P-Selectin Glycoprotein Ligand-1 Negatively Regulates T-Cell Immune Responses

Masanori Matsumoto, Masayuki Miyasaka and Takako Hirata
J Immunol December 1, 2009, 183 (11) 7204-7211; DOI: https://doi.org/10.4049/jimmunol.0902173

Abstract

Cell surface sialomucins often act as antiadhesive molecules by virtue of their extended structure and negative charge. CD43 is one such sialomucin, expressed on most leukocytes. P-selectin glycoprotein ligand-1 (PSGL-1) is another sialomucin expressed by leukocytes. It serves as a major selectin ligand, but no antiadhesive role for it has been described. In this study, we showed that PSGL-1-deficient T cells, like CD43-deficient T cells, exhibited increased adhesion and proliferation compared with wild-type cells. The loss of both PSGL-1 and CD43 led to a further increase in T cell adhesion and proliferation. The reexpression of full-length PSGL-1 or CD43 in double-deficient CD4+ T cells reversed their increased adhesion and proliferation phenotype. Using chimeric constructs of human CD8 and either PSGL-1 or CD43, we demonstrated that the intracellular domain of PSGL-1 or CD43 is required for suppressing proliferation but not adhesion. Furthermore, in a mouse model of inflammatory bowel disease induced by the adoptive transfer of naive T cells into RAG-deficient hosts, a PSGL-1 deficiency exacerbated the development of inflammation. These results reveal a novel regulatory role for PSGL-1 in T cell adhesion and proliferation and suggest that PSGL-1 negatively regulates T cell immune responses in vivo.

Cell surface sialomucins represent a family of glycoproteins that are highly glycosylated with sialylated O-linked glycans. Because of their extended structure and the negative charge imparted by their sialic acid residues, some sialomucins can provide a repulsive barrier around a cell that inhibits cell-cell interactions. CD43 is one such molecule (1). It is expressed by most hematopoietic cells, including T cells. The extracellular domain of both human and mouse CD43 contains more than 80 serine or threonine residues, most of which are glycosylated by O-linked glycans that are heavily sialylated; this domain has a rod-like structure that is predicted to extend 45 nm from the lipid bilayer (2). Supporting evidence for its antiadhesive role is observed in vitro by the enhanced homotypic adhesion and proliferation of CD43-deficient (CD43−/−) T cells (3), and in vivo by their increased migration into secondary lymphoid organs (4). CD43−/− neutrophils also show enhanced rolling and adhesion both in vitro and in vivo (5, 6). These antiadhesive functions, however, are not evident when CD43 interacts with its specific ligands (7). Several potential ligands for CD43 have been reported, including ICAM-1 (8), galectin-1 (9), and sialoadhesin (10). In particular, we and others reported that CD43 on mouse Th1 cells and human T lymphoblasts is modified with sialylated and fucosylated carbohydrate structures to interact with E-selectin (11, 12). This interaction plays an important role in mediating the migration of activated T cells to inflamed skin (13, 14).

P-selectin glycoprotein ligand-1 (PSGL-1;3 CD162) (3) is another sialomucin expressed by most leukocytes (15, 16). The extracellular domain of both human and mouse PSGL-1 contains >40 potential O-glycosylation sites. Like CD43, PSGL-1 is a highly extended molecule: its ectodomain is estimated to be 50-nm long (17). PSGL-1 was originally identified as the major ligand for P-selectin, and subsequent studies showed that it also binds the other two members of the selectin family, E- and L-selectin. Studies using PSGL-1-deficient (PSGL-1−/−) mice showed that, on activated T cells, PSGL-1 serves as the major ligand for P-selectin as well as being an E-selectin ligand (18, 19). Similarly, PSGL-1 on neutrophils binds P- and E-selectin to mediate their rolling on endothelial cells and migration into inflamed tissues (20, 21). These well-recognized functions of PSGL-1 are critically dependent on its glycosylation, which endows it with the ability to bind selectins. Thus, although PSGL-1 is abundantly expressed on most hematopoietic cells, its function in cells that are not equipped with the machinery to modify it to bind selectins, such as naive T cells, remains unknown.

