INSERM U345, Institut Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
Correspondence to: M. Papiernik; E-mail: papiernik{at}necker.fr
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Abstract |
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Keywords: CD4 CD25+, cytokines, IL-10, regulatory CD4 T cells, superantigen
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Introduction |
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In this study we examined IL-2R-deficient mice, which are devoid of CD4 CD25+ T cells and over-express various cytokines after bacterial SAG (bSAG) stimulation in vivo. We found that this abnormal cytokine expression could be controlled by in vivo injection of normal CD4 CD25+ T cells, but not by CD4 CD25+ T cells from IL-10 KO mice. We deduce that IL-10 produced by natural CD4 CD25+ T cells is needed for this regulatory function.
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Methods |
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Antibodies and immunofluorescence
The following antibodies were used: anti-CD4 (clone GK 1.5), anti-CD8 (clone 53-6.7), anti-CD25 (clone PC 61), anti-IL-10 receptor (IL-10R) (clone 1B1) (16) and anti-CD3 (clone 145-2C11). They were used purified, uncoupled or coupled to FITC, biotin or phycoerythrin (PE). Biotinylated antibodies were revealed with streptavidinCyChrome (Becton Dickinson). Subpopulations of labeled cells were analyzed in a FACScan cytometer (Becton Dickinson, Mountain View, CA) with the Lysys II program for data acquisition and analysis.
Cell separation
Peripheral lymph nodes were removed and used to prepare cell suspensions in sterile conditions. CD4 T cells were prepared by negative selection. Briefly, lymph node cells were incubated with purified anti-CD8 antibody (clone 53-6.7), and then with anti-rat antibody-coated magnetic beads to remove CD8 T cells and with anti-mouse Ig-coated beads to remove B cells (Dynabeads; Dynal, Oslo, Norway). CD8 and B cells coated with magnetic beads were then depleted with a magnet. Purified CD4 T cells were labeled with biotinylated anti-CD25 (clone PC61). CD4 CD25+ T cells were positively selected using MACS microbeads (Miltenyi Biotech, Paris, France). Briefly, CD4 CD25- cells were first eluted from the column, then CD4 CD25+ T cells (retained on the column) were eluted after removing the column from the magnetic field. CD4 CD25+ T cells were usually 9095% pure.
In vivo superantigen treatment and cytokine assays
Normal B6 and IL-2R KO mice were injected with 50 µg of staphylococcal enterotoxin B (SEB; Sigma, France) i.p. Serum was recovered 1, 2, 3, 4 and 5 h after the injection. In cell transfer experiments with irradiated mice, serum was recovered 2 h after SEB injection.
IL-2, IL-10, IFN- and tumor necrosis factor-
were assayed in serum with ELISA kits (R & D Systems Europe, Abingdon, UK) according to the manufacturer's recommendations. Results are expressed as pg/ml serum.
Cell transfer
IL-2R KO mice 57 weeks old were sublethally irradiated (600 rad) and injected i.v. on the same day with 106 purified CD4 CD25+ or CD4 CD25- T cells. Control mice were irradiated but not injected. CD4 CD25+ and CD4 CD25- T cells were from normal B6 BA mice, congenic for the expression of Thy-1, which was used to follow donor cell reconstitution of irradiated mice.
In some experiments IL-2R KO mice were injected with 106 purified CD4 CD25+ or CD4 CD25- lymph node cells from IL-10 KO mice. The congenic marker was not available in this case.
Fifteen days after irradiation, mice were injected with 50 µg SEB i.p. and killed 2 h later. Serum was recovered and stored at 20°C. Lymph nodes and spleens were removed, and cell suspensions prepared. Absolute numbers of total CD4 T cells, and donor and host CD4 T cells were determined by cell counting, and anti-CD4 and anti-Thy-1 labeling when possible.
IL-10R blockade in vivo
Normal B6 mice were injected i.p. (3x500 µg) with purified anti-IL-10R antibody (clone 1B1) (16) or saline. The second injection was given 24 h after the first and the third 2 h later. SEB (50 µg) was injected 30 min after the last antibody injection. Mice were killed 2 h later and serum was stored at 20°C until cytokine assay.
In vitro culture
CD4 CD25- T cells from B6 BA mice were purified and cultured (106 cells/well) in 100 µl of culture medium (RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1% sodium pyruvate, 10-5 M merceptoethanol and 10% FCS). CD4 CD25+ T cells (105 or 2x105) from B6 BA or IL-10 KO mice were added to CD4 CD25- T cells. Cells were cultured in wells coated overnight with anti-CD3 (10 µg/ml, 50 µl/well). Phorbol myristate acetate (PMA; 50 ng/ml) was added at the initiation of the cultures. Cells were cultured for 48 h at 37°C with 5% CO2.
