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Costimulation via TCR and IL-1 Receptor Reveals a Novel IL-1-Mediated Autocrine Pathway of Th2 Cell Proliferation¹

Magdalena Huber^*, Horst U. Beuscher^*, Peter Rohwer, Roland Kurrle, Martin Röllinghoff^* and Michael Lohoff^2,*

^* Institut für Klinische Mikrobiologie und Immunologie, and Medizinische Klinik III, Friedrich Alexander Universität Erlangen, Erlangen, Germany; and Hoechst-Marion-Roussel Rheumatologie, Wiesbaden, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Previous studies have shown that triggering of Th2 cells via the TCR is sufficient for production of IL-4 but not for proliferation of these cells. Proliferation of Th2 cells occurs only in the additional presence of a costimulatory signal delivered by IL-1. For the majority of Th2 cell clones, this type of proliferation was found to be independent of IL-4. Here, we further investigated the mechanism of IL-4-independent proliferation. We demonstrate that, after costimulation via TCR and IL-1R, but not via either receptor alone, Th2 cells are triggered to produce cell-associated IL-1, as detected at the level of function, protein, and mRNA expression. In the presence of the TCR signal, autocrine IL-1 is then able to costimulate IL-4-independent proliferation of Th2 cells and to further enhance its own production. Thus, our results point to a novel, IL-4-independent, self-amplifying autocrine pathway of Th2 cell proliferation that requires a signal via the TCR and a costimulatory signal via IL-1R. This pathway may explain frustrating results in experimental models that attempted to treat established Th2-mediated diseases in vivo with IL-4-neutralizing agents alone.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Thelper 1 (Th1) and Th2 cells differ in the lymphokines they produce and in their requirements for costimulatory signals. While Th1 cells require costimulation via the TCR and CD28 for production of their autocrine growth factor IL-2 (1), Th2 cells produce their growth factor IL-4 after stimulation via the TCR alone (2). In the absence of TCR stimulation, Th1 and Th2 cells proliferate in response to their respective growth factors, but their proliferation is blocked in the continuous presence of the TCR signal (3, 4). When the TCR-mediated signal is present, Th2 cells require a costimulatory signal for further proliferation, which is mediated by IL-1 (5, 6, 7). In contrast, a comparable costimulatory signal for Th1 cells is unknown to date and is not delivered via CD28 (8). Furthermore, it has been shown that costimulation of Th2 cells via TCR and CD28 induces proliferation mediated by autocrine IL-1 (9).

As mentioned, IL-4 is a growth factor for Th2 cells and is already produced by these cells after stimulation via the TCR alone, although proliferation of TCR-triggered Th2 cells strictly requires the additional presence of IL-1. This strict requirement has been explained as a capacity of IL-1 to render TCR-stimulated Th2 cells sensitive to proliferation induced by IL-4. Hence, the IL-1-induced proliferation of Th2 cells was interpreted to be dependent on IL-4. In a recent study, however, we demonstrated that in a majority of Th2 cell clones, triggering via the TCR and IL-1R led to a type of proliferation which, surprisingly, was independent of endogenous IL-4 (5). In the present study, we further elucidate the mechanism of IL-4-independent proliferation. We identify a novel, self-amplifying autocrine loop for Th2 cell proliferation that is triggered by two simultaneous signals via the TCR and IL-1R and leads to autocrine production of IL-1 and IL-4-independent proliferation in response to IL-1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Immunologic reagents

Purified recombinant murine (rm)³ IL-2 and rmIL-4 were kindly provided by Dr. E. Schmitt, University of Mainz, Germany. Recombinant human (rh)IL-1, rhIL-1ß, and rmIL-1 were provided by Dr Haag (Boehringer, Mannheim, Germany), Dr. D. Boraski (Sclavo, Siena, Italy), and Dr. I. G. Otterness (Pfizer, Groton, MA), respectively. Soluble rhIL-1RII (solIL-1RII) was provided by Behringwerke, Marburg, Germany. Purified anti-IL-4 mAb was obtained by passage of 11B11 hybridoma (10) cell supernatants through protein G columns (Pharmacia, Freiburg, Germany). Anti-mouse IL-1 mAb (11) was purchased from Genzyme (Rüsselsheim, Germany). Polyclonal anti-human (h)IL-1 antiserum was purchased from Endogen (distributed by Biozol, Eching, Germany). Anti-murine CD3 mAb was purified from hybridoma 145-2C-11 (12) cell supernatants using protein A columns (Pharmacia).