To explore the function of PSGL-1 independent of its role as a selectin ligand, we exploited our previous findings that the related sialomucin CD43 has dual functions: it is usually antiadhesive, but when appropriately glycosylated in certain cell types, it can become a proadhesive selectin ligand (5, 6). We investigated whether the PSGL-1 expressed on T cells has antiadhesive or regulatory roles, using T cells isolated from wild-type (WT), PSGL-1−/−, CD43−/−, and PSGL-1/CD43 double-knockout (DKO) mice. We show that PSGL-1−/− T cells exhibited enhanced adhesion and proliferation. Loss of both PSGL-1 and CD43 resulted in a further increase in T cell adhesion and proliferation. The increased adhesion and proliferation of the DKO T cells were reversed by the reintroduction of PSGL-1 or CD43. The intracellular domain of PSGL-1 or CD43 was required for the reversal of the increased proliferation, but not for reducing the adhesion. Moreover, in vivo, the PSGL-1 deficiency exacerbated the development of T cell-mediated colitis. Thus, in addition to its well-characterized role as a selectin ligand, these results point to a novel role for PSGL-1 in negatively regulating T cell adhesion and proliferation and in controlling T cell-mediated immune responses in vivo.

Materials and Methods

Mice

C57BL/6 (B6) mice were purchased from CLEA Japan. PSGL-1−/− mice on a B6 background were provided by B. Furie (Harvard Medical School, Boston, MA). RAG-1-deficient (RAG-1−/−) mice on a B6 background and CD43−/− mice on a B6 × 129S4/SvJae background were purchased from The Jackson Laboratory. CD43−/− mice were back-crossed more than six generations onto the B6 genetic background. DKO mice were generated by breeding PSGL-1−/− mice with CD43−/− mice. All mice were used when 6 to 10 wk old. The mice were housed at the Institute of Experimental Animal Sciences at Osaka University Medical School. All studies and procedures were approved by the Ethics Review Committee for Animal Experimentation of the Osaka University Graduate School of Medicine.

Isolation of T cells

Total T cells and CD4+ T cells were isolated from the spleen and the peripheral and mesenteric lymph nodes using a BD IMag Mouse T Lymphocyte Enrichment Set and CD4 T Lymphocyte Enrichment Set (both from BD Biosciences), respectively. Isolated CD4+ T cells were stained with anti-CD45RB-FITC (C363-16A; BioLegend), anti-CD25-PE (PC61; BD Biosciences), and anti-CD4-allophycocyanin (RM4-5; BD Biosciences), and naive CD4+ T cells (CD4+CD25−CD45RBhigh) were sorted by FACSAria (BD Biosciences).

Construction of retroviral vectors

Full-length mouse PSGL-1 (PSGL-1FL) cDNA was inserted into the pMXs-IG vector (22), provided by T. Kitamura (University of Tokyo, Tokyo, Japan), at the BamHI and XhoI sites. The viral long terminal repeats of this vector drive the expression of PSGL-1 and enhanced GFP via a bicistronic mRNA containing an internal ribosomal entry site element. cDNAs for full-length mouse CD43 (CD43FL) and full-length human CD8 (CD8FL) were amplified from mouse Th1-cell cDNA and human thymus cDNA, respectively. To produce chimeric constructs in which the extracellular and transmembrane domain of mouse PSGL-1 or CD43 was fused to the intracellular domain of human CD8 (PSGL-1/CD8 or CD43/CD8), or the extracellular and transmembrane domain of human CD8 was fused to the intracellular domain of mouse PSGL-1 or CD43 (CD8/PSGL-1 or CD8/CD43), the corresponding regions were amplified and ligated into the pMXs-IG vector.