After culture the absolute number of living cells per well was determined in the Trypan blue exclusion test. Pooled supernatants from several wells in each group were stored at 20°C for cytokine assays.
Statistical analysis
Results are expressed as means ± SEM. Comparisons were made using Student's t-test and differences were considered significant when P < 0.05.
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Results |
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Thus, after SAG administration, mice lacking CD25 expression escape the normal control of cytokine production and produce much higher levels of IL-2, IFN- and TNF-
, as well as the immunoregulatory cytokine IL-10.
Reconstitution of IL-2R KO mice by natural CD4 CD25+ regulatory T cells corrects the burst of SAG-induced cytokine production
Sublethally irradiated IL-2R KO mice were injected with 106 natural CD4 CD25+ or CD4 CD25- T cells purified from normal mice lymph nodes. Fifteen days later they were injected with SEB and serum was collected 2 h later. At this time point the absolute number of total CD4 T cells (1520x106 in the spleen plus lymph nodes) was identical whether mice were simply irradiated or both irradiated and injected with donor CD4 T cells (CD25+ or CD25-) (Fig. 2
). Importantly, the absolute number of donor CD4 T cells was identical in mice injected with CD4 CD25+ or CD4 CD25- T cells (
2.5x106 in the spleen plus lymph nodes). Similar numbers of CD8 T cells were also found in mice injected with CD4 CD25+ or CD4 CD25- T cells (data not shown). Injection of CD4 CD25- T cells from normal B6 mice to IL-2R
KO mice did not modify the elevated cytokine secretion (mean values 16298 pg IL-2/ml serum in CD4 CD25- reconstituted mice compared to 14492 pg in control mice, 2452 pg IFN-
versus 1896 pg in control mice and 264 pg IL-10 versus 272 pg in control mice). Reconstitution with CD4 CD25+ T cells significantly reduced cytokine production compared to both control and CD4 CD25- T cell injected mice (mean values: 3676 pg IL-2/ml of serum, P < 0.004; 60 pg of IFN-
, P < 0.01; and 90 pg IL-10, P < 0.01) (Fig. 3A
).
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Natural CD4 CD25+ T cells from IL-10, KO mice do not regulate SAG-induced cytokine production by IL-2R KO mice in vivo
In the model of IBD induction by transfer of CD4 CD45RBhigh cells, transforming growth factor (TGF)-ß and especially IL-10 mediate the suppression of the inflammatory disease by regulatory CD45RBl°w cells (1719). As we found that IL-2R KO mice produced high levels of IL-10 after SEB injection and that these high levels were reduced by natural CD4 CD25+ T cell transfer, IL-10 may not have been involved in the control of cytokine production in this model. Alternatively, IL-10 produced by CD4 CD25+ cells during cellcell interactions may have been required. IL-10 is produced by numerous cell types. Circulating IL-10 produced by cell types excluding CD4 CD25+ T cells, as it is the case in IL-2R
KO mice (Fig. 1
), may be ineffective. To test these two hypotheses, CD4 CD25+ and CD4 CD25- T cells from IL-10 KO mice were injected into irradiated IL-2R
KO mice. Two weeks later, IL-2, IFN-
and IL-10 levels were measured in serum 2 h after SEB injection. As shown in Fig. 3
(B), CD25+ T cells from IL-10 KO mice did not down-regulate IL-2, IFN-
and IL-10 production in IL-2R
KO mice (mean values: 8143 pg IL-2/ml serum in CD4 CD25+ T cell reconstituted mice compared to 9242 pg in control mice, 1364 pg IFN-
compared to 1113 pg in control mice and 372 pg IL-10 versus 245 pg in control mice). In terms of the absolute number of T cells expressing CD25 (expressed only on donor T cells), CD4 CD25+ T cell numbers in recipients were similar after injection of natural CD4 CD25+ T cells from either normal B6 or IL-10 KO mice, showing that their reconstitution capacity was identical (not shown). Interestingly, CD4 CD25- T cells from IL-10 KO mice induced enhanced production of IL-2 in the serum of their IL-2R
KO recipient mice (27311 pg/ml serum) and of IFN-
(8323 pg/ml serum) compared to control mice (P < 0.04 and P < 0.004 respectively). This is linked probably to the capacity of cells from IL-10 KO mice to produce high levels of these cytokines after SEB stimulation in vivo (20). Indeed, IL-10 which they did not produce was not enhanced (289 versus 245 pg in the serum of control mice). However, a stimulatory effect of IL-10 KO mouse CD4 T cells on recipient cells cannot be excluded.