T cells

Cloned D10.G4.1 Th2 cells (13) were restimulated (1 x 10⁶ cells/well) every 3 wk in 12-well flat-bottom culture plates (Costar, Cambridge, MA) with 100 µg/ml conalbumin (Sigma, Deisenhofen, Germany) and 5 x 10⁶ irradiated (25 Gy) splenocytes from C3H/HeJ mice (Charles River, Sulzfeld, Germany) in 3 ml Click’s/RPMI (Seromed, Berlin, Germany) medium supplemented as described (14). One week later, the cells were restimulated with fresh medium containing rmIL-2 and rmIL-4 (each at 1 ng/ml). Additionally, 5 to 6 days later, the cells were harvested, extensively washed, and used for the experiments. At this stage, D10G.4.1 cells had a purity of 99.9% as revealed by anti-CD4 staining and FACS analysis.

For the in vivo induction of Th2 cells, BALB/c mice (Charles River) at 6 wk of age were infected with 2 x 10⁷ Leishmania major parasites, as described (14). Three weeks later, the lesion-draining popliteal lymph nodes (LNs) were removed and single-cell suspensions were obtained. Selection of CD4⁺ LN cells was performed using the MACS system and anti-CD8, anti-B220, and anti-Mac-1 mAb conjugated to magnetic beads (all from Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s specifications (15). Enrichment of Th2 cells within the MACS-sorted cells was accomplished by a novel method described in detail elsewhere (16). Briefly, the method uses FACS sorting of cells according to their capacity to extrude the fluorescent dye Fluo-3. MACS-purified LN cells were loaded with Fluo-3 AM (Sigma) and incubated to allow secretion of the dye as described (16). Thereafter, cells were stained with either anti-CD4 Tri-Color mAb (Medac, Hamburg, Germany) or anti-CD4 mAb conjugated to phycoerythrin (PharMingen, Hamburg, Germany). CD4⁺ cells showing decreased Fluo-3 fluorescence (3% of total MACS-sorted cells) were sorted using an EPICS XL cell sorter and Elite software (Coulter, Krefeld, Germany). We have shown in the article cited above (16) that within the LNs of L. major-infected BALB/c mice, this procedure leads to strong selection for T cells of the Th2 phenotype. After sorting, the dye-extruding cells used in the experiments described therein contained 99.9% CD4⁺ T cells and 99.3% dye-extruding cells.

"Stimulator cell" lysates and proliferation assays

Stimulator D10.G4.1 cells were cultured (1 x 10⁶/well in 2 ml of medium) in 12-well plates with or without rhIL-1ß (300 U/ml) or rhIL-1 (10 U/ml) and immobilized (8) anti-CD3 mAb (coating concentration, 2 µg/ml). After 24 h, the cells were harvested, washed, and added (2 x 10⁴/well) in a volume of 40 µl of fresh medium into 96-well flat-bottom microtiter plates (Costar) either coated or uncoated with anti-CD3 mAb (2 µg/ml). Ex vivo-isolated stimulator Th2 cells (2 x 10⁴/well) were prepared by activating the EPICS-sorted cells described above in a total volume of 200 µl medium in 96-well flat-bottom microtiter plates with or without immobilized anti-CD3 mAb and rhIL-1, as described for D10.G4.1 stimulator cells. After 24 h, these plates were washed extensively. To obtain cell lysates, all microtiter plates containing activated stimulator cells (D10.G4.1 or ex vivo-purified cells) were shock frozen at -70°C and thawed.

To all plates containing stimulator cell lysates, resting D10.G4.1 cells (2 x 10⁴/well) were added as "responder cells." Where indicated, wells also received anti-IL-4 mAb (20 µg/ml), solIL-1RII (2 µg/ml), anti-hIL-1 (30 µg/ml), or anti-mIL-1 mAb (5 µg/ml) in a total volume of 200 µl of medium. Responder cells were cultured for 48 h, pulsed with 1.85 x Bq/well [³H]thymidine (New England Nuclear, Dreieich, Germany) for 16 h, and processed for ß-scintillation counting.

