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The Journal of Immunology, 2001, 167: 4476-4484.
Copyright © 2001 by The American Association of Immunologists

IL-12, STAT4-Dependent Up-Regulation of CD4+ T Cell Core 2 {beta}-1,6-n-Acetylglucosaminyltransferase, an Enzyme Essential for Biosynthesis of P-Selectin Ligands1

Yaw-Chyn Lim*, Huijuan Xie*, Carolyn E. Come*, Stephen I. Alexander*, Michael J. Grusby*,{dagger}, Andrew H. Lichtman* and Francis W. Luscinskas2,*

* Vascular Research Division, Departments of Pathology, Brigham and Women’s Hospital and Harvard Medical School; and {dagger} Department of Immunology and Infectious Diseases, Harvard School of Public Health and Department of Medicine, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR activation of naive T cells in the presence of IL-12 drives polarization toward a Th1 phenotype and synthesis of P- and E-selectin ligands. Fucosyltransferase VII (Fuc-T VII) and core 2 {beta}-1,6-N-acetylglucosaminyltransferase (C2GnT) are critical for biosynthesis of selectin ligands. P-selectin glycoprotein ligand-1 is the best characterized ligand for P-selectin and also binds E-selectin. The contributions of TCR and cytokine signaling pathways to up-regulate Fuc-T VII and C2GnT during biosynthesis of E- and P-selectin ligands, such as P-selectin glycoprotein ligand 1, are unknown. IL-12 signals via the STAT4 pathway. Here, naive DO11.10 TCR transgenic and STAT4-/- TCR transgenic CD4+ T cells were stimulated with Ag and IL-12 (Th1 condition), IL-4 (Th2), or neutralizing anti-IL-4 mAb only (Th0). The levels of Fuc-T VII and C2GnT mRNA in these cells were compared with their adhesive interactions with P- and E-selectin in vitro under flow. The data show IL-12/STAT4 signaling is necessary for induction of C2GnT, but not Fuc-TVII mRNA, and that STAT4-/- Th1 cells do not traffic normally to sites of inflammation in vivo, do not interact with P-selectin, and exhibit a partial reduction of E-selectin interactions under shear stress in vitro. Ag-specific TCR activation in CD4+ T cells was sufficient to trigger induction of Fuc-TVII, but not C2GnT, mRNA and expression of E-selectin, but not P-selectin, ligands. Thus, Fuc-T VII and C2GnT are regulated by different signals during Th cell differentiation, and both cytokine and TCR signals are necessary for the expression of E- and P-selectin ligands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cytokine milieu at the time of initial Ag stimulation strongly influences naive T cell differentiation into effector T cell subsets (1, 2). In the presence of IL-12, a cytokine produced by APCs, naive T cells differentiate into Th1 effector cells, whereas IL-4 drives T cells toward a Th2 effector phenotype. Th1 cells, which produce predominately IFN-{gamma} and TNF-{beta}, are involved in cell-mediated immune responses (1, 2, 3, 4, 5). Th2 cells produce IL-4, IL-5, and IL-13 cytokines and participate in immediate hypersensitivity and regulatory responses (1, 2, 3, 4, 5). The molecular mechanisms for the differential Th subset recruitment to sites of inflammation resides in part in the selective expression of surface adhesion molecules that recognize surface counter-receptors on inflamed endothelium.

Functional E- and P-selectin ligands are expressed on most Th1 cells, significantly fewer Th2 cells, and not on naive cells (6). Our recent study (6) and that of Wagers and coworkers (7) have demonstrated that the cytokine milieu can influence Th subset expression of selectin ligands because IL-12 promotes the expression of both P- and E-selectin ligand(s), whereas IL-4 down-regulates E-selectin ligand expression. However, the molecular mechanisms in Th1 subsets that coordinate TCR activation and cytokine-driven differentiation with surface expression of selectin ligands are not completely understood.

In general, the physiological selectin ligands are sialylated, fucosylated, and, in certain instances, sulfated carbohydrate structures generated by post-translational modifications (reviewed in Refs. 8 and 9). The importance of glycosylation enzymes in the post-translational modification of selectin ligands is further illustrated through experiments performed in mice deficient in fucosyltransferase VII (Fuc-T VII)3 or core 2 {beta}-1,6-N-acetylglucosaminyltransferases (C2GnT) (10, 11). Mice with a null mutation in the Fuc-T VII gene show loss of all selectin ligands and striking defects in leukocyte trafficking and immune surveillance (10). Mice deficient in C2GnT exhibit a partial deficiency in leukocyte E-, P-, and L-selectin ligands and have impaired neutrophil trafficking to inflamed tissues; however, T cell subset trafficking has not been examined (11). A recent analysis has suggested that the presence of a threshold level of Fuc-T VII controls the expression of P- and E-selectin ligand expression. These authors (12) concluded that the expression of selectin ligands correlated closely with elevated levels of Fuc-T VII mRNA as assessed by RT-PCR. Previously we had reported that murine naive T cells express very low or undetectable levels of Fuc-T VII and low levels of C2GnT mRNA, lack P- and E-selectin ligand expression, and do not traffic to the inflamed peritoneum (6, 13). After naive T cells were exposed to an Ag and IL-12 in vitro, they expressed elevated levels of Fuc-T VII and C2GnT mRNA, expressed selectin ligands, and accumulated at sites of inflammation in murine models (6, 7, 13, 14, 15). In the presence of Ag and IL-4, no increase in T cell Fuc-T VII mRNA occurred, but C2GnT mRNA levels were significantly increased. These T cells interacted poorly with E- and P-selectin (6, 7) and failed to traffic to the inflamed peritoneum (13). In addition, we have shown that an E-selectin ligand(s) can be induced without induction of P-selectin ligand when T cells are stimulated with Ag alone in the presence of anti-IL-4 mAb (without added cytokines) (6). To date, however, the cytokine-dependent mechanism(s) that regulates the level of these glycosylation enzymes during Ag-driven CD4+ T cell differentiation remains undefined.