Retroviral infections

The improved retrovirus packaging cells, PLAT-E (23), were transfected by calcium phosphate precipitation with 20 μg of the pMXs-IG vector containing one of the constructs described above. At 12 h post transfection, the calcium phosphate-containing medium was replaced, and the cells were cultured for an additional 48 h. The virus-containing supernatant from the PLAT-E cells was harvested, added to a six-well plate coated with 20 μg/ml recombinant fibronectin fragment CH-296 (RetroNectin; Takara), and then centrifuged at 1,500 × g for 2 h. To infect T cells, purified CD4+ T cells from WT or DKO mice were stimulated with plate-immobilized anti-CD3ε (10 μg/ml; 145-2C11; BD Biosciences) plus anti-CD28 (10 μg/ml; 37.51; BD Biosciences) for 48 h and added to the retrovirus-coated plate in the presence of 10 ng/ml IL-2 (R&D Systems). After 48 h of infection, the cells were washed, transferred to uncoated dishes, and cultured for an additional 3 days.

Homotypic aggregation assays

Isolated T cells (1 × 105 cells), naive CD4+ T cells (1 × 105 cells), or retrovirus-transduced CD4+ T cells (1 × 104 cells) were added to 96-well culture plates and incubated with PMA (100 ng/ml) for 5 h in the presence of calcium or EDTA. In some experiments, T cells from DKO mice were added to the wells and incubated with PMA in the presence of 10 μg/ml anti-CD11a (M17/4; BD Biosciences), anti-ICAM-1 (KAT-1; provided by K. Kato, Juntendo University, Tokyo, Japan), anti-CD18 (GAME-46; BD Biosciences), anti-CD11b (M1/70; BD Biosciences), or isotype controls. To examine the effect of neuraminidase treatment, the isolated T cells from WT, PSGL-1−/−, CD43−/−, and DKO mice were incubated with or without neuraminidase from Clostridium perfringens (25 mU/ml; Sigma-Aldrich) for 1 h and cultured with PMA for 5 h. The number of unaggregated cells was determined from the samples before they were dissociated. To determine the total number of cells in each sample, the aggregates were dissociated to the single-cell level by the addition of EDTA. The percentage of cells that had aggregated (percent aggregation) was calculated as the number of cells in aggregates (total number of cells minus the number of single cells) over the total number of cells.

Cell adhesion assays

Isolated T cells or naive CD4+ T cells (1 × 105 cells) were stimulated with PMA (100 ng/ml), and then added to 96-well culture plates coated with 10 μg/ml fibronectin (Sigma-Aldrich) in the presence of calcium or EDTA. In blocking experiments, PMA-stimulated T cells from DKO mice were added to the wells in the presence of anti-CD49d (R1-2; BD Biosciences), anti-CD29 (Ha2/5; BD Biosciences), anti-β7 integrin (FIB27; BD Biosciences), or isotype controls. In some experiments, the isolated T cells were incubated with or without neuraminidase, stimulated with PMA, and added to 96-well plates coated with fibronectin. The plates were incubated for 20 min, unbound cells were removed, and the number of bound cells was determined by photographing the cells and counting them.

Proliferation assays

Isolated T cells or naive CD4+ T cells (1 × 105 cells) were labeled with 3.3 μM CFSE for 10 min at 37°C and stimulated with plate-immobilized anti-CD3ε (1 μg/ml) and anti-CD28 (1 μg/ml) for 48 h. The cells were stained with anti-CD4-PE (RM4-5; BD Biosciences) and anti-CD8-APC (53.6.7; BD Biosciences) and then analyzed on a FACSCalibur. Daughter generations of CD4+ and CD8+ T cells were determined by measuring the CFSE dilution. In some experiments, retrovirus-transduced CD4+ T cells (1 × 104 cells) were stimulated with plate-immobilized anti-CD3ε (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence of 1 μCi/well of [3H]thymidine (GE Healthcare) for 24 h. Cells were harvested onto a UniFilter-96 GF/C (PerkinElmer) using a UniFilter-96 Harvester (PerkinElmer), and the incorporated radioactivity was measured using a TopCount NXT scintillation counter (PerkinElmer).