Flow cytometry

D10.G4.1 cells (1 x 10⁶/well) were cultured for 4 h in the presence of Brefeldin A (10 µg/ml, Sigma) in 12-well plates (2 ml medium/well) with or without rhIL-1ß (300 U/ml) and immobilized anti-CD3 mAb (coating concentration 2 µg/ml). Thereafter, cells were harvested, washed in PBS, fixed in 75 µl of solution A (Fix & Perm kit, Medac) for 15 min at 25°C, washed again, and incubated in 75 µl of solution B containing digoxigenin (DIG)-conjugated polyclonal rabbit anti-mouse IL-1 Ab (1:100) (17) for 15 min at 25°C. After washing, cells were incubated for additional 15 min at 25°C in 75 µl of solution B containing FITC-conjugated sheep anti-DIG Fab fragments (2 µg/ml) (Boehringer). After washing, cell fluorescence was analyzed in a FACScan using Lysis II software (Becton Dickinson, Heidelberg, Germany). In control samples, anti-IL-1 Ab were preincubated for 1 h with rmIL-1 (10 µg/ml) or rmIL-2 (10 µg/ml) before staining.

Northern blot analysis

D10.G4.1 cells (1 x 10⁶/well) were activated for 6 h in 12-well plates with or without rhIL-1ß and anti-CD3 mAb, as described above. Peritoneal exudate cells were stimulated with LPS (10 µg/ml) for 4 h. After stimulation, total RNA was prepared using the RNeasy Total RNA Kit (Qiagen, Hilden, Germany). Equal amounts of RNA (20 µg/lane) were separated on 1.2% agarose gels, and a Northern blot was performed to detect mRNA for mIL-1 and ß-actin as described (18). The autoradiographs were scanned using an Epson GT 8000 and ScanPack software (Biometra, Göttingen, Germany).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Th2 cells activated via TCR and exogenous IL-1 mediate proliferation of resting Th2 cells independently of IL-4 and exogenous IL-1

Recently, we demonstrated that the majority of Th2 cell clones proliferate independently of IL-4 when they are simultaneously costimulated via TCR and IL-1R (5). In the current paper, we investigated whether this type of Th2 cell proliferation was induced directly by exogenous IL-1 or whether costimulation via TCR and exogenous IL-1 led to endogenous synthesis of a mitogenic molecule by the Th2 cells. To test for this possibility, we stimulated Th2 cells in a first culture phase via TCR (using immobilized anti-CD3 mAb) and IL-1R (using rhIL-1ß) for 24 h. Thereafter, the cells (called stimulator cells) were harvested, washed, lysed, and tested for their ability to induce an IL-4-independent proliferation of responder Th2 cells. For this purpose, we cocultured the stimulator cell lysates with fresh, viable Th2 responder cells of the same Th2 cell clone. Residual rhIL-1ß from the first culture phase and endogenous IL-4 were neutralized by solIL-1RII and anti-IL-4 mAb, respectively. As a control, responder cells were cultured in parallel without stimulator cell lysates but in the presence of anti-CD3 mAb and rhIL-1ß. The results of a representative experiment are depicted in Figure 1.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Effect of lysates of activated Th2 cells on the proliferation of resting responder Th2 cells. Stimulator D10.G4.1 Th2 cells were activated as indicated on the y axis, washed, and added into 96-well microtiter plates either coated (A) or uncoated (B) with anti-CD3 mAb. To obtain cell lysates, the plates were shock frozen immediately. After thawing, resting D10.G4.1 Th2 responder cells and, where indicated, anti-IL-4 mAb, solIL-1RII, or anti-IL-4 mAb and solIL-1RII were added. Where indicated, responder cells were stimulated in the absence of lysates, but the presence of rhIL-1ß (300 U/ml), with (A) or without (B) immobilized anti-CD3 mAb. After 48 h of culture, the responder cells were pulsed with [3H]thymidine, further incubated for 16 h, and processed for ß-scintillation counting. Data represent cpm ± SD of triplicate cultures.

Not only the production of this activity but also the proliferative response to it was dependent on costimulation. This is shown by the finding that responder cells were only able to respond to this activity when they were simultaneously triggered via the TCR (compare Fig. 1, A and B). In the absence of the TCR signal, the proliferation of the responder cells was entirely blocked by anti-IL-4.