The STAT proteins consist of a family of latent transcription factors that become activated in response to cytokines or growth factors, translocate to the nucleus, and stimulate the transcription of specific genes (16). IL-12 activation of STAT4 is critical for Th1 differentiation, whereas IL-4 activation of STAT6 is important for Th2 differentiation (17, 18). Mice deficient in STAT4 have impaired IL-12-induced functions, including poor Th1 differentiation and NK cell cytotoxicity, and were incapable of mounting delayed-type hypersensitivity responses (17, 18, 19).

In this study we used in vitro differentiated CD4+ T cells from DO11.10 TCR transgenic mice to address how IL-12 and IL-4 influence the induction and expression of Fuc-T VII and C2GnT mRNA levels and, as a functional correlate, the expression of both P- and E-selectin ligands during Ag-driven T cell activation. By comparing the levels of glycosyltransferases in in vitro differentiated STAT4-/- and STAT4+/+ DO11.10 TCR transgenic CD4+ T, we determined whether the IL-12-induced functional P-selectin ligand is mediated via the STAT4 pathway.


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

BALB/c mice, 4–6 wk of age, were purchased from Taconic Farms (Germantown, NY). DO11.10 TCR transgenic mice specific for chicken OVA peptide OVA323–339 plus MHC class II molecule I-Ad (20) were obtained from Dr. D. Loh (Hoffmann-LaRoche, Nutley, NJ). STAT4-deficient mice have been reported previously (18). STAT4-/- mice were bred with DO11.10 TCR transgenic mice to generate DO11.10 x STAT4-/- mice. DO11.10/STAT4+/+ (wild type (WT)) and DO11.10/STAT4-/- mice were bred in our pathogen- and viral-free facility in accordance with guidelines of the committee on animals of the Harvard Medical School, and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (Washington, D.C.).

In vitro differentiation of CD4+ cells

Purified CD4+ T cells from DO11.10 STAT4+/+ (WT) and DO11.10 STAT4-/- mice were differentiated in the presence of OVA peptide, APCs (APC-T cell ratio, 10:1), and IL-12 plus neutralizing anti-IL-4 mAb (Th1), IL-4 (Th2), or neutralizing anti-IL-4 mAb only without exogenous cytokines (Th0) as reported previously (6). IL-2 (10 U/ml, final concentration) was added to the cultures on day 3. The cells were harvested on day 5 and centrifuged over a density gradient to remove dead APCs and cell debris. In the experiments presented in Fig. 6Go, purified CD4+ T cells from DO11.10 WT mice were activated in the presence of OVA peptide and APCs only until day 3, at which time cytokines and Abs were added as follows: IL-12 (10 ng/ml) plus neutralizing mAb specific for IL-4 (0.5 µg/ml) and IFN-{gamma} (1 µg/ml), IL-4 (1000 U/ml) plus neutralizing mAb to both IL-12 (2 µg/ml, final concentration), and IFN-{gamma}; or IFN-{gamma} (500 U/ml) plus neutralizing mAb to both IL-12 and IL-4 to different wells.



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  		FIGURE 6. IL-12 maintains a high level of Fuc-T VII mRNA and directly induces an increase in C2GnT mRNA transcripts. Naive T cells were activated in the presence of OVA peptide and APCs as described in Materials and Methods. On day 3 IL-12, IFN-{gamma}, or IL-4 together with their respective cocktail of neutralizing Abs, as indicated, were added to different wells. Medium alone or a combination of the three neutralizing Abs was added to control wells. Cells were harvested, and their expression of Fuc-T VII and C2GnT mRNA was detected by RNase protection assay as described above. The protected RNA fragments were separated on a 5% polyacrylamide gel and were visualized by phosphorimaging (A); the density of the protected RNAs was quantified using ImageQuant software and was normalized to the mean RNA levels of housekeeping genes L32 and GAPDH (B). The gel and graph shown are a representative experiment of two separate experiments. Flow cytometric data show the correlation between IL-12-induced P-selectin binding and mAb 1B11 epitope expression (C).

 
Parallel plate flow chamber analysis

The differentiated WT or STAT4-/- T cells were resuspended in Dulbecco’s PBS containing 0.1% human serum albumin and 20 mM HEPES, pH 7.4, at 37°C (5 x 105 cells/ml). T cells were drawn over E-selectin-Ig or P-selectin-Ig fusion protein-coated coverslips at an initial flow rate of 0.52 ml/min (estimated shear stress, 1 dyne/cm2) for 3 min and subsequently decreased to 0.26 ml/min (0.5 dyne/cm2) for another 3 min as previously reported (6, 21). Human P-selectin-Ig (20 µg/ml) or E-selectin-Ig (60 µg/ml) fusion proteins (gifts from Dr. Raymond, Genetics Institutes, Cambridge, MA) were captured on glass coverslips using goat F(ab')2 anti-human Fc Ab as previously detailed (21). This concentration was found to be saturating by performing dose-response adhesion assays (6, 21). T cell accumulation was determined after the final minute of each flow rate by counting the number of interacting cells in four different fields. The effects of anti-P-selectin glycoprotein ligand-1 (PSGL-1) mAb were assessed by preincubating T cells with a saturating concentration of mAb 2PH1 (10 µg/ml IgG; BD PharMingen, San Diego, CA) for 15 min on ice before assay.