Mouse colitis model

We adapted a mouse model for colitis in which colitis is induced in immunodeficient mice by the transfer of naive T cells (24, 25). Naive CD4+ T cells (5 × 105) sorted from WT, PSGL-1−/−, CD43−/−, and DKO mice were injected via the tail vein into RAG-1−/− mice. The body weight of the recipient mice was monitored throughout the experiment. The mice were analyzed 6 wk after the T cell transfer. They were assigned a disease activity score that was the sum of four parameters: hunching and wasting were scored 0 or 1, colon thickening 0–3, and stool consistency 0–3, as described (26). Tissue samples from the middle colon of untreated or T cell recipient RAG-1−/− mice were prepared for histological staining with H&E. Individual sections were scored blind, according to the following independent criteria: cell infiltration 0–3, crypt elongation 0–3, and number of crypt abscesses 0–3 (26).

Isolation of cells infiltrating the large intestine lamina propria

Cells in the large intestine lamina propria were prepared by digesting the intestinal tissue fragments depleted of intraepithelial lymphocytes with 400 U/ml collagenase D (Roche) and 10 μg/ml DNase I (Roche) for 30 min at 37°C. Lymphocytes were further enriched by centrifugation on 40/75% Percoll (GE Healthcare). Total cell numbers were determined with a hemocytometer. The percentage of CD4+ T cells was determined by staining the cells with anti-CD4-APC and analyzing them on a FACSCalibur (BD Biosciences).

Statistical analysis

Data are presented as the mean ± SEM. Statistical analyses were performed using the 2-tailed unpaired Student’s t test.

Results

T cells deficient in PSGL-1 and/or CD43 show an increase in homotypic aggregation and adhesion

To examine the role of PSGL-1 as an antiadhesive molecule on T cells, we first tested T cells isolated from WT, PSGL-1−/−, CD43−/−, and DKO mice for homotypic aggregation upon PMA stimulation. In accordance with a previous report (3), CD43−/− T cells showed an increase in homotypic aggregation compared with WT cells (Fig. 1⇓A). The percentage of aggregated PSGL-1−/− T cells was also increased, and the loss of both PSGL-1 and CD43 led to a further increase in aggregation (Fig. 1⇓A). The addition of EDTA completely abolished the aggregation of cells of all genotypes, confirming that the aggregation was calcium dependent (data not shown). The percentage of CD4+ and CD8+ T cells or those with a naive or memory phenotype was not significantly different among the four genotypes (data not shown). Similar results were obtained when naive CD4+ T cells from the four genotypes were tested for homotypic aggregation (supplementary Fig. S1A).4 To determine which adhesion molecules mediate the aggregation, we examined the effect of Ab treatment on the homotypic aggregation of PMA-stimulated DKO T cells. The percent aggregation was significantly reduced in the presence of an anti-αL (CD11a), anti-ICAM-1, or anti-β2 (CD18) mAb (Fig. 1⇓B), but not in the presence of an anti-αM (CD11b) mAb (data not shown), indicating that the aggregation was mediated by the interaction between LFA-1 and ICAM-1. Thus, PSGL-1 and CD43 both prevented the homotypic aggregation of PMA-stimulated T cells, which was mediated by LFA-1 and ICAM-1.

FIGURE 1.
FIGURE 1.

Loss of PSGL-1 and CD43 increases T cell adhesion. A, Homotypic aggregation of PMA-stimulated T cells. T cells isolated from WT, PSGL-1−/−, CD43−/−, and DKO mice were incubated with PMA (100 ng/ml) for 5 h. The percentage of cells within aggregates was determined. B, Effects of Ab treatment on the homotypic aggregation of PMA-stimulated T cells. T cells from DKO mice were incubated with PMA for 5 h in the presence of anti-CD11a, anti-ICAM-1, anti-CD18, or isotype controls. C, Adhesion of PMA-stimulated T cells to fibronectin. The isolated T cells were stimulated with PMA (100 ng/ml), added to 96-well plates coated with fibronectin, and incubated for 20 min. Unbound cells were removed, and the number of bound cells was determined. D, Effects of Ab treatment on T cell adhesion to fibronectin. PMA-stimulated T cells from DKO mice were added to fibronectin-coated plates and incubated in the presence of anti-CD49d, anti-CD29, anti-β7 integrin, or isotype controls. Results represent one of three similar experiments. Data are presented as means ± SEM. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

