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The Journal of Immunology, 2006, 177: 7673-7679.
Copyright © 2006 by The American Association of Immunologists, Inc.

Microenvironment-Dependent Requirement of STAT4 for the Induction of P-Selectin Ligands and Effector Cytokines on CD4+ T Cells in Healthy and Parasite-Infected Mice1

Uta Syrbe2,*,{dagger}, Ute Hoffmann*, Kerstin Schlawe*, Oliver Liesenfeld{ddagger}, Klaus Erb3,§ and Alf Hamann*

* Charité, Campus Mitte, Experimentelle Rheumatologie, c/o Deutsches Rheumaforschungszentrum, Berlin, Germany; {dagger} Charité, Campus Benjamin Franklin, Medizinische Klinik I, Berlin, Germany; {ddagger} Charité, Campus Benjamin Franklin, Institut für Mikrobiologie und Hygiene, Berlin, Germany; and § Klaus Erb, Zentrum für Infektionsforschung, Universität Würzburg, Würzburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T effector cells require selectin ligands to migrate into inflamed regions. In vitro, IL-12 promotes induction of these ligands as well as differentiation of CD4+ T cells into IFN-{gamma}-producing Th1 but not Th2 cells. STAT4 is strongly involved in these processes. However, the presence of selectin ligands on various T effector cell subsets in vivo points to more complex regulatory pathways. To clarify the role of the IL-12/STAT4 signaling pathway, we analyzed the impact of STAT4 deficiency on the expression of P-selectin ligands (P-lig) on CD4+ T cells in vitro and in vivo, including conditions of infection. In vitro, we found significant expression of P-lig upon activation not only in the presence, but also in the absence, of IL-12, which was independent of STAT4. TGF-beta, an alternative inducer of selectin ligands in human T cells, was not effective in murine CD4+ T cells, suggesting a role of additional signaling pathways. In vivo, a significant impact of STAT4 for the generation of P-lig+CD4+ T cells was observed for cells from peripheral lymph nodes, but not for those from spleen or lung. However, upon infection with the Th2-inducing parasite Nippostrongylus brasiliensis, P-lig expression became dependent on STAT4 signaling. Interestingly, also the frequency of IL-4-producing cells was greatly diminished in absence of STAT4. These data reveal a hitherto unknown contribution of STAT4 to the generation of Th2 cells in parasite infection and suggest that signals inducing inflammation-seeking properties in vivo vary depending on environmental conditions, such as type of organ and infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The endothelial E- and P-selectin plays a major role for the recruitment of leukocytes into inflamed regions. They interact with ligands generated by posttranslational modification of a few protein carriers such as P-selectin glycoprotein ligand 1 (PSGL-1)4 with sialyl Lewis X oligosaccharides (1). A critical step in the synthesis of these epitopes is the addition of a terminal fucose to the carbohydrate side chains by the activity of distinct fucosyltransferases (FucT), especially FucT-VII, as demonstrated by the almost complete lack of ligands for either P- or E-selectin in FucT-VII–/– effector T cells (2). Furthermore, branching of the oligosaccharide chains conducted by core 2 beta1,6-glucosaminyltransferase (C2GlcNAT)-I is also essential for induction of P-selectin binding activity (3).

On T cells, selectin binding epitopes are induced upon activation and differentiation into effector cells. Initial in vitro data suggested that culture conditions favoring Th1 development, notably the presence of IL-12, are required for the induction of selectin ligands (4). IL-12 also promoted the expression of the cutaneous lymphocyte Ag, an E-selectin binding epitope on PSGL-1 similar to P-selectin binding epitopes, and both correlated with FucT-VII induction (5, 6). The synthesis of ligands for either P-or E-selectin has been shown previously to be largely regulated in parallel, with P-selectin ligands (P-lig) being the more sensitive indicators of FucT-VII activity (7, 8).