RNase protection assay

T cells (5 x 106) from each condition were lysed in TRIzol reagent (Life Technologies, Grand Island, NY), and the lysate was stored at -80°C. On the day of assay, RNA was purified from lysates according to the manufacturer’s instructions. A template probe set specific for unique and selected sequences of the Fuc-T IV (GenBank accession no. U33457; location sequence, 1296–1615), Fuc-T VII (GenBank accession no. U45980; location sequence, 2467–2753), and C2GnT (GenBank accession no. U19265; location sequence, 803-1058) mRNA was custom made by BD PharMingen. 32P-labeled RNA probe synthesis, RNA hybridization, and subsequent RNase protection assays were conducted according to the manufacturer’s instruction. The protected RNA was separated on a 5% denaturing polyacrylamide gel, and the quantity of protected RNAs was determined using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA) (22). For quantification, the densities of the bands were corrected for background levels, and the RNA levels were normalized to the mean levels of the housekeeping gene GAPDH and L32. Because the mRNA levels of L32 and GAPDH in naive cells are different from those in differentiated cells, the RNA levels in naive cells were normalized to only housekeeping gene L32.

C2GnT enzyme assay

Th1, Th2, Th0, and naive CD4+ DO11.10 T cells were generated and were harvested on day 5 as described above. The cells were washed, pelleted by centrifugation (2.5 x 107 cells/pellet), and stored at -80°C until assayed. Core 2 enzymatic activity was determined by incorporation of [3H]UDP-N-acetylglucosamine substrate into acceptor molecule p-nitrophenyl 2-acetamido-2-deoxy-3-O-({beta}-D-galactopryanosyl)-{alpha}-D-galactopyranoside (Sigma) as described by Kumar and coworkers (23). All samples were assayed in duplicate, and the control for each consisted of a reaction run in the absence of acceptor, which was subtracted to adjust for background activity.

FACS analysis

Single- or double-color staining of T cells was conducted as previously described (6). CD4+ T cells (0.8–1.0 x 104) were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Analyses were performed on CD4+-gated cells. The expression of the 130-kDa CD43 isoform on the CD4+ T cells was monitored using anti-CD43 mAb 1B11 (BD PharMingen) (24, 25). During CD4+ T cell activation, tightly regulated C2GnT-dependent O-glycosylation of CD43 results in the expression of a 130-kDa species that is recognized by mAb 1B11 (24, 25, 26). 1B11 is also reported to bind to resting CD8+ cells (24), and a recent study using CD43-deficient mice showed that the Ab recognizes a novel epitope of CD45RB expressed on CD8+ T cells, but not on CD4+ T cells (26). Previous studies demonstrated that the expression of 1B11 correlates directly with the level of C2GnT enzyme activity in T cells (25) and that C2GnT-deficient myeloid and CD4+ T cells lack this epitope (11, 27). Thus, the epitope recognized by mAb 1B11 is a reasonable marker of C2GnT activity in the activated CD4+ T cells examined in this study.

Adoptive transfers and immunization

In vitro activated DO11.10 WT or STAT4-/- T cells were harvested on day 5 or 6 and centrifuged over Ficoll density gradient before adoptive transfer (13). T cells (15–20 x 106) were introduced into normal BALB/c recipients by tail vein injection. Twenty-four hours later mice were injected i.p. with CFA (Difco, Detroit, MI) emulsified in PBS or with PBS alone. Seventy-two hours after adoptive transfer of cells, the spleen and peritoneal cells were harvested, and cell suspensions were prepared and stained with anti-CD4 and the clonotypic Ab, KJ126, as previously described (13, 28).

Statistical analysis

All results are expressed as the mean ± SD. Statistical analysis by unpaired t test was performed using Microsoft Excel 5.0 (Microsoft, Redmond, WA), and differences were considered statistically significant at p <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of Fuc-T VII and C2GnT mRNA transcripts precedes P- and E-selectin adhesion under flow

To gain insight into the mechanism by which IL-4 and IL-12 regulate the level and/or activity of Fuc-TVII and C2GnT enzymes during Ag-stimulated T cell differentiation, the initial experiments determined the time course of induction of Fuc-T VII and C2GnT using a quantitative RNase protection assay (Fig. 1GoA). In parallel, we also determined the ability of these cells to interact with immobilized P- and E-selectin as previously detailed under flow (Fig. 1GoB) (6, 29, 30). Naive CD4+ T cells express low or undetectable levels of Fuc-T IV, Fuc-T VII, or C2GnT mRNA transcripts as determined by normalizing mRNA levels to control mRNA transcript L32 (Naive T panel, Fig. 1GoA). After 3 days of culture under Th1 conditions, both Fuc-T VII and C2GnT mRNA transcripts are elevated. The peak expression of mRNA transcripts for both enzymes occurs between days 3 and 4 and then gradually declines (Fig. 1Go, A and B, panel Th1) as determined by normalizing mRNA levels to two control genes, GAPDH and L32. In contrast, only low expression of Fuc-T VII mRNA transcripts is detected under Th2 conditions when normalized to control mRNAs. Both Th1 and Th2 conditions lead to a significant increase in C2GnT mRNA transcripts. The low expression levels and lack of Fuc-T IV up-regulation by cytokines suggest that the relative level of Fuc-T IV mRNA does not have a critical role in the differential expression of selectin ligands in this murine system (Fig. 1GoB). Evidence for a role of Fuc-T IV in producing selectin ligands is conflicting in other systems (8, 31, 32) and thus may vary depending on the specific molecule as well as the species and experimental model under study. Th0 cells bind E-selectin, but not P-selectin (6). Under Th0 conditions, the levels of Fuc-T VII mRNA were elevated and comparable to those in Th1 cells, whereas levels of Fuc-T IV and C2GnT were modestly elevated above those in naive T cells on day 3 and declined toward baseline levels by day 5 (Fig. 1Go, A and B).