The novel costimulatory activity for Th2 cell proliferation is cell associated

The uncharacterized activity could have been represented by a cell-associated or a released molecule. To differentiate between these two possibilities, stimulator cells were again activated by anti-CD3 mAb and exogenous rhIL-1ß. Cell lysates and supernatants were obtained and compared for their capacity to mediate responder cell proliferation independently of IL-4 and exogenous rhIL-1ß. The results demonstrate (Fig. 2) that upon simultaneous triggering of their TCR, responder cells were able to detect the activity (black bars) only within the lysates, but not within the supernatants of stimulator cells. In contrast, in the absence of the TCR signal, responder cells readily proliferated in response to IL-4 present within lysates or supernatants of stimulator cells. Thus, the activity was cell associated and not released by the cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Fragments but not culture supernatants of anti-CD3- and rhIL-1ß-activated Th2 cells costimulate the rhIL-1ß- and IL-4-independent proliferation of responder Th2 cells. Stimulator D10.G4.1 Th2 cells were activated with rhIL-1ß and immobilized anti-CD3 mAb. After 24 h, culture supernatants and stimulator cells were harvested and added separately into 96-well microtiter plates (A, 2 x 104 cells per well; B, 40 µl supernatant/well) with or without immobilized anti-CD3 mAb, and the plates were shock frozen and thawed. D10.G4.1 responder cells and, where indicated, anti-IL-4 mAb or solIL-1RII were added, and the test was processed as described for Figure 1.

Endogenous IL-1 costimulates Th2 cell proliferation independently of IL-4 and exogenous rhIL-ß

Three main features of this activity are very reminiscent of IL-1: first, the proliferative response to IL-1 also requires costimulation via the TCR (5, 6, 7); second, in a recent report, Th2 cells were also shown to produce IL-1 only after costimulation via two different signals, namely via TCR and CD28 (9); third, IL-1 has been reported to be produced in T cells in a cell associated, but not released form (19). Therefore, we investigated whether the activity was mediated via endogenous IL-1. To do this, we used several IL-1 antagonists (11, 20), the specificity of which is depicted in Figure 3: anti-mIL-1 mAb specifically blocked the proliferative response of anti-CD3-triggered responder cells to rmIL-1, but not to rhIL-1 or rhIL-1ß. In contrast, solIL-1RII blocked the activity of rhIL-1ß, but not of rhIL-1 or rmIL-1, whereas anti-hIL-1 blocked the activity of rhIL-1, but not of rhIL-1ß or rmIL-1.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Specificity of anti-IL-1 agents. D10G4.1 Th2 cells were cultured with immobilized anti-CD3 mAb and either rmIL-1 (100 pg/ml) or rhIL-1 (10 U/ml) or rhIL-1ß (300 U/ml). Where indicated, cultures also received solIL-1RII (2 µg/ml), anti-hIL-1 (30 µg/ml), or anti-mIL-1 (5 µg/ml). After 48 h of culture, the responder cells were pulsed with [3H]thymidine, further incubated for 16 h, and processed for ß-scintillation counting. Data represent cpm ± SD of triplicate cultures.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Th2 cells produce IL-1 after combined stimulation via TCR and IL-1R. Stimulator D10.G4.1 Th2 cells were activated with immobilized anti-CD3 mAb and rhIL-1 or rhIL-1ß as indicated. Lysates were obtained and added into microtiter plates (all coated with anti-CD3) as described for Figure 1. D10.G4.1 responder cells and, where indicated, anti-IL-4 mAb, solIL-1RII, anti-hIL Ab, or anti-mIL-1 mAb were added. The test was further processed as described for Figure 1.

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Ex vivo-isolated Th2 cells produce IL-1 after combined stimulation via TCR and IL-1R. Ex vivo-isolated Th2 cells were obtained as described in Materials and Methods. They were activated (2 x 104/well) with immobilized anti-CD3 mAb as described above. After 24 h, the plates containing the cells were extensively washed and, to obtain cell lysates, were shock-frozen at -70°C and thawed. D10.G4.1 responder cells and, where indicated, anti-IL-4 mAb, solIL-1RII, or anti-mIL-1 mAb were added. The test was further processed as described for Figure 1.

Combined stimulation of Th2 cells via TCR and IL-1R induces the expression of IL-1 protein and mRNA

So far, only the functional activity of IL-1 has been identified. The next set of experiments was performed to detect the expression of IL-1 at the protein and mRNA level. For detection at the protein level, we adapted the method of intracellular cytokine staining (22) and FACS analysis for IL-1. Again, cloned Th2 cells were stimulated with or without anti-CD3 mAb or exogenous rhIL-1ß. Then, 24 h later, cells were processed for intracellular staining of IL-1. A representative result is shown in Figure 6: IL-1 protein was detected in individual cells, provided that they had been triggered by a combination of anti-CD3 mAb plus rhIL-1ß but not by either of the reagents alone. Thus, staining at the single-cell level perfectly confirmed the functional data described above. The staining was specific for IL-1 for several reasons: first, an affinity purified antiserum was used that reacts specifically with murine IL-1, but not with IL-1ß (17); second, no staining was obtained when this antiserum was omitted and only the FITC-conjugated secondary Ab was applied (Fig. 6B); and third, staining was blocked by preincubation of anti-IL-1 antiserum with rmIL-1, but not with rmIL-2 (Fig. 6B).