STAT4 is the most critical mediator of IL-12-induced intracellular signaling. It is preferentially expressed in Th1 cells and was initially thought to primarily regulate IL-12Rbeta2 expression in a positive feedback loop. STAT4-knockout mice phenotypically resemble in most respects mice lacking IL-12 or IL-12R subunits (9, 10). Accordingly, STAT4–/– mice have impaired Th1 differentiation, IFN-{gamma} production, and cell-mediated immunity (11, 12). IFN-{gamma} induction is independent from its ability to up-regulate IL-12Rbeta2, as expression of the Rbeta2 chain in the absence of STAT4 was not sufficient to support Th1 differentiation (13, 14).

As shown by White et al. (15), IL-12 induces FucT-VII as well as C2GlcNAT-I. In STAT4-deficient mice, induction of C2GlcNAT-I was dependent on STAT4 signaling, but not that of FucT-VII. Minor differences in the regulation of P- and E-lig were found in STAT4-deficient mice, as E-selectin ligand expression was only partially impaired in STAT 4–/– cells treated with IL- 12, suggesting the requirement of a transferase other than C2GlcNAT-I for E-lig generation (15). Recently, also T-bet was shown to contribute to the formation of selectin ligands on T cells and to regulate C2GlcNAT-I as well as FucT-VII expression. Furthermore, it induces expression of sialyltransferase VI, which also appears to be involved in the generation of selectin-binding epitopes in T cells (16).

Whereas these initial in vitro studies suggested a link between IL-12 driven differentiation into Th1 cells and selectin ligand expression, other in vitro studies observed expression of selectin ligands after activation in the absence of IL-12 (7, 17, 18, 19, 20, 21). In the human system, also TGF-beta has been shown to be a further mediator capable to induce P-lig expression on lymphocytes in a p38-dependent manner (22).

In vivo, the link between Th1 differentiation and selectin ligand expression was much less clear; in fact, selectin ligands were found to be expressed on both IFN-{gamma} and IL-4-producing T cells in men and mice (8, 17, 23). Recent studies emphasized the importance of tissue-dependent factors for the regulation of selectin ligands. Campbell et al. (23) reported a preferential induction on CD4+ T cells activated within peripheral lymph nodes, whereas cells activated within mesenteric lymph nodes did not up-regulate P-lig. However, strong inflammatory stimuli were found to induce selectin ligands even in mucosal compartments (24, 25).

These studies left the question open which signaling pathways are involved in induction of selectin ligands on CD4+ T cells in vivo, especially under conditions of inflammation or infection. In the present study, we investigated whether STAT4, commonly seen as a signaling pathway associated with Th1 development, might play a broader role under in vivo circumstances, especially in case of Th2-dominated parasite infection. We provide evidence, that STAT4 contributes under homeostatic conditions only to P-lig expression in peripheral lymph nodes but not in the spleen or lung. Surprisingly, upon infection, a major role of STAT4 was not only observed for P-lig expression on CD4+ T cells within the inflamed tissues but also for the generation of IL-4-producing effector cells.


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

Wild-type (WT) BALB/c mice (Bundesinstitut für Risikobewertung) and STAT4–/– mice (The Jackson Laboratory) were bred under specific pathogen-free conditions in our animal facility. All animal experiments were performed in accordance with institutional, state and federal guidelines.

Nippostrongylus infection

WT or STAT4–/– mice were subcutaneously infected with 750 L3 larvae of the helminth Nippostrongylus brasiliensis.

Isolation of primary cells for cytometric detection of P-lig expression and cytokine production

For lymphocyte isolation from lymph nodes or spleen, organs were removed and teased through stainless steel meshes. After washing, cells, erythrocytes, and cell debris were removed by density centrifugation (Histopaque-1083; Sigma-Aldrich).

Mononuclear cells from lung cell suspensions were prepared as described previously (26). Briefly, lung tissue was perfused via the right ventricle of the heart with 10–20 ml of PBS. Subsequently, the organs were passed through sieves to obtain single-cell suspensions, and cells were washed. Lung suspensions were subjected to a 40/70% Percoll (Amersham Biosciences) gradient (26) and washed subsequently with PBS containing 0.2% BSA.