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  		FIGURE 1. Th1 cell binding to P- and E-selectin under flow conditions correlates with elevations in Fuc-T VII and C2GnT mRNA transcripts. Naive DO11.10 T cells were activated with OVA peptide in the presence of APCs (APCs to T cell ratio, 10:1) and IL-12 plus anti-IL-4-neutralizing mAb (Th1), IL-4 (Th2), or anti-IL-4 neutralizing mAb only (Th0) as detailed in Materials and Methods. T cells were harvested on days 3–6 and tested for their ability to bind to immobilized P- and E-selectin under flow conditions as previously described (6 ). In parallel, total RNA was purified from each cell population and used in the RNase protection assay as described in Materials and Methods. The protected RNA fragments were separated on a 5% polyacrylamide gel and were visualized by phosphorimaging (A). Quantification of the protected RNAs was conducted using the ImageQuant software, and then the levels were normalized to the mean RNA levels of housekeeping genes L32 and GAPDH. The total number of T cells rolling on P- or E-selectin at 1.0 dyne/cm2 was correlated with the normalized Fuc-T VII and C2GnT levels (B). The gel and graph shown are representative of two or three different experiments. N.D., not done.

 
Th1 cells accumulate P- and E-selectin as early as day 3, and maximum adhesive interactions occur by day 5. From Fig. 1GoB it is apparent that mRNA levels and accumulation of P- or E-selectin parallel one another, and that the peak level in mRNA transcripts for both enzymes precede by 1 day cell binding to selectins. Time points earlier than day 3 could not be tested due to insufficient number of cells. Under Th2 conditions, cells exhibit few adhesive interactions with either selectin. Based on these experiments, Th1 cells exhibit maximal interactions with E- and P-selectins by day 5, and both Fuc-T VII and C2GnT peak ~24 h before this. Thus, a direct correlation exists between the time course of induction of Fuc-T VII and C2GnT mRNA and the acquisition of functional selectin ligands on CD4+ Th1 cells activated in vitro.

PSGL-1 expressed by Th1 cells is a major ligand for both E- and P-selectin

It has been shown previously (6, 7, 15) that IL-12 is essential for functional P-selectin ligand expression on Th subsets. The best characterized ligand for P-selectin is PSGL-1, which is expressed abundantly on T cells, and from several lines of evidence requires post-translational modification by Fuc-T VII and C2GnT as well as sulfation to bind ligand (10, 11, 12, 33, 34). Preliminary experiments showed that PSGL-1 is expressed uniformly at high levels on Th1, Th2, Th0, and naive T cell subsets by flow cytometry using mAb 2PH1, but that only Th1 cells display significant adhesion to P- and E-selectin in an in vitro flow assay (data not shown). Pretreatment of Th1 cells with saturating concentrations of mAb 2PH1 (anti-PSGL-1) significantly inhibits (50–60%) accumulation of both P-selectin and E-selectin (50% inhibition) across a range of shear stress (Fig. 2Go). These data clearly establish PSGL-1 as an important Th1 cell ligand for both P- and E-selectin. This conclusion is also consistent with the recent findings (35) that murine Th1 cells from PSGL-1-deficient mice exhibited no binding to P-selectin and diminished binding to E-selectin in in vivo and in vitro models.



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  		FIGURE 2. Pretreatment of Th1 cells with blocking anti-PSGL-1 mAb inhibits both P- and E-selectin interactions under flow. Th1 cells were pretreated with blocking anti-PSGL-1 mAb (2PH1, 10 µg/ml) for 15 min on ice before flow assay. Th1 cell accumulation on P- and E-selectin was assessed as described in Materials and Methods. Data are the mean ± SD from three different experiments. *, p <= 0.05 compared with medium-treated Th1 cells.

 
IL-12-stimulated STAT4-/- DO11.10 CD4+ T cells do not roll on P-selectin and exhibit impaired interactions with E-selectin under flow

To examine the role of the IL-12-STAT4 signaling pathway in the induction of functional PSGL-1, naive T cells from DO11.10 STAT4-/- transgenic mice were activated for 5 days with Ag (OVA peptide) in the presence of APCs and IL-12 (Th1 condition), IL-4 (Th2 condition), or no added cytokines plus a neutralizing Ab to IL-4 (termed Th0 condition) as detailed in Materials and Methods (6). STAT4-/--naive CD4+ T cells activated in the presence of IL-12 (Th1 condition) and restimulated had a decreased amount of cytosolic IFN-{gamma} and a lower percentage of cells that produced IFN-{gamma} compared with WT cells, whereas STAT4-/- cells activated under Th2 conditions and restimulated with Ag had IL-4 levels somewhat higher than those in WT cells (data not shown), which is the consistent with previous studies in STAT4-/- mice (18).

We next assessed the ability of WT and STAT4-/- T cells to interact with P- or E-selectin under defined flow conditions. Strikingly, fewer IL-12-stimulated STAT4-/- cells interact with P-selectin at either 1.0 and 0.5 dyne/cm2 (Fig. 3Go, top) compared with control WT cells. Essentially no IL-4-stimulated (Th2 condition) or anti-IL-4 mAb-treated (Th0 condition) WT or STAT4-/- T cells interacted with P-selectin at 1.0 dyne/cm2, and only minimum interactions were detected at 0.5 dyne/cm2. Interestingly, on E-selectin, although total interactions of IL-12-, IL-4-, or anti-IL-4-treated STAT4-/- cells are comparable to the WT controls at the lower shear of 0.5 dyne/cm2, there is a significant reduction in interactions between IL-12-treated WT and STAT4-/- T cells at the higher shear stress (Fig. 3Go). The mechanisms accounting for the loss of binding at higher shear stress are not known. Consistent with our previous report (6), anti-IL-4-treated (i.e., Th0 conditions) WT and STAT4-/- cells also interact with E-selectin over the range of shear stress studied, albeit the number of cells rolling on E-selectin at 1.0 dyne/cm2 is significantly less than that seen with Th1 cells (IL-12-stimulated). In addition, few IL-4-stimulated WT or STAT4-/- T cells interact with E-selectin. We conclude that STAT4 signaling is essential for IL-12-mediated expression of functional ligands (e.g., functional PSGL-1) that support interactions with P-selectin (36, 37).



