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The Journal of Immunology, 2006, 176: 2857-2863.
Copyright © 2006 by The American Association of Immunologists

Self-Limitation of Th1-Mediated Inflammation by IFN-{gamma}1

Markus Feuerer2,*, Katharina Eulenburg*, Christoph Loddenkemper{dagger}, Alf Hamann* and Jochen Huehn3,*

* Experimentelle Rheumatologie, Charité Universitaetsmedizin Berlin, Campus Mitte, Berlin, Germany; and {dagger} Institut fuer Pathologie, Charité Universitaetsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IFN-{gamma} is an effector cytokine of cell-mediated immunity that plays an essential role in both innate and adaptive phases of an immune response. Interestingly, in several Th1-dependent autoimmune models, lack of IFN-{gamma} is associated with an acceleration of disease. To distinguish the influence of IFN-{gamma} on the polarization of naive precursors from the influence on effector cells, we used an adoptive transfer model of differentiated Ag-specific Th1 cells. In this study, IFN-{gamma} displayed a dual function in a Th1-dependent immune reaction. In the early phase, IFN-{gamma} accelerated the inflammation, whereas in the late phase it mediated the process of self-limitation. We demonstrated that IFN-{gamma} limits the number of Th1 effector cells after Ag challenge. Studies using IFN-{gamma}R–/– mice as recipients showed that IFN-{gamma} acts indirectly via host cells to regulate the pool size of Th1 cells. NO was a downstream effector molecule. Transfer experiments of Th1 cells into IFN-{gamma}–/– mice revealed that Th1 cells control both themselves and the corresponding inflammation by the release of IFN-{gamma}. Thus, the proinflammatory cytokine IFN-{gamma} can act as a negative feedback regulator to control Th1-mediated immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Thelper CD4+ cells are typically classified into two subsets, Th1 and Th2. Th1 cells secrete IFN-{gamma}, IL-2, TNF-{alpha}, and TNF-beta which are critical for cell-mediated immunity. Th2 cells produce the cytokines IL-4, IL-5, IL-6, and IL-13 which are essential for optimal Ab production (reviewed in Ref.1). The phenotype acquired by naive CD4+ T cells is strongly influenced by the cytokine milieu during the primary T cell activation. IFN-{gamma} and IL-12 are prototypic cytokines directing Th1 differentiation during the primary immune response, whereas IL-4 leads to Th2 polarization. CD4+ T cells can also mediate pathologic immune responses. Oversupply of Th1-type cytokines has been associated with tissue destruction found in autoimmune diseases, whereas excessive Th2-type cytokines have been implicated in atopy and allergic diseases.

There is only limited knowledge of how Th1-mediated immune responses are controlled. One way to terminate effector responses is elimination of expanded effector cells to restore homeostasis (2). It has been proposed that there is a concomitant regulation of T cell activation and homeostasis and that homeostasis is regulated through feedback interactions (3). Recent publications describing a delayed contraction of CD8+ T cells after infection with Listeria monocytogenes in IFN-{gamma} knockout (KO) 4 mice point toward IFN-{gamma} as an important cytokine in T cell regulation (4, 5).

IFN-{gamma} is a pleiotropic cytokine that plays an essential role in both innate and adaptive phases of an immune response. It is the principal effector cytokine of cell-mediated immunity. One essential role of IFN-{gamma} is to activate macrophages resulting in increased phagocytosis, increased expression of MHC class I and II, and induction of IL-12, NO, and superoxide (6). Besides Th1 cells, NK and CD8+ T cells are the most potent, but not the only sources of IFN-{gamma}. Macrophages, dendritic cells, and even B cells have been described to produce IFN-{gamma} (1, 7). A large body of evidence has accumulated over the years indicating that IFN-{gamma} also plays a previously unexpected protective role in autoimmune models (8). A protective influence was observed in studies of experimental autoimmune encephalitis (EAE) (9), collagen-induced arthritis (10, 11), experimental autoimmune uveoretinitis (12), experimental autoimmune myasthenia gravis (13), and experimental autoimmune thyroiditis (14). In other autoimmune models, IFN-{gamma} has been shown to contribute to disease severity (7, 8, 15), suggesting diverse functions of IFN-{gamma} in inflammation. All these studies were performed by immunizing mice with disrupted IFN-{gamma} or IFN-{gamma}R genes. Hence, a discrimination of the observed effects between differentiation and effector phase of the immune response was not possible.