Cytometric analysis

P-selectin binding ligands were detected with a P-selectin-human IgG chimeric protein and PE- or cytochrome 5-conjugated anti-human IgG Ab (F(ab')2) (Dianova) as secondary reagent as described (27). In brief, staining was conducted in HBSS supplemented with Ca2+ and Mg2+ and with 10 mM HEPES. The optimal concentration of P-selectin-IgG for staining was determined by titration of the chimeric protein for each batch. Background staining was determined by appropriate controls, including staining of FucT-VII-deficient T cells (data not shown) and staining of P-lig+ cells in the presence of 5 mM EDTA.

CD4+ T cells were stained with anti-CD4-PerCP (BD Pharmingen). Cytometric analysis was performed using FACSCalibur and CellQuest software, both from BD Biosciences.

Detection of P-lig expression and cytokine production of CD4+ T cells ex vivo

After preparing single-cell suspensions, cells were stimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml) for 4 h. Brefeldin A was added at 10 µg/ml for the last 2 h of stimulation to prevent secretion of cytokines. After 4 h, cells were stained with anti-CD4 mAb and for P-lig expression as described before. After staining, cells were fixed in HBSS containing Ca2+, Mg2+ and PFA 2%, permeabilized by 0.5% saponin, and stained with FITC-conjugated anti-IFN-{gamma} (AN18.17.24), anti-IL-4-PE (BD Pharmingen), or anti-IL-10-PE (BD Pharmingen).

Purification of naive CD62L+CD4+ cells for in vitro culture

CD4+ T cells were purified from pooled peripheral and mesenteric lymph nodes by panning using anti-CD8 (53-672), anti-CD25 (PC/6), anti-Mac-1 (M1/70) and anti-FcR II/III (2.4G2) Abs or by anti-CD4-FITC (GK1.25) and anti-FITC-Multisort Beads (Miltenyi Biotec) to a purity of ≥98%. Multisort beads were released according to the manufacturer’s suggestion. Naive CD4+CD62L+ cells were positively selected by high-gradient MACS with anti-CD62L microbeads (Miltenyi Biotec) to a purity of ≥98%. APCs were generated by depletion of spleen cells from CD90+ lymphocytes using anti-CD90 microbeads (Miltenyi Biotec).

Cell culture

For effector cell generation, naive T cells were cultured at 1 x 106 cells per ml in complete RPMI 1640 medium, containing 10% FCS and 10 µM 2-ME (Life Technologies) and activated with plate-bound anti-CD3 (1 µg/ml) and soluble anti-CD28 (1 µg/ml). For culture under Th1 conditions, recombinant murine IL-12 (R&D Systems) at 5 ng/ml, IFN-{gamma} (R&D Systems) at 20 ng/ml, and neutralizing anti-IL-4 Abs at 5 µg/ml (11B11; own production) was added. Th2 cultures contained 30 ng/ml IL-4 (R&D Systems), and 5 µg/ml anti-IFN-{gamma} and 5 µg/ml anti-IL-12 (AN 18.17.24 and C17.8.6; own production). In some experiments, anti-TGF-beta Ab (R&D Systems) was added at a concentration of 1 µg/ml. Th0 cultures contained 10 ng/ml IL-2, 5 µg/ml anti-IL-12, 5 µg/ml anti-IFN-{gamma}, and 5 µg/ml anti-IL-4 and were supplemented as indicated with recombinant human TGF-beta (R&D Systems) at either 2 or 8 ng/ml.

Statistical analysis

Data are shown as mean and 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
 
Role of IL-12/STAT4 signaling for the induction of selectin ligands in vitro

In vitro data suggested that IL-12 induces P-lig expression on CD4+ T cells in a STAT4-dependent manner, whereas IL-4 was rather inhibitory (5, 6). When naive CD4+CD62L+ T cells from STAT4-deficient or from WT mice were activated by anti-CD3/anti-CD28 under Th1 promoting conditions, i.e., in the presence of IL-12, IFN-{gamma}, and anti-IL-4, induction of P-lig was indeed reduced on STAT4-deficient cells (Fig. 1A); however, the reduction was only partial (50%). Under the conditions used here, T cell activation in the presence of IL-4 and Abs to IL-12 and IFN-{gamma}, i.e., culture conditions promoting Th2 development, also resulted in generation of a varying, but significant number of P-lig-expressing cells if analyzed 4 days after activation. This was found for both WT and STAT4–/–-deficient cells; induction in the absence of IL-12 was occasionally even higher in STAT4–/– cells than in WT CD4+ T cells (Fig. 1A). Mean fluorescence intensity (MFI) as a measure of the density of P-lig molecules was comparable among P-lig+ Th1 and Th2 cells (Fig. 1A).