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  		FIGURE 3. Accumulation of differentiated WT and STAT4-/- cells on P-selectin and E-selectin under defined flow conditions. After 5 days in culture, WT ({blacksquare}) and STAT4-/- ({square}) T cells differentiated in the presence of IL-12 alone, IL-4 alone, or neutralizing mAb to IL-4 (anti-IL-4) were tested for their ability to bind to P-selectin or E-selectin-IgG chimeras at 0.5 (left side) or 1.0 (right side) dyne/cm2. Data are the mean ± SD from three different experiments. *, p <= 0.05, WT vs STAT4-/- T cells; {ddagger}, p <= 0.05 compared with IL-12-stimulated WT T cells.

 
IL-12-stimulated STAT4-/- T cells are less efficiently recruited in vivo

Based on the above differences seen in in vitro assays, we tested whether IL-12-stimulated WT or STAT4-/- T cells differ in their ability to enter an inflammatory site using a previously described adoptive transfer protocol (13, 28). Briefly, IL-12-stimulated WT and STAT4-/- cells were adoptively transferred into syngeneic BALB/c recipient mice by tail vein injection, the mice were challenged with CFA, and the number of adoptively transferred CD4+KJ126+ cells in the spleen and peritoneum were determined after 3 days. In control mice (without CFA), the numbers of CD4+ T cells in the peritoneum from the WT and STAT4-/- groups were similar (Fig. 4GoA, top panels). Of these, 2.8% from the WT group and 2.4% from the STAT4-/- group were identified as the adoptively transferred CD4+/KJ126+ double-positive cells. In contrast, ~2.5 times as many WT cells as STAT4-/- cells migrated into the peritoneum of mice injected with CFA (5.7% in WT vs 2.2% in STAT4-/-). In three separate experiments, migration was 4.9-fold (p < 0.05) higher for WT cells (recruited cells, 7.3 ± 3.1 x 105) than STAT4-/- cells (1.5 ± 1.4 x 105). There was no difference in the number of CD4+ KJ126+ T cells from WT compared with the STAT4-/- groups that were recruited to spleen or lymph nodes (Fig. 4GoB and data not shown) in either the absence or the presence of CFA treatment. The less efficient trafficking of STAT4-/- CD4+ T cells in vivo is consistent with their lack of functional selectin ligand expression during differentiation in vitro in response to IL-12 treatment (Fig. 3Go). However, this deficiency did not appear to affect the normal recirculation of T cells through lymphoid tissues. This finding is consistent with our previous report (13) and with a recent study by Tietz et al. (38) that only CD4+ T cells that expressed functional ligand for E- and for P-selectin were capable of homing to inflammatory sites.



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  		FIGURE 4. IL-12-stimulated STAT4-/- DO11.10 T cells are less efficiently recruited into the inflamed peritoneum. CD4+-naive DO11.10 WT and STAT4-/- T cells were activated in vitro using standard Th1 differentiation conditions as previously detailed (13 ). On day 6 equal numbers of activated cells (2 x 107 cells) were adoptively transferred into BALB/c mice. After 24 h the mice were either left untreated (-CFA) or were treated with PBS-CFA (+CFA) i.p. Three days later, peritoneal (A) and spleen (B) cells were collected and stained for CD4 (PE) and the DO11.10 TCR Ag KJ126 (CyC) expression. The numbers adjacent to the upper right corner of each panel are the percentage of the total gated cells that are CD4+KJ126+. Results from one of three comparable experiments are shown.

 
TCR-activation alone is sufficient to induce Fuc-T VII mRNA, but the STAT4-IL-12 cytokine signal is required for the induction of C2GnT mRNA

Because the above results show that STAT4 signaling is necessary for the induction of P-selectin ligand, but not E-selectin ligand, and other investigators have found that PSGL-1-dependent binding to either E- or P-selectin is regulated through post-translational glycosylation (27, 34, 39, 40), we compared the levels of C2GnT and Fuc-T VII mRNAs in cultured WT and STAT4-/- T cells by quantitative RNase protection assay. IL-12-stimulated WT cellsexpressed elevated levels of both Fuc-T VII and C2GnT mRNA, whereas WT IL-4-stimulated cells had an increase in C2GnT mRNA, but not Fuc-T VII (Fig. 5GoA, compare lane 2 to lane 3 and normalized data in Fig. 5GoB). WT cells activated with Ag only in the presence of anti-IL-4 (Th0 condition) expressed elevated levels of Fuc-T VII mRNA, but low amounts of C2GnT mRNA (lane 4), which correlates with rolling on E-selectin, but not P-selectin, as shown in Fig. 3Go. IL-12-stimulated STAT4-/- cells expressed high levels of Fuc-T VII, but diminished levels of C2GnT, a profile similar to that of WT anti-IL-4 cells (Fig. 5GoA, compare lanes 4 and 5). In three independent experiments, the normalized C2GnT mRNA levels were 2- to 5-fold lower in IL-12-stimulated STAT4-/- compared with those in IL-12-stimulated WT cells (Fig. 5GoB). In contrast, there was no difference in the induction of Fuc-T VII and C2GnT mRNA between WT and STAT4-/- in IL-4- or anti-IL-4-treated cells (Fig. 5GoA, lanes 3 and 6 and lanes 4 and 7, and normalized results in Fig. 5GoB). However, the presence of IL-4 (Th2 condition) consistently decreased the expression of Fuc-T VII in both WT and STAT4-/- T cells compared with that in either Th1 or Th0 conditions. In addition, a higher level of Fuc-T VII mRNA transcripts was detected in the IL-12-stimulated WT than in STAT4-/- T cells.