To address the question of whether an Ag-specific immune response of differentiated Th1 cells is controlled by IFN-{gamma}, we used an adoptive transfer model of TCR-transgenic Th1 cells. Lack of IFN-{gamma} impaired the process of self-limitation in a delayed-type hypersensitivity (DTH) model. Transfer of Ag-specific Th1 cells from wild-type (WT) mice into IFN-{gamma}, IFN-{gamma}R, and inducible NO synthase (iNOS)-deficient mice revealed that Th1 cells autonomously regulate their cell number after Ag challenge by IFN-{gamma} acting most likely via induction of NO in host cells. Therefore, Th1 cells can also act as regulatory effector cells by negative feedback regulating in an IFN-{gamma}-dependent manner and thereby self-adjusting their proinflammatory effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c, C57BL/6, IFN-{gamma} KO mice (16), iNOS KO (17), OT-II, and DO11.10 mice were bred at the Bundesinstitut für Risikobewertung (BfR) or purchased from Charles River Laboratories and used at 6–12 wk of age. IFN-{gamma}R KO mice (18), provided by T. Blankenstein, (Max-Delbrueck Center, Berlin, Germany), were bred at the Max-Delbrueck Center animal facility. All genetically engineered mice have been backcrossed onto the C57BL/6 strain for more than six generations. In all experiments performed in this study, parental WT inbred strains were used as controls. All animal experiments were performed age- and sex-matched and in accordance with institutional, state, and federal guidelines.

Abs, staining, and sorting reagents

The following Abs were produced in our laboratory: anti-FcR II/III (2.4G2), anti-IFN-{gamma} (R4.6.A2) for depleting experiments, FITC-labeled anti-IFN-{gamma} (AN-18.17.24), FITC- and Cy5-labeled anti-CD4 (GK1.5), and Cy5-labeled anti-OVA-TCR (KJ1.26). The following fluorochrome-conjugated Abs and secondary reagents were purchased from BD Pharmingen: anti-CD62L (Mel-14), anti-IFN-{gamma} (AN-18), anti-TNF-{alpha} (MP6-XT22), anti-IL-4 (11B11), anti-IL-10 (JES5-16E3), streptavidin, and appropriate isotype controls. Rat IgG control was obtained from Jackson ImmunoResearch Laboratories. All microbeads were purchased from Miltenyi Biotec.

Generation of Th1 cells

Th1 cells were generated in vitro from OT-II or DO11.10 mice as described previously (19). Briefly, CD4+ lymph node (LN) T cells were enriched by depletion of B cells, CD8+ T cells, macrophages, and CD25+ cells (clones: 2.4G2, Tib105, M1/70, PC6.1). Naive CD4+CD62Lhigh T cells were sorted using anti-CD62L microbeads and the AutoMACS magnetic separation system (Miltenyi Biotec). Sorted cells were stimulated for 6 days with CD90-depleted and irradiated spleen cells (30 Gy) together with 2 µg/ml OVA323–339 peptide (synthesized at the Department of Biochemistry, Humboldt University, Berlin, Germany), anti-IL-4 (5 µg/ml), IFN-{gamma} (20 ng/ml; R&D Systems), and IL-12 (5 ng/ml; R&D Systems). Cell culture was done with RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FCS (Sigma-Aldrich). To analyze cytokine production of in vitro-generated Th1 cells, cells were harvested at day 6 and restimulated with PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich) for 4 h at 37°C. After 1 h, 1 µg/ml brefeldin A (Sigma-Aldrich) was added for the last 3 h and intracellular expression of IFN-{gamma}, TNF-{alpha}, and IL-4 was measured by flow cytometry. In general, >70% of the cells produced IFN-{gamma}, >95% TNF-{alpha}, and <1% IL-4.

Th1-mediated DTH model

A total of 5 x 105 Th1 cells generated from OT-II or DO11.10 mice were injected i.v. into naive C57BL/6, IFN-{gamma}-R KO, IFN-{gamma} KO, and iNOS KO or BALB/c, respectively, and 24 h later the DTH response was induced by s.c. injection of 250 ng of OVA323–339 peptide together with IFA (Sigma-Aldrich) into the left footpad. As a control, the right footpad was injected with PBS/IFA. Starting from 20 h after Ag injection, the inflammatory reaction was measured using an Oditest micrometer gauge in a blinded fashion (Kröplin Längenmesstechnik). {Delta}Footpad thickness was calculated by subtracting footpad thickness measured before induction of the DTH response from footpad thickness measured during the kinetic from each individual mouse. In some experiments, 1 mg of blocking anti-IFN-{gamma} Ab (R4.6.A2) or control rat IgG was injected i.p. 5 h before the DTH was induced. In other experiments, the blocking anti-IFN-{gamma} Ab was injected i.v. 30 min before DTH induction.