Figure 1
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  		FIGURE 1. Effect of STAT4 deficiency on P-lig induction on CD4+ T cells in vitro. CD4+CD62L+ cells from WT or STAT4–/– mice were isolated from peripheral and mesenteric lymph nodes and stimulated by anti-CD3/anti-CD28 under Th1 (IL-12, IFN-{gamma}, anti-IL-4) or Th2 (IL-4, anti-IL-12, anti-IFN-{gamma}) priming conditions. A, The percentage of P-lig+ cells/CD4+cells (left) and MFI of P-lig-positive cells (right) was determined on day 4 after activation. The mean + SD from four independent experiments is shown. B, After restimulation with PMA/ionomycin for 4 h and addition of brefeldin A for the last 2 h, the percentage of IFN-{gamma}+/CD4+ cells was determined. C, Representative histogram plots of WT and STAT4–/– T cells activated under the conditions indicated for 7 days. Dashed line represents background staining, i.e., P-lig staining in the presence of EDTA, bold line shows positive staining. D, WT T cells were activated as above for 4 or 7 days either under Th1 or Th2 priming conditions or without cytokine supplement (Th0). Either anti-TGF-beta Abs (aTGF-beta) or rTGF-beta were added as indicated. One representative of two individual independent experiments is shown. E, WT and STAT4–/– T cells were activated for 7 days under the indicated conditions and the frequency of P-lig+ cells was determined. One representative of two independent individual experiments is shown.

 
Analyzing IFN-{gamma} production by T cells induced under Th1 conditions we found a strong inhibition of IFN-{gamma}-induction cells in STAT4–/– T cells, confirming the defect of STAT4–/– mice in the generation of a Th1 response (Fig. 1B). Induction of cytokine production appeared to be more impaired than induction of selectin ligands in STAT4–/– mice, compared with WT mice.

To look for the kinetics of P-lig induction, prolonged cultures were performed and cells were analyzed on days 4 and 7 after activation. Fig. 1C shows representative histogram plots from T cells cultured for 7 days under Th1 and Th2 conditions from WT and STAT4–/– mice. Effector cells from STAT4–/– mice induced under Th2-promoting conditions expressed similar levels of P-lig as WT T cells on day 7 after activation, suggesting that STAT4 is not involved in the induction of P-lig under these conditions. In contrast, under Th1 conditions P-lig expression remained lower on STAT4–/– T cells than on WT T cells (Fig. 1C).

Comparing P-lig expression on days 4 and 7 after activation we found that, under Th1 conditions, P-lig expression reaches maximal expression already on day 4, whereas under Th2-promoting conditions, P-lig expression further increases up to day 7 after activation (Fig. 1D). These data suggest that an IL-4-dominated environment can be permissive for P-lig induction in vitro.

In the human system, TGF-beta-1 was found to induce P-lig expression on CD4+ T cells by an alternative pathway (22). We therefore tested whether the presence of TGF-beta could contribute to the induction of P-lig observed under Th2 conditions. Neither blockade of TGF-beta under conditions promoting Th2 development nor the addition of TGF-beta to conditions containing no further cytokine supplement (Th0) had major effects on P-lig induction in WT or STAT4–/– cells analyzed on day 4 (Fig. 1D and data not shown) and day 7 after activation (Fig. 1E). This suggests that TGF-beta alone does not play a significant role for P-lig induction on murine T cells.

