6 Todd, M. D., Grusby, M. J., Lederer, J. A., Lacy, E., Lichtman, A. H. and Glimcher, L. H. 1993. Transcription of the interleukin 4 gene is regulated by multiple promoter elements. J. Exp. Med. 177:1663. 7 Szabo, S. S., Gold, J. S., Murphy, T. L. and Murphy, K. M. 1993. Identification of cis-acting regulatory elements controlling interleukin-4 gene expression in T cells: role for NF-Y and NFATc. Mol. Cell. Biol. 13:4793. 8 Barve, S. S., Cohen, D. A., De Benedetti, A., Rhoads, R. E. and Kaplan, A. M. 1994. Mechanism of different regulation of IL-2 in murine Th1 and Th2 cell subsets. I. Induction of IL-2 transcription by up-regulation of transcription factors with the protein synthesis initiation factor 4E. J. Immunol. 152:1171. 9 Lederer, J. A., Liou, J. S., Todd, M. D., Glimcher, L. H. and Lichtman, A. H. 1994. Regulation of cytokine gene expression in T helper cell subsets. J. Immunol. 152:77. 10 Brombacher, F., Scha¨fer, Th., Weissenstein, U., Tschopp, C., Andersen, E., Bu¨rki, K. and Baumann, G. 1994. IL-2 promoter driven lacZ expression as a monitoring tool for IL-2 expression in primary T cells of transgenic mice. Int. Immunol. 6:189. 11 Chuvpilo, S., Schomberg, Ch., 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 interleukin4 promoter. Nucleic Acids Res. 24:5694. 12 Lohoff, M., Matzner, C. and Ro¨llinghoff, M. 1988. Polyclonal B-cell stimulation by L3/T41 T-cells in experimental leishmaniasis. Infect. Immunol. 56:2120. 13 Kaye, J. S., Gillis, S. B., Mizel, S. B., Shevach, E. M., Malek, T. R., Dinarello, C. A., Lachman, L. B. and Janeway, C. A. 1984. Growth of a cloned helper T cell line induced by a monoclonal antibody specific for the antigen receptor: Interleukin 1 is required for the expression of receptors for Interleukin 2. J. Immunol. 133:1339. 14 Schmidt, E., v. Brandwijk, R., v. Snick, J., Siebold, B. and Ru¨de, E. 1989. TCGF III/P40 is produced by naive murine CD41 T cells but is not a general T cell growth factor. Eur. J. Immunol. 19:2167. 15 Lohoff, M., Steinert, M., Weiss, A., Ro¨llinghoff, M., Balderas, R. S. and Theophilopoulos, A. N. N. A. 1994. Vβ gene repertoires in T cells expanded in local self- healing and lethal systemic murine leishmaniasis. Eur. J. Immunol. 24:492 . 16 Solbach W., Forberg, K., Kammerer, E., Bogdan, C. and Ro¨llinghoff, M. 1986. Suppressive effect of cyclosporin A on the development of Leishmania major-induced lesions in genetically susceptible BALB/c mice. J. Immunol. 137:702. 17 Harwell, L., Skidmore, B., Marrack, P. and Kappler, J. 1980. Concanavalin A inducible, interleukin-2-producing T-cell hybridoma. J. Exp. Med. 152:893. 18 Geering, G., Old, L. J. and Boyse, E. A. 1969. Antigens of leukemias induced by naturally occuring murine leukemia virus: their relation to the antigen gross virus and other murine leukemia viruses. J. Exp. Med. 124:753. 19 Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. and Bluestone, J. A. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl Acad. Sci. USA 84:1374. 20 Lohoff, M., Dirks, M., Rohwer, P. and Ro¨llinghoff, M. 1989. Studies on the mechanism of polyclonal B cell stimulation by TH2 cells. Eur. J. Immunol. 19:77. 21 Watson, J. 1979. Continuous proliferation of murine antigenspecific T lymphocytes in culture. J. Exp. Med. 150:1510. 22 Lohoff, M., Sommer, F., Solbach, W. and Ro¨llinghoff, M. 1989. Coexistence of antigen-specific TH1 and TH2 cells in genetically susceptible BALB/c Mice infected with Leishmania major. Immunobiology 179:412. 23 van Snick, J., Cayphys, S., Vink, A., Uyttenhove, C., Coulie, M. R., Rubira, M. R. and Simpson, R. J. 1986. Purification and NH2terminal amino acid sequence of a T-cell derived lymphokine with