Ag-specific in vivo challenge

To analyze the Ag-specific response on a cellular level, a 10-fold higher number of Th1 cells was adoptively transferred in the in vivo challenge experiments: 5 x 106 Th1 cells generated from OT-II mice were injected i.v. into C57BL/6, IFN-{gamma} KO, or IFN-{gamma}R KO mice. Twenty-four hours later, mice were challenged by s.c. injection of 25 µg of OVA protein in IFA into the footpad. Mice were analyzed at indicated time points for OVA-reactive Th1 cells from spleen and draining LNs (see below). In some experiments, IFN-{gamma} was blocked by injecting 1 mg of blocking Ab i.p. 5 h before mice were challenged with OVA protein. The local inflammation was monitored by measuring footpad swelling with an Oditest micrometer gauge in a blinded fashion (Kröplin Längenmesstechnik).

Ag-specific in vitro restimulation of Th1 cells

Total spleen and popliteal LN cells were isolated at the indicated time points and 1 x 106 cells were cultured in the presence of 10 µg of OVA323–339 peptide for 6 h at 37°C. After 1 h, 1 µg/ml brefeldin A (Sigma-Aldrich) was added for the last 5 h. Then, cells were harvested, fixed with 2% paraformaldehyde, and stained with indicated Abs in the presence of saponin (Sigma-Aldrich).

Flow cytometry

Cytometric analysis was performed as previously described (19) using a FACSCalibur (BD Biosciences) and CellQuest software. Dead cells were excluded by propidium iodide or diamidophenylindole staining (Sigma-Aldrich).

Immunohistochemistry

For immunostaining, 4-µm thick sections of the formalin-fixed, paraffin-embedded tissues were cut, deparaffinized, and subjected to a heat-induced epitope retrieval step. Sections were immersed in sodium citrate buffer solutions at pH 6.0 and heated in a high-pressure cooker for 2 min. After this treatment, slides were rinsed in cool running water, placed in 3% hydrogen peroxide to block endogenous peroxidase and washed in TBS (pH 7.4) before incubation with the polyclonal rabbit Ab against iNOS (Calbiochem; 1/1000 dilution) for 30 min. For detection of iNOS, an indirect immunoperoxidase method using the EnVision peroxidase kit compatible only with rabbit primary Abs (K4010; DakoCytomation) was used. Peroxidase was developed with a high sensitive diaminobenzidine chromogenic substrate for ~10 min. Negative controls were performed by omitting the primary Ab.

Statistics

Data are presented as mean ± SD. Significance was determined by Mann-Whitney U test. Differences were considered statistically significant with p ≤ 0.05 and highly significant with p ≤ 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IFN-{gamma} is involved in the self-limitation of a Th1-dependent DTH response

To dissect the impact of IFN-{gamma} on the outcome of an immune reaction between the priming and the effector phase of a Th1 response, we generated Ag-specific Th1 cells from OVA TCR-transgenic mice. Naive CD4+ T cells from OT-II mice were polarized in vitro toward Th1 cells. To analyze the in vivo function of committed Th1 effector cells, we established an Ag-specific DTH model that is based on the adoptive transfer of 5 x 105 OVA-specific Th1 cells into C57BL/6 recipients followed by Ag injection in IFA into the footpad. The oil emulsion was chosen to keep the Ag localized at the injection site. PBS in IFA was used to control the Ag specificity. Typically, 1 day after s.c. immunization footpad swelling reached its maximum, which was followed by a gradual decline during the next 2 wk until the swelling had completely vanished (Fig. 1). This process of spontaneous regression could be described as a self-limitation of the inflammation. Surprisingly, if we injected blocking Abs against IFN-{gamma} shortly before the induction of the DTH response the self-limitation was abrogated leading to a sustained inflammation (Fig. 1). Thus, IFN-{gamma} is involved in the regulation of the effector phase of a Th1 response.