Reduced frequency of P-lig+ T cells from lymph nodes of STAT4–/– mice

To determine the impact of STAT4-dependent IL-12 signaling on P-lig induction in vivo, we analyzed the frequency of P-lig-expressing cells among CD4+ lymphocytes within peripheral lymph nodes and the spleen from 6-mo-old STAT4–/– mice and WT mice. Fig. 2A shows representative histogram plots of P-lig staining from spleen CD4+ T cells from WT and STAT4–/– mice. As shown in Fig. 2B, the frequency of P-lig+ cells was lower in peripheral lymph node CD4+ T cells from STAT4–/– mice, compared with WT cells. In spleen, the difference was marginal and did not reach significance. MFI of P-lig+ cells appeared to be lower within the spleen, compared with peripheral lymph nodes; however, there was no difference among WT and STAT4–/– cells (Fig. 2B). A stronger effect of STAT4 deficiency was consistently seen for the IFN-{gamma} production (Fig. 2C), confirming the importance of the STAT4 pathway for the induction of Th1 responses in vivo. This indicates that STAT4 signaling is involved in the induction of selectin ligands only under certain environmental conditions.


Figure 2
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  		FIGURE 2. Reduced frequency of P-lig+ CD4+ T cells in STAT4–/– mice ex vivo within lymph nodes. Cells from peripheral lymph nodes or spleen from WT and STAT4–/– mice were stimulated with PMA/ionomycin for 4 h, and brefeldin A was added for the last 2 h of culture. After surface staining for CD4 and P-lig cells were fixed, permeabilized, and stained for intracellular cytokines and cytometrically analyzed. A, Individual histogram plots from WT and STAT4–/– mice from CD4+ T cells isolated from the spleen. Gray area under dashed line represent control staining, bold line reflects positive staining. B, A summarization of data of P-lig expression on CD4+ T cells from six WT and six STAT4–/– mice analyzed in two independent experiments. Bars represent the mean. C, A summarization of data from IFN-{gamma} expression on CD4+ T cells from six WT and six STAT4–/– mice. Bars represent the mean. Mann-Whitney U test for unpaired samples was used.

 
Reduced induction of P-lig in STAT4–/– mice after infection with the helminth N. brasiliensis

The above in vitro data suggest a partial role of STAT4 for induction of P-lig on Th1, but not on Th2 cells. However, previous data demonstrated a discordance of findings regarding ligand expression in vitro and in vivo; especially expression on ex vivo Th2 effector/memory cells was unexpectedly high and a major impact of immune reactions against infection became evident (8).

We therefore investigated the role of STAT4 signaling under conditions of infection with a Th2-inducing helminth N. brasiliensis. Whereas IFN-{gamma}-producing cells were reduced in uninfected STAT4–/– mice as discussed above, the frequency of IL-4- and IL-10-producing CD4+ cells is unimpaired under homeostatic conditions in the spleen (data not shown).

After s.c. infection with infective third-stage larvae of N. brasiliensis, parasitic larvae migrate via the blood stream into the lung where they are coughed up and swallowed within 2 days. As shown in Fig. 3A, a strong Th2-biased immune response occurs within the lung associated with the appearance of large numbers of IL-4- and IL-10-producing cells. Furthermore, a strong increase in the frequency of P-lig-expressing cells is observed within the lung (Fig. 3B). The frequency of P-lig+ cells increased among all subsets of effector cells present within the lung and reached up to 60% among IL-4- and among IL-10-producing cells (Fig. 3C).


Figure 3
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  		FIGURE 3. Kinetic of CD4 T cell infiltration of the lung after infection with the helminth N. brasiliensis. The frequency (mean + SD) of cytokine-producing cells (A) and P-lig expression (B) among CD4+ T cells was determined after PMA/ionomycin restimulation of cells isolated from the lung before (d0) and at different time points after infection of WT mice with N. brasiliensis. C, The frequency of P-lig+ T cells among cytokine-producing cells before and at different time points after infection was determined.

 
In STAT4–/– mice, infection with N. brasiliensis also resulted in an increase in the frequency of P-lig-expressing CD4+ T cells within the lung, compared with uninfected mice; however, the increase was much lower than in WT mice (Fig. 4, A and B). Surprisingly, reduced frequencies were also found for IL-4-producing Th2 cells (Fig. 4B), unraveling an hitherto unknown role of STAT4 signaling in the induction of Th2 cells in vivo.