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  		FIGURE 5. Effects of cytokines and TCR activation on Fuc-T VII and C2GnT mRNA induction. Total RNA was purified from WT and STAT4-/- T cells differentiated in culture medium containing IL-12 alone, IL-4 alone, or with neutralizing anti-IL-4 mAb ({alpha}-IL-4 mAb). On day 5 the cells were harvested and used in the RNase protection assay as described in Materials and Methods. The protected RNA fragments were separated on a 5% polyacrylamide gel and visualized by phosphorimaging (A). Quantification of the protected RNAs was conducted using ImageQuant software and normalized to the mean RNA levels of housekeeping genes L32 and GAPDH (B). The gel and graph shown are representative of results from three separate experiments. WT and STAT4-/--naive T cells and T cells stimulated in presence of IL-12, IL-4, and anti-IL-4 neutralization mAb were analyzed for cell surface expression of 1B11 epitope, which recognizes the 130 isoform of CD43. The numbers represent the mean channel fluorescence of 95–100% of the CD4+ gated cells for each condition. Data are representative of five to nine different experiments for a–d and three different experiments for e–h (C).

 
We next confirmed the lack of C2GnT activity in Th1 STAT4-/- cells by monitoring surface expression of the mAb 1B11 epitope, which in CD4+ T cells is absolutely dependent on this enzyme (11, 24, 25, 26, 27). As shown in Fig. 5GoC, flow cytometric analysis showed that 1B11 is weakly expressed on WT or STAT4-/- naive T cells. Under Th1 conditions, WT T cells showed a striking elevation of 1B11 epitope (>80-fold over naive), whereas STAT4-/- cells had a markedly attenuated expression (17-fold). This held true when both WT and STAT4-/- cells were evaluated at earlier time points, suggesting the reduction in 1B11 epitope expression is not due to altered kinetics of C2GnT enzyme (data not shown). Taken together, these results demonstrate an essential role for IL-12 signaling via STAT4 for the induction of C2GnT mRNA transcripts and C2GnT activity, and that TCR activation alone (Th0 conditions) is sufficient to induce transcription of Fuc-T VII, but not C2GnT, genes. However, the ability of IL-4 and IL-12 to exert opposing effects on Fuc-T VII mRNA levels implies a further level of regulation by the exogenous cytokine milieu (see below and Fig. 6Go). Moreover, the elevated levels of C2GnT mRNA found in both IL-4-stimulated WT and STAT4-/- T cells, which are comparable to those in IL-12-stimulated WT cells, suggest that C2GnT can also be induced by an IL-4-dependent, IL-12/STAT4-independent mechanism.

In the course of these studies we noted that WT Th2 cells have a diminished up-regulation of 1B11 epitope (17-fold over naive) despite the relative abundance of C2GnT mRNA transcripts. The explanation appears to reflect a significantly diminished C2GnT enzymatic activity in WT Th2 cells compared with Th1 cells (Table IGo). This lack of correlation between mRNA level and activity for C2GnT is interesting and may reflect diminished translation of the mRNA, post-translational modifications that attenuate C2GnT activity or the presence of an inhibitor(s) in differentiating Th2 cells.


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  		Table I. Correlation between Fuc-T VII and C2GnT mRNA expression with 1B11 epitope, C2GnT enzyme activity, and ability to interact with P- and E-selectin under flow1

 
IL-12 induces C2GnT mRNA in Ag-activated T cells, which correlates with C2GnT activity and P-selectin binding

In the absence of STAT4, IL-12 failed to induce C2GnT mRNA, whereas IL-4 induced equivalent levels of C2GnT in both STAT4-/- and WT T cells. To test whether IL-12 or IL-4 could directly induce C2GnT mRNA transcripts, we used a modified in vitro T cell differentiation protocol. Th0 cells (Ag stimulation in absence of added cytokines) were prepared from naive CD4+ T cells, and IL-12, IL-4, or IFN-{gamma} was added on day 3 with appropriate cytokine-neutralizing mAb. As shown in Fig. 6GoA and the normalized data in Fig. 6GoB, treatment with Ag alone induced Fuc-T VII, but not C2GnT, mRNA. Addition of IL-12, but not IL-4 or IFN-{gamma}, induced C2GnT expression within 24 h (day 4), which was maintained for at least 48 h (day 5). The rapid induction of C2GnT by IL-12 correlates with high 1B11 epitope expression and binding P-selectin (Fig. 6GoC). Surprisingly, under these specific assay conditions, IL-4 did not induce C2GnT mRNA expression; however, IL-4 rapidly decreased the expression of Fuc-T VII. In the normalized data in Fig. 6GoB, IL-4 decreases Fuc-T VII mRNA expression compared with control treatment through an as yet unidentified mechanism. In contrast, addition of IL-12 sustained Fuc-T VII mRNA transcripts (Fig. 6Go, A and B, compare Fuc-T VII levels in medium with/without Abs to +IL-12 conditions on days 4 and 5). This result is consistent with the previous report by Blander et al. (14) that Fuc-T VII mRNA is induced by TCR activation, and that the relative level expressed is maintained by IL-12 and decreased by IL-4. In comparison, IFN-{gamma} exerted little effect on either enzyme compared with medium. We conclude that in the presence of IL-12, Ag-activated CD4+ T cells express abundant C2GnT mRNA that generates the C2GnT-dependent 1B11 epitope. These conditions also sustain Fuc-T VII expression, which together with C2GnT generate P-selectin ligands.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous in vivo and in vitro studies have shown that generation of E-selectin ligands require post-translational modification by Fuc-T VII, whereas P-selectin ligands require both C2GnT and Fuc-T VII (10, 11, 34, 39, 41). The main findings in this study are that the expression and activity of glycosyltransferases essential for generation of E- and P-selectin ligands can be dramatically affected by the cytokine milieu that influences CD4+ Th subset differentiation (6). Initially, we focused on Th1 cell polarization by IL-12; the data show a direct correlation between the induction of Fuc-T VII and C2GnT mRNA transcripts with the acquisition of P- and E-selectin binding. To dissect the contributions of the STAT4/IL-12 signaling pathway in the generation of selectin ligands and the induction of C2GnT and Fuc-T VII, STAT4-/- mice were used. During Th1 differentiation, C2GnT induction was absolutely dependent on IL-12/STAT4 signaling. In contrast, Fuc-T VII could be induced by TCR activation alone in the absence of IL-12/STAT4. The functional correlates of glycosylation gene expression were as follows. 1) STAT4-/- Th1 cells expressed very low levels of P-selectin ligands as demonstrated by minimal rolling interactions with P-selectin under fluid shear stress conditions. 2) These Th1 cells exhibited a partial defect in E-selectin ligands under elevated fluid shear stress and not at lower levels. 3) Th1 STAT4-/- cells exhibited significantly less trafficking to sites of inflammation, but normal homing to lymphoid tissues in vivo. Taken together, we conclude that IL-12/STAT4 signaling is required for the induction of C2GnT and generation of P-selectin ligands. Furthermore, that the IL-12/STAT4 signaling, together with Ag-TCR-induced FucT-VII gene expression are essential for the synthesis of functional ligands for P-selectin and are required for optimal expression of E-selectin ligands.