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Costimulation via TCR and rhIL-1ß induces expression of IL-1 protein in Th2 cells. D10.G4.1 cells were stimulated for 4 h, as indicated. Thereafter, the cells were fixed, permeabilized, and stained intracellularly using rabbit anti-mouse IL-1-DIG Ab and sheep anti-DIG-FITC Fab fragments (A). B, Control staining was done, as indicated, using either sheep anti-DIG-FITC Fab fragments alone or rabbit anti-mouse IL-1-DIG Ab preincubated with rmIL-1 or rmIL-2 followed by sheep anti-DIG-FITC Fab fragments.

View larger version (63K): [in this window] [in a new window]   FIGURE 7. Costimulation via TCR and rhIL-1ß induces expression of IL-1 mRNA in Th2 cells. Total RNA was prepared from D10.G4.1 cells that had been stimulated for 6 h as indicated. The RNA together with control RNA from LPS-activated peritoneal exudate cells was analyzed by Northern blotting as described in Materials and Methods. Scans of the autoradiographs are depicted.

Our results establish for the first time a self-amplifying autocrine loop for Th2 cell proliferation; triggering of their TCR by an APC in conjunction with small amounts of IL-1 or IL-1ß leads to amplification of IL-1 production by the Th2 cells and their IL-4 independent proliferation in response to it (Fig. 8). These findings help to explain the differences in the literature with regard to IL-1 production by Th2 cells. While it was reported in several studies that a signal via the TCR alone is sufficient to induce IL-1 production by these cells (19, 23), a recent study did not confirm this result (9). Rather, in that study, a costimulatory signal delivered by CD28 was found to be strictly required for production of IL-1. However, the amounts of IL-1 mRNA produced appeared to be quite low, because they were only detectable using the very sensitive PCR technology. In our study, using the same Th2 cell clone, we detected substantial amounts of IL-1 mRNA by Northern blotting, i.e., without requiring amplification. In the cited studies, Th2 cells have probably been analyzed in different states of activation. TCR triggering of Th2 cells that are not sufficiently resting may by itself lead to production of small amounts of IL-1 in the absence of a costimulus. Such IL-1 then self-amplifies further production of IL-1, particularly when the cell density is high. If the cells are resting, however, they are critically dependent on costimulation, e.g., via CD28, to produce IL-1. Yet, the amounts of IL-1 detected after costimulation via TCR and CD28 are very small and may serve only as a starter for production of higher amounts by the self-enhancing loop described in our article. In the absence of a B7-CD28 interaction, small amounts of IL-1 derived from other cellular sources, such as APC or other already-activated Th2 cells, may serve to costimulate IL-1 production of TCR-triggered Th2 cells and initiate their autocrine proliferative loop.

View larger version (14K): [in this window] [in a new window]   FIGURE 8. A model that illustrates a novel pathway of Th2 cell proliferation. Triggering of Th2 cells via Ag-peptide presented by an APC (Signal 1) and IL-1 or B7 expressed by the APC (Signal 2) leads to the self-amplifying production of IL-1 by Th2 cells and IL-4-independent proliferation in response to IL-1.

	Acknowledgments

 
We acknowledge the assistance of Mrs. Susanne Bischof and the technical help of Mrs. Claudia Feulner at our institute. We thank Dr. G. Schuler and Dr. D. Ferrick for critical revision of the manuscript.

	Footnotes

 
¹ This work was supported by Grant SFB 263 from the Deutsche Forschungsgemeinschaft and by the Johannes und Frieda Marohn Stiftung, Erlangen, Germany.

² Address correspondence and reprint requests to Dr. Michael Lohoff, Institut für Klinische Mikrobiologie und Immunologie der Universität Erlangen, Wasserturmstrasse 3, 91054 Erlangen, Germany. E-mail address:

³ Abbreviations used in this paper: rm, recombinant murine; rh, recombinant human; LN, lymph node; solIL-1RII, soluble rhIL-1RII; DIG, digoxigenin.

Received for publication October 15, 1997. Accepted for publication January 5, 1998.

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