Figure 4
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  		FIGURE 4. Impaired induction of P-lig and cytokine production on effector cells in STAT4–/– mice after infection with N. brasiliensis. P-lig expression and effector cytokine production was determined after PMA/ionomycin restimulation of CD4+ cells infiltrating the lung of WT vs STAT4–/– mice. A, Representative histogram plots from cells isolated on day 13 after N. brasiliensis infection from the lung of WT and STAT4–/– mice. Gray area under dashed line represents control staining using EDTA, bold line represents positive staining. B, The frequency of cytokine-producing CD4+ T cells and of P-lig+CD4+ cells before (left) and on day 13 (right) after infection with N. brasiliensis is shown. C, The MFI of P-lig+ cells determined in B is given. Data represent the mean + SD from six mice within each group summarized from two independent experiments. Mann-Whitney U test was used for statistical analysis.

 
Although the frequency of P-lig+CD4+ T cells was strongly reduced, MFI on the remaining P-lig+ cells was comparable among WT and STAT4–/– cells (Fig. 4C). The frequency of P-lig-expressing cells among individual populations of cytokine-producing cells was not significantly reduced (data not shown), and also the total number of mononuclear cells recovered from the lung did not significantly differ among WT and STAT4–/– mice (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Studies on the regulation of selectin ligand expression on CD4+ effector cells have yield contradictory results: On one site, different in vitro studies observed a strongly biased expression of selectin ligands on in vitro generated Th1 effector cells but not on Th2 cells (4, 19) while in vivo, no restriction of P-lig expression to Th1 cells was seen (8, 28).

Furthermore, recent data showed a tissue-dependent induction in vivo (23). This raised the question which factors regulate P-lig expression in vivo and what role the IL-12/STAT4 signaling pathway plays, which was shown to up-regulate FucT-VII and C2GlcNAT-I in vitro (5, 29, 30). In a Th1-dominated transfer model using transgenic CD4+ T cells and i.p. application of specific Ag in CFA, a reduced induction of Ag-specific P-lig+ T cells was observed after IL-12 blockade (24). Similarly, in a the Th1-dominated Leishmania major infection model, pretreatment of mice with anti-IL-12 also reduced the frequency of E- and P-lig+CD4+ T cells and suppressed induction of FucT-VII (31), suggesting a major role of the IL-12/STAT4-pathway for the induction of selectin ligands in vivo.

The present study focuses on the question whether STAT4 is indeed a dominant regulator of the synthesis of selectin ligand epitopes and what role distinct conditions in vivo such as organ environment or activation in the course of an infection do play. Especially the role of STAT4 for the differentiation of Th2 cells had not been addressed so far.

The data of this study confirm a major, although not indispensable, function for the in vitro generation of P-lig+ effector cells under Th1-inducing conditions, i.e., in the presence of IL-12. In vivo, P-lig expression only on T cells from peripheral lymph nodes was (partly) dependent on STAT4, whereas spleen cells were not significantly affected. Thus, the contribution of the STAT4 signaling pathway to selectin ligand induction is less dominant or even lacking in some compartments, in contrast with its more prominent role for the development of IFN-{gamma} secretion, as shown here or in previous studies (13, 14). The identity of alternative stimuli inducing selectin ligand expression on T cells remains unclear; our data suggest that, in the murine cells, TGF-beta apparently does not play a role, in contrast with the human cells (22).

It has been reported that FucT-VII, one of the critical ligand-synthesizing enzymes, is induced by IL-12 in a partly STAT4-independent way. However, we found considerable expression of P-lig even in absence of IL-12, i.e., under Th2-inducing conditions; the frequency of P-lig+Th2 cells is, dependent on culture conditions, variable but marked. The expression of selectin ligands on Th2 cells was completely unaffected by a lack of STAT4. A reduced but significant expression of P-lig was also observed for ex vivo CD4+ cells of IL-12–/– mice (H. Hess, unpublished observation) and expression was independent of IL-12 in spleen CD8+ T cells ex vivo (21).

These data underline the importance of IL-12/STAT4-independent pathways of the induction of selectin ligands in T cells.