C2GnT catalyzes the addition of branched O-glycan side chains to PSGL-1 (34, 42) and to CD43-generating epitope 1B11 (11, 24, 25, 26, 27). When comparing WT and STAT4-/- cells stimulated under Th2 conditions, we found unexpectedly that the level of C2GnT mRNA in STAT4-/- T cells was comparable to that in WT T cells, and that the extent of expression was similar to that in WT Th1 cells (Fig. 1Go, A and B; Fig. 5Go, A and B; and Table IGo). Despite the abundance of mRNA transcripts in WT and STAT4-/- Th2 cells, these cells expressed significantly lower levels of mAb 1B11 epitope, the 130-kDa O-glycosylated form of CD43 (Fig. 5GoC). Possible explanations for this finding are that the mRNA was not translated to protein, or that the enzyme was in some way impaired. Because we lacked an Ab for detection of the enzyme protein, we evaluated the C2GnT activity in WT Th2 cells and found a decrease in enzyme activity in these cells (Table IGo). Nevertheless, the results with STAT4-/- Th2 cells (Fig. 5Go) support the hypothesis that IL-4 can induce C2GnT mRNA independently of the IL-12-STAT4 signaling pathway. Unlike IL-12, IL-4 induction of C2GnT appears to require a simultaneous TCR activation signal because adding IL-4 3 days after Ag stimulation failed to induce C2GnT mRNA (compare Fig. 1Go to Fig. 6Go). Consistent with these results, putative binding sites for STAT4 are present in the nucleotide sequence for mouse C2GnT (43, 44). Further work is necessary to identify the STAT4-DNA binding sites in the C2GnT promoter.

Function blocking mAb studies showed that on Th1 cells, PSGL-1 was a major ligand for both E- and P-selectin in an in vitro flow adhesion assay. In addition, the data strongly support the idea that there also exists a unique and PSGL-1-independent E-selectin ligand on Th1 cells. These data are consistent with other reports in a variety of experimental systems showing that PSGL-1 is a ligand for both E- and P-selectin (9, 34, 45, 46). Recent studies showed that Th1 cells from PSGL-1-/- mice failed to interact with P-selectin in vivo or in vitro (35). Interestingly, PSGL-1-/- Th1 cell interactions with E-selectin in vitro and in vivo were reduced, but not abolished, demonstrating the existence of other ligands for E-selectin. One possibility is E-selectin ligand-1 (47, 48). E-selectin ligand-1 was purified from murine neutrophils and a mouse myeloid cells line (32Dcl3) by affinity isolation with an E-selectin-IgG chimera fusion protein. However, E-selectin ligand-1 expression in murine or human T cells subsets has yet to be examined. In this study we also found that the partial defect in E-selectin ligands in the IL-12-stimulated STAT4-/- cells is evident under elevated, but not at lower, fluid shear stress. Under these conditions, STAT4-/- Th1 cells lack sufficient C2GnT activity, which, in turn, may lead to impair the function of PSGL-1 at high shear stress. This observation is consistent with a recent report by Sperandio et al. (49), who studied E- and P-selectin-mediated leukocyte rolling in venules of C2GnT-deficient mice. These authors concluded that E-selectin-mediated rolling in vivo at wall shear rates >300 s-1 is significantly reduced compared with WT controls. This relationship between C2GnT-dependent E-selectin ligand generation and differential rolling/attachment at high and low fluid shear stress in vivo and in vitro is interesting and merits further studies.

Recent studies have identified two additional C2GnT loci from the human genome survey, C2GnT-M and C2GnT3, in addition to the original C2GnT gene found in leukocytes now designated C2GnT-L (50, 51). C2GnT-M was identified from expressed sequence tag (dbEST) and exhibits both C2GnT and C4GnT enzymatic activities. Its expression is localized to the colon, small intestine, trachea, and stomach, where mucins are produced (50). C2GnT3, which exhibits exclusively C2GnT activity, was identified by BLAST analysis (51). However, C2GnT3 transcript appear to be expressed selectively in the thymus (51). C2GnT-M has 48% homology with C2GnT-L at the amino acid levels (50). In light of this, one potential caveat of the RNase protection assay analysis for murine C2GnT mRNA is that the probe (designed for murine C2GnT-L) may also detect C2GnT mRNA transcribed from other C2GnT genes. At this juncture, we are unable to determine whether our probe will cross-hybridize with murine C2GnT-M or C2GnT3 because to the best of our knowledge the murine C2GnT orthologs have not been identified or cloned. However, if murine analogs exist and exhibit a similar level of homology as reported for the human genes (~50%), the RNase protection assay probe would be unlikely to detect mRNA transcribed from these analogs, because the probes must hybridize and bind 90 contiguous nucleotides to be protected from RNase digestion according to the manufacturer.