These results suggest that IL-10 is involved in the regulation of cytokine over-production by IL-2R KO mice transferred by CD4 CD25+ regulatory T cells. Moreover, IL-10 has to be produced by CD4 CD25+ regulatory T cells themselves.
IL-10 is also involved in the control of cytokine production by normal T cells both in vivo and in vitro
The results described above lead to the conclusion that normal CD4 CD25+ regulatory T cells producing IL-10 can correct the abnormal SAG-induced production of cytokine in mice unable to express a high-affinity IL-2R. We need to demonstrate that IL-10 and regulatory CD4 CD25+ T cells are also involved in the regulation of cytokine production by normal T cells.
In the first set of experiments, we blocked the IL-10R by repeated injections of anti-IL-10R antibody in normal mice, before SEB stimulation in vivo. As shown on Table 1 the in vivo production of IL-2, IFN-
and TNF-
was enhanced by inhibiting IL-10IL-10R interaction. It indeed suggests that IL-10 is also involved in the control of cytokine production in normal mice. Interestingly, in that experimental model, IL-10 production in the serum was not significantly modified.
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It has already been shown that regulatory CD4 CD25+ T cells suppress the proliferation of CD4 CD25- T cells in vitro by a cellcell interaction mechanism which involves the inhibition of IL-2 production. However, the role of IL-10 in this effect has not been demonstrated (8,1012). We attempted to reconcile these findings with our observation of the regulatory role of IL-10 in vivo (Fig. 2 and Table 1
). Purified CD4 CD25- T cells were stimulated by plastic-bound anti-CD3 and PMA. CD4 CD25+ T cells were added at a physiologic ratio (105 or 2x105 CD25+ cells/106 CD25- cells). Production of IL-2, IFN-
and TNF-
was assayed in the culture supernatant 48 h later. Cytokine production was inhibited when B6 CD25+ and B6 CD25- T cells were co-cultured, and the percentage inhibition was proportional to the number of CD25+ T cells. Production of both IL-2 and IFN-
was nearly abrogated by 2x105 CD25+ T cells. TNF-
production was less sensitive to inhibition. We then examined the involvement of IL-10 production by natural CD4 CD25+ regulatory T cells in vitro. Combinations of B6 CD25- cells with CD25+ T cells from IL-10 KO mice were used. As shown in Table 2
natural CD4 CD25+ T cells from IL-10 KO mice can also inhibit the production of cytokine by activated normal CD4 CD25- T cells, but this effect is poor compared to the inhibition induced by normal CD4 CD25+ T cells.
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Collectively, these results suggested that CD4 CD25+ T cells inhibited not only IL-2 as previously shown by others (10,12) but also IFN- and TNF-
production by normal CD4 CD25- T cells activated by anti-CD3 in vitro. The lesser inhibition produced by CD4 CD25+ T cells from IL-10 KO mice suggested that IL-10 was involved in the inhibitory process, although additional mechanisms were involved, at least in vitro.
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Discussion |
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IL-2R KO mice were injected with the bSAG SEB. IL-2, IFN-
and TNF-
production was elevated in the hour following SEB injection in IL-2R
KO mice compared to normal B6 mice. IFN-
and TNF-
are the main cytokines involved in the tissue damage characteristic of IBD. Indeed, injection of anti-IFN-
and anti-TNF-
inhibits the development of IBD in SCID mice injected with pathogenic CD4 CD45RBhigh T cells as efficiently as the injection of CD4 CD45RBl°w regulatory T cells (25,26).
Natural CD4 CD25+ or CD4 CD25- T cells were injected in vivo to irradiated IL-2R KO mice, and serum IL-2 and IFN-
levels were then measured. Contrary to CD4 CD25- T cells, transfer of CD4 CD25+ T cells induced a reduction in IL-2 and IFN-
production after SAG stimulation in vivo.
It has been shown that IL-10 is the most effective cytokine in controlling induced IBD, although TGF-ß is also involved (3,25). Indeed, regulatory T cells from IL-10 KO mice (which themselves normally develop IBD in conventional environment) do not prevent inflammatory disease onset, while injection of anti-IL-10 or anti-IL-10R antibody in vivo abrogates the protective effect of regulatory CD4 CD45RBl°w cells against IBD (19). IL-10 production by regulatory CD4 CD25+ T cells in vivo was also involved in the control of cytokine production in our model. Indeed, transfer of natural CD4 CD25+ T cells from IL-10 KO mice into IL-2R KO mice did not down-regulate their elevated production of IL-2 and IFN-
. Although circulating IL-10 was very low in B6 mice injected with SEB, IL-10 may also be involved in the regulation of cytokine production in normal mice. We found that IL-10R blockade by repeated injection of anti-IL-10R antibody in normal mice led to increased circulating levels of SAG-induced IL-2, IFN-
and TNF-
, suggesting that IL-10 is currently involved in the control of cytokine production in vivo.