Figure 1
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  		FIGURE 1. Blocking of IFN-{gamma} results in disturbed self-limitation of a Th1-mediated DTH response. A total of 5 x 105 Th1 cells generated from OT-II mice were injected i.v. into C57BL/6 mice, and 24 h later the DTH response was induced by s.c. OVA peptide/IFA injection into the left footpad (filled symbols). As control, the right footpad was injected with PBS/IFA (open symbols). Before DTH induction, indicated groups received 1 mg of blocking anti-IFN-{gamma} Ab or rat IgG. Shown is {Delta}footpad thickness over time (mean ± SD; n = 5) of one representative experiment of two.

 
Blocking of IFN-{gamma} during Ag challenge augments frequency of Th1 cells

To elucidate whether the observed effect was due to an interference of IFN-{gamma} with the frequency of Th1 cells after Ag challenge, we transferred 5 x 106 Th1 cells from OT-II mice into C57BL/6 recipients and measured the number of Th1 cells in spleen and draining LN 7 days after Ag challenge. To identify Ag-reactive Th1 cells from the organs, we performed a restimulation with the cognate OVA peptide and analyzed the frequency of IFN-{gamma}-producing cells among total CD4+ T cells. Strikingly, blocking of IFN-{gamma} during the in vivo challenge resulted in a significant increase of IFN-{gamma}-producing CD4+ T cells in the spleen and the draining LN compared with control mice (Fig. 2, A and B). No decrease of CD25+CD4+ regulatory T cell numbers and no increase of IL-4- or IL-10-producing CD4+ T cells were observed under these conditions, indicating that blocking of IFN-{gamma} neither led to a depletion of suppressor cells nor induced an immune deviation in transferred T cells (our unpublished data). Calculation of the total number of IFN-{gamma}-producing CD4+ T cells also revealed a significant increase of Ag-reactive Th1 cells in spleen and LN of the anti-IFN-{gamma}-treated group compared with controls (Fig. 2C).


Figure 2
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  		FIGURE 2. Blocking of IFN-{gamma} during Ag challenge augments frequency of Th1 cells. A total of 5 x 106 Th1 cells generated from OT-II mice were injected i.v. into C57BL/6 mice, and 24 h later mice were challenged s.c. with OVA protein/IFA. Before Ag challenge, indicated groups received 1 mg of blocking anti-IFN-{gamma} Ab or rat IgG. Control groups did not receive Th1 cells (no Th1 cells). After 7 days, spleen (SP) and draining LN (dLN) cells were restimulated with OVA peptide and stained for intracellular IFN-{gamma}. A, Representative dot plots for indicated groups are shown. Numbers indicate frequency of IFN-{gamma}+ cells among CD4+ T cells. B, Comparison of the frequency of IFN-{gamma}+ cells among CD4+ T cells between indicated groups. Each symbol indicates data from one mouse and lines indicate mean from each group (*, p < 0.05; n = 4–8). C, Comparison of the total number of CD4+IFN-{gamma}+ T cells per organ in the indicated groups (*, p < 0.05; mean ± SD; n = 4). One representative experiment of two is shown.

 
Pronounced inflammatory reaction in IFN-{gamma}R-deficient hosts

It has been described that Th1 cells down-regulate their functional IFN-{gamma}R upon activation to avoid downstream signaling (20, 21), making direct, autocrine mechanisms of self-limitation by IFN-{gamma} less likely. To investigate whether the observed IFN-{gamma}-dependent effects were mediated indirectly via IFN-{gamma}R expressing host cells, we adoptively transferred Th1 cells into IFN-{gamma}R KO mice (18). One day later, mice were challenged with OVA and analyzed 7 days later as described above. Approximately four times more cytokine-producing Th1 cells could be isolated from spleens of IFN-{gamma}R KO mice compared with transfer experiments into WT mice (Fig. 3A). The majority of these cells coexpressed IFN-{gamma} and TNF-{alpha}, whereas we could not detect IL-4- or IL-10-producing T cells above background (our unpublished data).