The current data also demonstrate a more intricate interrelationship than previously appreciated between the cytokine- and TCR-mediated signals that regulate the biosynthesis of functional selectin ligands. Ag-specific TCR activation alone can induce levels of Fuc-T VII mRNA sufficient to support E-selectin ligand expression, presumably sialylated Lewisx-related epitopes. The presence of IL-4 during cell activation appears to rapidly diminish the TCR activation-induced increase in Fuc-T VII mRNA, which, in turn, has a direct negative influence on the expression of functional E-selectin ligand(s). Interestingly, our data further show that IL-12 retards the normal decay of Fuc-T VII mRNA expression seen after TCR activation (Fig. 6GoB). This finding is consistent with a previous report that Fuc-T VII mRNA was detected in naive T cells as early as 48 h following TCR activation and then was rapidly down-regulated in the presence of IL-4 but maintained in the presence of IL-12 (14). These findings support our working hypothesis that cytokine signals that drive Th cell polarization also significantly influence the levels of Fuc-T VII and C2GnT mRNA, which are critical for E- and P-selectin ligand synthesis and dictate the ability of the Th cells to emigrate to peripheral inflammatory sites. A working model emerges suggesting that binding of IL-12 to IL-12R (or IL-4 to IL-4R) together with Ag-driven TCR activation induce differentiation of naive T cells to effector cytokine-producing Th1 or Th2 cells. This cytokine signal also induces and modulates the levels of two glycosyltransferases, Fuc-T VII and C2GnT, which are required for optimal expression of both selectin ligands. The mechanism underlying the STAT pathway regulation of C2GnT transcription and regulation of enzymatic activity and TCR activation-induced transcription of Fuc-T VII emerge as very timely questions that need to be addressed by future experiments.

In summary, the current findings show IL-12/STAT4 signaling is essential for induction of C2GnT, and that STAT4-/- Th1 cells do not interact with P-selectin, have a partial defect in rolling on E-selectin, and do not traffic normally to sites of inflammation in vivo. Furthermore, that Ag-specific TCR activation is sufficient to trigger induction of Fuc-TVII, but not C2GnT mRNA, and expression of E-selectin, but not P-selectin, ligands. Thus, both cytokine and TCR signals are necessary for proper biosynthesis of functional E- and P-selectin ligands and recruitment to tissues in a murine experimental model of inflammation.


   Acknowledgments
 
We thank Drs. Raymond Camphausen and Ravi Kumar (Genetics Institute, Cambridge, MA) for providing purified human selectin-Ig chimeras and assistance with the C2GnT enzyme assay, and members of the Vascular Research Division and the Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital, for their helpful discussions and support during this study.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants HL36028 (to A.H.L.) and HL53993 and HL65090 (to F.W.L.). M.J.G. is a Scholar of the Leukemia Society of America and is supported by National Institutes of Health Grant AI40171 and a gift from the Mathers Foundation. H.X. was supported by an Arthritis Foundation postdoctoral fellowship, and Y.-C.L. was supported by an American Heart Association postdoctoral fellowship. Back

2 Address correspondence and reprint requests to Dr. Francis W. Luscinskas, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, MA 02115. E-mail address: fluscinskas{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: Fuc-T VII, fucosyltransferase VII; PSGL-1, P-selectin glycoprotein ligand-1; C2GnT, core 2 {beta}-1,6-N-acetylglucosaminyltransferase; WT, wild type. Back

Received for publication September 14, 2000. Accepted for publication August 15, 2001.


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 Materials and Methods
 Results
 Discussion
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G. An, H. Wang, R. Tang, T. Yago, J. M. McDaniel, S. McGee, Y. Huo, and L. Xia
P-Selectin Glycoprotein Ligand-1 Is Highly Expressed on Ly-6Chi Monocytes and a Major Determinant for Ly-6Chi Monocyte Recruitment to Sites of Atherosclerosis in Mice
Circulation, June 24, 2008; 117(25): 3227 - 3237.
[Abstract] [Full Text] [PDF]

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 Int ImmunolHome page
N. Harakawa, A. Shigeta, M. Wato, G. Merrill-Skoloff, B. C. Furie, B. Furie, T. Okazaki, N. Domae, M. Miyasaka, and T. Hirata
P-selectin glycoprotein ligand-1 mediates L-selectin-independent leukocyte rolling in high endothelial venules of peripheral lymph nodes
Int. Immunol., March 1, 2007; 19(3): 321 - 329.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
U. Syrbe, U. Hoffmann, K. Schlawe, O. Liesenfeld, K. Erb, and A. Hamann
Microenvironment-Dependent Requirement of STAT4 for the Induction of P-Selectin Ligands and Effector Cytokines on CD4+ T Cells in Healthy and Parasite-Infected Mice
J. Immunol., December 1, 2006; 177(11): 7673 - 7679.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
V. R. Taqueti, N. Grabie, R. Colvin, H. Pang, P. Jarolim, A. D. Luster, L. H. Glimcher, and A. H. Lichtman
T-bet Controls Pathogenicity of CTLs in the Heart by Separable Effects on Migration and Effector Activity
J. Immunol., November 1, 2006; 177(9): 5890 - 5901.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
Z. Ni, J. J. Cam