Little is known about the conditions leading to the generation of inflammation-seeking effector cells by up-regulation of selectin ligands during inflammation, especially in Th2-dominated types of infection. We therefore studied the in vivo induction of selectin ligands in presence or absence of STAT4 signaling in infection by the helminth N. brasiliensis.

Infection with the nematode results in the induction of high numbers of IL-4- as well as IL-10-producing T cells, whereas IFN-{gamma} producers are only moderately enhanced throughout infection. Most of the cytokine-producing CD4+ T cells are found within the lung. IL-12 appeared to be a major counterregulator in this setting, since administration of rIL-12 to Nippostrongylus-infected mice promoted survival of the worm and suppressed IL-4 response (32). Surprisingly, we found both a reduced frequency of P-lig-expressing cells among CD4+ T cells within the lung of infected STAT4–/– mice and a reduced frequency of cytokine-producing CD4+ T cells especially in IL-4-producing CD4+ T cells.

Thus, STAT4 signaling appears to be necessary under the conditions of infection for generation of CD4+ effector cells, even of the Th2 type, and for P-lig induction on most effector cells.

Previous evidence for a role of IL-12 in Th2-dominated responses came from a study of Ishikawa et al. (33) who characterized the cytokine responses during Nippostrongylus infection. Within 2 days, after worms reached the intestine, mesenteric lymph node cells were found to respond with a rather Th1-like response encompassing an increase in IFN-{gamma} production. Furthermore, IFN-{gamma} has been shown recently to enhance in vitro and in vivo priming for IL-4 production (34). Raman et al. (35) observed suppression of a Th2-dominated airway inflammation in STAT4–/– mice, suggesting that, in particular, pulmonary Th2 immune responses might be modulated by IL-12. Thus, even under Th2-dominated conditions some IL-12–STAT4 signaling might be produced and might occur and play, together with induced IFN-{gamma}, a significant role in the generation even of IL-4- and IL-10-producing effector cells.

The data of this present study show that the contribution of the STAT4-mediated signaling for the generation of Th2 cells is remarkable, and also that the induction of P-lig expression on these cells under conditions of infection is much more dependent on this transcriptional regulator then the homeostatic expression on memory cells in noninfected animals. One model to reconcile the apparent contradiction of major effects of a Th1 cytokine in a Th2-dominated reaction is to assume sequential waves of cytokine action. As mentioned above, evidence for early IL-12 activity in Th2 reactions has been provided. If this time window is short enough, no stable cytokine imprinting can occur and a later IL-4-dominated phase is able to suppress IL-12R expression and IFN-{gamma}- production via up-regulation of GATA-3 (36). In our hands, IL-4, at least in the mouse, does not significantly down-regulate the expression of selectin ligands (our unpublished data). Accordingly, early induction of P-lig by IL-12 might last throughout a later conversion of the inflammation into a Th2 milieu and lead to the appearance of P-lig+ Th2 cells.

In conclusion, this study unravels an unforeseen complexity in the regulation of selectin ligands in vivo, and a major role of the STAT4 pathway in generation of both IL-4-producing T cells and of P-lig-expressing cells in a classical Th2-dominated infection model.


   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 Deutsche Forschungsgemeinschaft Grants SFB 366 and SY 31/2-1. Back

2 Address correspondence and reprint requests to Dr. Uta Syrbe, Experimental Rheumatology, c/o Deutsches Rheumaforschungszentrum, Schumannstrasse 21/22, 10117 Berlin, Germany. E-mail address: syrbe{at}drfz.de Back

3 Current address: Boehringer Ingelheim Pharma, 88397 Biberach an der Riss, Germany. Back

4 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand 1; P-lig, P-selectin ligand; FucT, fucosyltransferase; C2GlcNAT, core 2 beta1,6-glucosaminyltransferase; MFI, mean fluorescence intensity. Back

Received for publication October 13, 2005. Accepted for publication September 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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S. Jennrich, B. A. Ratsch, A. Hamann, and U. Syrbe
Long-Term Commitment to Inflammation-Seeking Homing in CD4+ Effector Cells
J. Immunol., June 15, 2007; 178(12): 8073 - 8080.
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