Figure 3
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  		FIGURE 3. IFN-{gamma} acts via IFN-{gamma}R on host cells to control Th1 homeostasis in vivo. A total of 5 x 106 Th1 cells generated from OT-II mice were injected i.v. into C57BL/6 (IFN-{gamma}R WT) or IFN-{gamma}R KO mice, and 24 h later mice were challenged s.c. with OVA protein/IFA. After 7 (A) or 16 days (B), spleen (SP) and draining LN (dLN) cells were restimulated with OVA peptide and stained for intracellular IFN-{gamma}, TNF-{alpha}, and IL-4. Isotype Abs served as a staining control. A, Comparison of the total number of cytokine (IFN-{gamma} plus TNF-{alpha}) producing CD4+ T cells per organ in the indicated groups (*, p < 0.05; mean ± SD; n = 4). Numbers in parentheses indicate fold difference between means of WT and IFN-{gamma}R KO mice from the same organ. One representative experiment of two is shown. B, Left, Comparison of the total number of cytokine (IFN-{gamma} plus TNF-{alpha}) producing CD4+ T cells per organ in the indicated groups. Right, Comparison of the frequency of cytokine (IFN-{gamma} plus TNF-{alpha}) producing CD4+ T cells per organ in the indicated groups. Numbers in parentheses indicate fold difference between means of WT and IFN-{gamma}R KO mice from the same organ (*, p < 0.05; mean ± SD; n = 8; pooled data from two independent experiments).

 
To investigate whether the accumulation of Th1 cells in the spleen and draining LN of IFN-{gamma}R KO mice was only due to a delay in the kinetic of Th1 homeostasis compared with WT hosts, we chose a late time point and analyzed the mice at day 16. Again, we observed a similar accumulation of Th1 cells in IFN-{gamma}R KO mice (Fig. 3B) demonstrating that the differences in the homeostasis of Th1 cells in IFN-{gamma}R KO and WT recipients after Ag challenge were not merely a result of different kinetics.

To test whether the observed differences in Th1 cell number correlate with an altered course of the immune response, we determined the grade of inflammation by measurement of footpad swelling after Ag injection. Interestingly, we found a strong increase in the footpad swelling of IFN-{gamma}R KO mice compared with WT mice. The enhanced inflammatory response could be documented on day 7 (Fig. 4A) and lasted >16 days after immunization (Fig. 4B). These data indicate that the IFN-{gamma}R expressed on host cells transmits signals leading to a reduction of Th1 cell number and thereby controlling Th1-mediated inflammation.


Figure 4
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  		FIGURE 4. Acceleration of inflammatory reaction in IFN-{gamma}R KO mice. A total of 5 x 106 Th1 cells generated from OT-II mice were injected i.v. into C57BL/6 (IFN-{gamma}R WT) or IFN-{gamma}R KO mice, and 24 h later mice were challenged s.c. with OVA protein/IFA. Shown is {Delta}footpad thickness 7 days (A) and 16 days (B) after Ag challenge. Pictures show footpads of one representative mouse from indicated groups 16 days after challenge. Each symbol indicates data from one mouse and lines indicate mean from each group (**, p < 0.01; n = 7–12).

 
One of the molecules being induced upon activation of APCs by IFN-{gamma} is iNOS (6). To investigate the role of iNOS in the context of the observed self-limitation, we first compared the expression of iNOS directly within the inflamed site between anti-IFN-{gamma}-treated mice and mice receiving rat IgG as a control. Interestingly, in control animals, we observed a clear induction of iNOS protein, which was almost completely absent in animals receiving anti-IFN-{gamma}-blocking Abs (Fig. 5A). To analyze whether IFN-{gamma}-induced iNOS is critically involved in the self-limitation, we performed the DTH experiment in iNOS KO mice. The DTH response in iNOS KO mice was significantly increased compared with WT mice (Fig. 5B). Interestingly, blocking of IFN-{gamma} during the DTH reaction did not further enhance the inflammation in iNOS KO mice indicating that the suppressive effect of IFN-{gamma} is dependent on iNOS.


Figure 5
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  		FIGURE 5. Self-limitation of Th1-mediated inflammation by IFN-{gamma} is dependent on iNOS. A, A total of 5 x 105 Th1 cells generated from OT-II mice were injected i.v. into C57BL/6 mice, and 24 h later the DTH response was induced by s.c. OVA peptide/IFA injection into the left footpad. Before DTH induction, indicated groups received 1 mg of blocking anti-IFN-{gamma} Ab or rat IgG. Three days after Ag injection, inflamed footpads were analyzed for iNOS expression by immunohistochemistry. Representative photomicrographs (original magnification, x400) show overt iNOS expression in rat IgG-treated animals, which was strongly reduced in mice receiving blocking anti-IFN-{gamma} Abs. B, A total of 5 x 105 Th1 cells generated from OT-II mice were injected i.v. into C57BL/6 (WT) or iNOS KO mice, and 24 h later mice were challenged s.c. with OVA peptide/IFA. Before DTH induction, indicated groups received 1 mg of blocking anti-IFN-{gamma} Ab or rat IgG. Shown is {Delta}footpad thickness 7 days after Ag challenge. Each symbol indicates data from one mouse and lines indicate mean from each group (**, p < 0.01; n = 7–11).