Coordinate regulation of Th2 lymphokine production 1353
24
25
26
27 28
29
30 31
32
33
growth factor activity for B-cell hybridomas. Proc. Natl Acad. Sci. USA 83:9679. Vink, A., Coulie, P. G., Wauters, P., Nordan, R. P. and van Snick, J. V. 1988. B cell growth and differentiation activity of interleukinHP1 and related murine plasmocytoma growth factors. Synergy with interleukin 1. Eur. J. Immunol. 18:607. Gessner, A., Drjupin, R., Lo¨hler, J., Lother, H. and LehmannGrube, F. 1990. IFN-γ production in tissues of mice during acute infection with Lymphocytic Choriomeningitis Virus. J. Immunol. 144:3160. Gessner, A., Schro¨ppel, K., Will, A., Enssle, K.-H., Lauffer, L. and Ro¨llinghoff, M. 1994. Recombinant soluble interleukin-4 (IL-4) receptor acts as an antagonist of IL-4 in murine cutaneous Leishmaniasis. Infect. Immun. 62:4117. Krug, M. S. and Berger, S. L. 1987. First strand cDNA synthesis primed with oligo(dT). Methods Enzymol. 152:316. Montgomery, R. A. and Dallman M. J. 1991 Analysis of cytokine gene expression during fetal thymic ontogeny using the polymerase chain reaction. J. Immunol. 147:554. Serfling, E., Barthelma¨s, R., Pfeuffer, I., Schenk, B., Zarius, S., Swoboda, R., Mercurio, R. and Karin, M. 1989. Ubiquitous and lymphocyte-specific factors are involved in the induction of the mouse interleukin 2 gene in T lymphocytes. EMBO J. 8:46529 Perez, V. C., Lederer, J. A., Lichtman, H. and Abbas, A. K. 1995. Stability of Th1 and Th2 populations. Int. Immunol. 7:869. Szabo, S. J., Jacobson, N. G., Dighe, A. S., Gubler, U. and Murphy, K. M. 1995. Developmental commitment to the Th2 lineage by extinction of IL-12 signaling. Immunity 2:665. Munoz, E., Zubiga, A., Olson, D. and Huber, B. T. 1989. Control of lymphokine expression in T helper 2 cells. Proc. Natl Acad. Sci. USA 86:9461. Bill, O., Garlisi, C. G., Grove, D. S., Holt, G. E. and Mastro, A. M.
34
35
36 37 38 39 40
41
1994. IL-2 mRNA levels and degradation rates change with mode of stimulation and phorbol ester treatment of lymphocytes. Cytokine 6:101. Gerez, L., Arad, G., Efrat, S., Ketzinel, M. and Kaempfer, R. 1995. Post-transcriptional regulation of human interleukin-2 gene expression at processing of precursor transcripts. J. Biol. Chem. 270:19569. Fox, F. E., Chernajovsky, Y. and Platsoucas, C. D. 1994. Inhibition of interleukin 2 production and alteration of interleukin 2 mRNA processing by human T–T cell hybridoma-derived suppressor factors. Hybridoma 13:343. Li-Weber, M., Eder, A., Krafft-Czepa, H. and Krammer, P. H. 1992. T cell-specific negative regulation of transcription of the human cytokine IL-4. J. Immunol. 148:1913. Tara, D., Weiss, D. L. and Brown, M. A. 1993. An activation responsive element in the murine IL-4 gene is the site of an inducible DNA–protein interaction. J. Immunol. 151:3617. Lederer, J. A., Liou, J. S., Todd, M. D., Glimcher, L. H. and Lichtman, A. H. 1994. Regulation of cytokine gene expression in T helper cell subsets. J. Immunol. 152:77. Kopf, M., Le Gros, G., Bachmann, M., Lamers, M. C., Bluethmann, H. and Ko¨hler, G. 1993. Disruption of the murine IL-4 gene blocks Th2 cytokine response. Nature 362:245. Cockerill, P. N., Shannon, M., F., Bert, A. G., Ryan, G. R. and Vadas, M. A. 1993. The granulocyte-macrophage colonystimulating factor/interleukin 3 locus is regulated by an inducible cyclosporin A-sensitive enhancer. Proc. Natl Acad. Sci. USA 90:2466. Siebenlist, U., Durand, D. B., Bressler, P., Holbrook, M. J., Norris, C. A., Kamoun, M., Kant, J. A. and Crabtree, G. R. 1986. Promoter region of the IL-2 gene undergoes chromatin structure changes and confers inducibility on chloramphenicol acetyltransferase gene during activation of T cells. Mol. Cell. Biol. 6:3042.