However, when IL-2R KO mice were injected with SEB, not only were IL-2, IFN-
and TNF-
levels increased compared to normal B6 mice, but the level of circulating IL-10 also increased markedly, for up to 2 h after SEB challenge. IL-10 is produced by various cell types, including macrophages (27), which can be stimulated by SAG. The circulating IL-10 in IL-2R
KO mice may originate from accessory cells, as CD4 regulatory T cells, which produce IL-10, are lacking in these mice. Thus, despite a high level of circulating IL-10, SEB-induced cytokine production is not properly controlled in IL-2R
KO mice. Contacts between CD4 CD25+ and CD4 CD25- T cells are required for the suppressive effect on IL-2 production and CD4 CD25- T cell proliferation in vitro (8,10). Similar cellcell interactions may be required for the inhibition of cytokine synthesis in vivo, between regulatory (IL-10-producing) CD4 CD25+ T cells and CD4 CD25- T cells. Interestingly, Rag-2 KO mice transferred with IL-10 KO T cells develop IBD and high-level IFN-
production, even though the tissues of Rag-2 KO mice contain macrophages and epithelial cells capable of producing IL-10 (28). Indeed, SAG-treated Rag-2 KO mice produce high levels of IL-10 (personal unpublished data). Collectively, these results suggest that the critical source of IL-10 is the regulatory CD4 CD25+ T cell population.
Interestingly, injection of natural CD4 CD25+ T cells to IL-2R KO mice regulated not only circulating IL-2 and IFN-
production induced by SAG, but also IL-10 production. One possible explanation is a feedback mechanism by which IL-10 produced by regulatory CD4 CD25+ T cells inhibits IL-10 synthesis by other IL-10-producing cells (29). Another is that CD4 CD25+ T cells use the circulating IL-10 available at the time of cell transfer for their own differentiation and/or maintenance. In that case, the target for IL-10 may be essentially the regulatory T cells themselves. Indeed, Groux and collaborators raised CD4 T cell clones by chronic antigen activation in vitro in the presence of IL-10. While they have been differentiated in vitro with IL-10, CD4 clones produced high levels of IL-10, and displayed regulatory functions both in vitro and in vivo (18). Whether IL-10 is also acting directly on the effector cells in vivo has to be demonstrated. Interestingly, we show here that IL-10 KO, CD4 CD25+ T cells transferred to IL-2R
KO mice do not down-regulate circulating IL-10. It suggests that even when they are unable to produce IL-10, CD4 CD25+ T cells cannot use circulating ones, in order to become regulatory. It may be that autocrine production of IL-10 is needed for the maturation of CD4 CD25+ T cells in vivo.
IL-10 may not be the only factor involved in the regulatory functions of CD4 CD25+ T cells. Although IL-10 was involved in the regulation of cytokine production in vivo, other factors (such as TGF-ß or IFN-) (3,25,30) may also play a role. These factors may be differently involved in different experimental protocols, both in vivo and in vitro. CD4 CD25+ T cells differentiated normally in vivo in IL-10 KO mice (data not shown). However, they cannot compensate their lack of IL-10 production in vivo to control SAG-induced cytokine over-production, but can partly overcome the absence of IL-10 to suppress in vitro. Indeed, we found that CD4 CD25+ T cells from IL-10 KO mice were able to inhibit CD4 CD25- T cell production of IL-2, IFN-
and TNF-
induced by anti-CD3 and PMA in vitro. However, the suppressive activity of natural regulatory CD4 CD25+ T cells was much less effective in the absence of IL-10. Different conditions of stimulation or CD25-/CD25+ T cell ratio may explain the discrepancy between our in vitro data and others, where the role of IL-10 was not obvious (8,1012). Higher quantities of regulatory T cell may compensate for the lack of IL-10 by the production of other regulatory cytokine.
CD4 CD25+ T cells variably express markers such as CD45RB, CD44, CD69, CD54 and CD62 ligand (8,15). Further studies are required to determine whether CD4 CD25+ T cells can be divided into different functional subpopulations using different regulatory strategies.
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Abbreviations |
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bSAG bacterial superantigen |
IL-2R![]() ![]() |
IL-10R IL-10 receptor |
KO knockout |
IBD inflammatory bowel disease |
PMA phorbol myristate acetate |
SAG superantigen |
SEB staphylococcal enterotoxin B |
TGF transforming growth factor |
TNF tumor necrosis factor |
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References |
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