 
IFN-{gamma} affects the number of Th1 effector cells

Our observations from the IFN-{gamma}-blocking studies and from the adoptive transfer experiments into IFN-{gamma}R KO and iNOS KO mice prompted us to investigate whether IFN-{gamma} simply leads to a reduction in the number of Th1 cells or whether IFN-{gamma} also affects the ability of Th1 cells to produce effector cytokines. To track the adoptively transferred Th1 cells, we performed the IFN-{gamma}-blocking experiments in BALB/c recipients using Th1 cells derived from DO11.10 mice, which can be identified by the use of the clonotypic Ab KJ1.26. The DTH response under nonblocking and blocking conditions showed a similar course than in C57BL/6 recipients (Figs. 6 and 1). We then measured the number of Th1 cells 12 days after immunization. At this time point, pronounced differences in footpad swelling between the anti-IFN-{gamma}-treated group and control mice are visible (Fig. 6). Evaluation of the number of TCR-transgenic T cells revealed an accumulation of Th1 cells in the anti-IFN-{gamma}-treated group compared with control mice (Fig. 6). However, when we compared the ability of the KJ1.26+ Th1 cells to produce effector cytokines no differences between the two groups were observed indicating that IFN-{gamma} solely affects the number of Th1 cells.


Figure 6
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  		FIGURE 6. IFN-{gamma} affects the number of Th1 effector cells. A total of 5 x 105 Th1 cells generated from DO11.10 mice were injected i.v. into naive BALB/c mice, and 24 h later the DTH response was induced by s.c. OVA peptide/IFA injection into the left footpad (filled symbols). As control, the right footpad was injected with PBS/IFA (open symbols). Before DTH induction, indicated groups received 1 mg of blocking anti-IFN-{gamma} Ab or rat IgG. Left panel, {Delta}Footpad thickness over time (mean ± SD; n = 5). Right panel, At day 12 after immunization, mice from the anti-IFN-{gamma}-treated group (filled symbols) and from the control group (open symbols) were sacrificed, and spleen (SP) and draining LN (dLN) cells were restimulated with OVA peptide and stained for KJ1.26, CD4, IFN-{gamma}, and TNF-{alpha}. Upper graph, The total number of CD4+KJ1.26+ T cells per organ. Lower graph, The frequency of cytokine (IFN-{gamma} plus TNF-{alpha}) producing cells among all CD4+KJ1.26+ T cells (*, p < 0.05; n.s., not significant; mean ± SD; n = 5).

 
Th1 cells self-limit their inflammatory activity by IFN-{gamma}

Having demonstrated that IFN-{gamma} limits the number of Th1 effector cells after Ag challenge resulting in a more restricted inflammation, we were interested to study whether Th1 cells themselves control that process by the release of IFN-{gamma}. To address that question, we adoptively transferred Th1 cells from OT-II mice into IFN-{gamma} KO mice (16). In this setting, the adoptively transferred Th1 cells were the only source of IFN-{gamma}. Indeed, IFN-{gamma} released by Th1 cells was sufficient to influence the inflammatory response measured by footpad swelling (Fig. 7A). In the early phase after challenge (20 h), IFN-{gamma} showed a proinflammatory capacity. Blocking was associated with a strong reduction of the footpad swelling (Figs. 7A, 1, and 6). In the later phase starting after 4 days, IFN-{gamma} had a suppressive effect on the immune response (Fig. 7A). Furthermore, blocking of IFN-{gamma} released by Th1 cells significantly increased the number of IFN-{gamma}-producing CD4+ T cells in spleen and draining LNs (Fig. 7B).


Figure 7
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  		FIGURE 7. Th1 cells self-limit their inflammatory activity by IFN-{gamma}. A total of 5 x 106 Th1 cells generated from OT-II mice were injected i.v. into IFN-{gamma} KO mice, and 24 h later mice were immunized s.c. into the left footpad with OVA protein/IFA. Before DTH induction, indicated groups received 1 mg of blocking anti-IFN-{gamma} Ab or rat IgG. A, Shown is {Delta}footpad thickness at indicated time points after immunization. Each symbol indicates data from one mouse and lines indicate mean from each group (*, p < 0.05; n = 4). One representative experiment of two is shown. B, At day 7, mice were sacrificed, and spleen (SP) and draining LN (dLN) cells were restimulated with OVA peptide and stained for CD4 and IFN-{gamma}. Total number of double-positive cells was calculated per organ (*, p < 0.05; mean ± SD; n = 4). One representative experiment of two is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study focuses on how Th1-mediated immune responses are controlled. Here, we demonstrate that beside their proinflammatory capacity Th1 cells can act as regulatory effector cells and limit their own effector response in a negative feedback loop by producing IFN-{gamma}.

Several studies using Th1-based experimental autoimmune models described an unexpected protective role of IFN-{gamma} (8). However, in other autoimmune models such a protective role could not be observed and IFN-{gamma} contributed to disease severity (7, 8, 15). This conflicting data can be explained by different features of IFN-{gamma}. In the current study, we also observed opposite effects of IFN-{gamma} during the time course of a DTH response even though committed Th1 cells already primed and differentiated in vitro were used to elicit the inflammation. In the early phase after challenge (20 h), IFN-{gamma} acted as a strong proinflammatory cytokine, whereas in the late phase (>4 days) its suppressive effect dominated the inflammatory reaction. Taking into account that the aforementioned autoimmune studies were performed in situations where IFN-{gamma} was not only lacking during the effector phase but also during priming and differentiation of CD4+ T cells, different outcomes between the models can be expected. Surprisingly, most of the autoimmune models described a protective role for IFN-{gamma} (8), emphasizing a dominant suppressive effect of IFN-{gamma} in autoimmune diseases.

In a recent publication, it has been proposed that homeostasis of T cells is regulated through feedback interactions (3). Several feedback control mechanisms, including competition for growth and viability signals (22), competition for access to APCs or peptide-MHC ligands (23), or suppression by specialized regulatory T cells (24) are thought to function to prevent both excessive expansion and exhausting differentiation when lymphocytes are repeatedly exposed to Ag (3). Our current data suggest that IFN-{gamma} contributes in that sense as a negative feedback regulator to control the pool size of Th1 cells after restimulation. Recently, it has been described that Ag challenge led to activation and elimination of Th1 memory cells in vivo (25). In that study, Th1 cells were adoptively transferred and 30–60 days later mice were challenged. A transient expansion of memory CD4+ T cells was followed by depletion. The memory cells responded to the Ag challenge with a burst of activation and cytokine production. Two hours after challenge, one-third of the memory cells produced large amounts of IFN-{gamma}. However, in that study the authors did not evaluate whether secreted IFN-{gamma} was responsible for the observed depletion of Th1 memory cells. One can speculate that IFN-{gamma} not only limits the number of Th1 effector cells as described in the present study, but also controls the homeostasis of Th1 memory cells upon restimulation.

Mice lacking CTLA-4, IL-2, and the common {gamma} chain spontaneously accumulate activated CD4+ T cells (26, 27, 28, 29), suggesting that these molecules are involved in the homeostasis of T cells independent of a specific immune response. In contrast, no spontaneous accumulation of activated CD4+ T cells was observed in IFN-{gamma} KO mice in the absence of bacterial infections or antigenic stimulation (30). This implicates that IFN-{gamma} is involved in CD4+ T cell homeostasis only during the effector phase of immune responses.

L. monocytogenes infection in IFN-{gamma} KO mice revealed that IFN-{gamma} does not only contribute to pathogen clearance but also plays a major role in CD8+ T cell homeostasis (4). In a follow-up study using antibiotic pretreated mice, the authors showed that decreased IFN-{gamma} production during early inflammation was correlated with an absence of the contraction phase of the primary CD8+ response (5). Furthermore, using parasite-specific CD4+ T cell lines another report showed that IFN-{gamma} indeed is involved in the depletion of effector cells during infections (31).

In the CD4+ T cell-mediated EAE model, it has been reported that IFN-{gamma} KO mice develop progressive and fatal EAE. Disease progression in IFN-{gamma} KO mice was correlated with an accumulation of CD4+ T cells in the CNS and the spleen compared with WT mice (32). These data are in accordance with the findings of the present study showing that blockade of IFN-{gamma} resulted in an increased number of cytokine-producing effector cells inducing an uncontrolled DTH response. However, the aforementioned study using the EAE model could not distinguish between the impact of IFN-{gamma} on the differentiation or effector phase. By adoptively transferring in vitro-generated Th1 cells, we can clearly show in the current study that IFN-{gamma} strongly influences the effector response of differentiated Th1 cells after an Ag challenge in vivo.

Regulation of the number of effector cells by IFN-{gamma} could be mediated either directly or indirectly. A recently published in vitro study proposed that IFN-{gamma} is required for activation-induced cell death of CD4+ T cells by stimulating caspases downstream of the FasR (33). In that study, naive CD4+ T cells were stimulated in the presence or absence of IFN-{gamma} for 3 days allowing IFN-{gamma} to act during the priming phase. The results indicated a direct effect of IFN-{gamma}. However, another report suggested an indirect effect of IFN-{gamma} on CD4+ T cell homeostasis during Mycobacterium bovis infection (30). CD4+ T cells from infected IFN-{gamma} KO mice were isolated and upon in vitro restimulation under the cover of IFN-{gamma}, an increased apoptosis rate among these activated CD4+ T cells was observed. The IFN-{gamma}-mediated apoptosis was abolished by depleting adherent cells or Mac-1+ cells (30). These data are in line with our experiments using IFN-{gamma}R KO mice showing that expression of IFN-{gamma}R on host cells, including APCs, is required to control both in vivo Th1 homeostasis and the immune response mediated by Th1 cells. Although it has been described that Th1 cells down-regulate their functional IFN-{gamma}R upon activation to avoid downstream signaling (20, 21), we cannot formally exclude that Th1-derived IFN-{gamma} acts directly on Th1 cells in an autocrine manner. However, given that transfer of WT Th1 cells into either IFN-{gamma}R KO or anti-IFN-{gamma}-treated recipients resulted in a similar course of the DTH response makes this mode of action rather unlikely.

It has been suggested by Dalton et al. (30) that IFN-{gamma} activates macrophages to produce NO, which then triggers apoptosis of CD4+ T cells. That idea is supported by a report describing that administration of IL-12 is protective in an experimental autoimmune uveitis model (34). The protection involved hyperinduction of IFN-{gamma}, which causes an up-regulation of iNOS and production of NO (34). In fact, in the present study, we were able to demonstrate that iNOS is involved in the limitation of a Th1-mediated DTH response and acts most likely as the effector molecule downstream of IFN-{gamma}.

However, none of the aforementioned studies specifically addressed the question of the source of IFN-{gamma}. Beside Th1 cells, NK cells, CD8+ T cells, macrophages, dendritic cells, and B cells can produce IFN-{gamma} (1, 7). The current study shows that IFN-{gamma} produced by Th1 cells is crucial for the self-limitation of a Th1-mediated immune response. Further analyses are required to determine whether Th1 cells can also regulate the immune response of other cell types such as CD8+ T cells in an IFN-{gamma}-dependent manner.

How Th1-mediated immune responses are regulated is a central issue not only for infectious immunology but also for autoimmune diseases or tumor immunology. Our results demonstrate that IFN-{gamma} is an important player in that regulatory process. Th1 cells can act as regulatory effector cells suppressing their own inflammatory action by producing IFN-{gamma}. Data from the present study can serve to explain the underlying mechanism from the unexpected protective role of IFN-{gamma} in a number of autoimmune models. The demonstrated self-limiting potential of IFN-{gamma} may provide approaches for manipulating unwanted immune responses but it also has to be considered when designing vaccination strategies.


   Acknowledgments
 
We thank T. Blankenstein (MDC, Berlin, Germany) for providing us with IFN-{gamma}R KO mice, Simone Spieckermann for technical assistance, D. Huscher for statistical analyses, and H. Schliemann, H. Hecker, and T. Geeske for providing us with mAbs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB421, 633, and 650). Back

2 Current address: Section on Immunology and Immunogenetics, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215. Back

3 Address correspondence and reprint requests to Dr. Jochen Huehn, Experimental Rheumatology, Medical Clinic Rheumatology and Clinical Immunology, Charité University Medicine, Berlin, c/o Deutsches Rheumaforschungs-Zentrum, Schumannstrasse 21/22, 10117 Berlin, Germany. E-mail address: huehn{at}drfz.de Back

4 Abbreviations used in this paper: KO, knockout; DTH, delayed-type hypersensitivity; EAE, experimental autoimmune encephalitis; iNOS, inducible NO synthase; LN, lymph node; WT, wild type. Back

Received for publication July 18, 2005. Accepted for publication December 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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