To determine whether PSGL-1 and CD43 regulate integrin-mediated adhesive processes in general, we tested the adhesion of PMA-stimulated T cells to fibronectin immobilized on plastic plates. Consistent with the results of the homotypic aggregation assays (Fig. 1⇑A), the adhesion of PMA-stimulated PSGL-1−/− T cells to fibronectin was greater than that of WT cells (Fig. 1⇑C). The adhesion of DKO T cells was further increased compared with cells deficient in either PSGL-1 or CD43 (Fig. 1⇑C). We confirmed that the deficiency of PSGL-1 and/or CD43 also increased the binding of PMA-stimulated naive CD4+ T cells to fibronectin (supplementary Fig. S1B). The adhesion was largely mediated by α4β1 integrin, because it was blocked by treatment of the cells with an anti-α4 (CD49d) or anti-β1 (CD29) mAb (Fig. 1⇑D).

To examine whether the deficiency of PSGL-1 and/or CD43 affects the expression of other adhesion molecules, the cell surface expression of PSGL-1, CD43, and integrin chains on T cells was measured by flow cytometry. The expression level of PSGL-1 on CD43−/− cells and of CD43 on PSGL-1−/− cells was comparable to that on WT cells (supplementary Fig. S2). The expression of integrin chains αL, α4, β1, β2, and β7, as well as ICAM-1, was also comparable among the four genotypes (supplementary Fig. S2 and data not shown). These results suggest that the increased aggregation and adhesion of T cells deficient in PSGL-1 and/or CD43 is not due to alterations in the expression of other adhesion molecules, but rather is a direct effect of their deficiency.

T cells deficient in PSGL-1 and/or CD43 show enhanced proliferative responses

We next investigated the proliferative responses of T cells from WT, PSGL-1−/−, CD43−/−, and DKO mice. The isolated T cells were labeled with CFSE and stimulated with anti-CD3ε plus anti-CD28 for 2 days. The proliferative responses of both the CD4+ and CD8+ T cells were determined by measuring the dilution of the CFSE. In agreement with published reports (3, 27), both the CD4+ (Fig. 2⇓A) and the CD8+ (Fig. 2⇓B) T cells from CD43−/− mice exhibited increased proliferative responses as did the CD4+ and CD8+ T cells from the PSGL-1−/− mice (Fig. 2⇓). The DKO T cells showed an additional enhancement in proliferation (Fig. 2⇓). Similar results were obtained with naive CD4+ T cells (supplementary Fig. S3). These data suggest that both PSGL-1 and CD43 function to regulate the proliferative responses of T cells.

FIGURE 2.
FIGURE 2.

Loss of PSGL-1 and CD43 increases T cell proliferation. The isolated T cells from WT, PSGL-1−/−, CD43−/−, and DKO mice were labeled with CFSE and stimulated with plate-immobilized anti-CD3ε and anti-CD28 for 48 h. Daughter generations of CD4+ (A) and CD8+ (B) T cells were determined by measuring the dilution of CFSE. Results represent one of three similar experiments. Data are presented as means ± SEM. ∗∗, p < 0.01 vs WT.

The extracellular/transmembrane domain of PSGL-1 or CD43 is responsible for the reversal of increased T cell aggregation

To verify that the deficiency of PSGL-1 and/or CD43 was responsible for the increased adhesion and proliferation, we reintroduced either PSGL-1 or CD43 into DKO T cells using a retroviral gene transfer system. DKO CD4+ T cells were infected with a GFP-containing control retrovirus vector (pMXs-IG) or with vectors encoding full-length PSGL-1 (PSGL-1FL) or CD43 (CD43FL). Transduced cells were sorted based on their GFP expression level, so that the sorted cells would express levels of reintroduced PSGL-1 or CD43 that were similar to the endogenous expression levels in WT cells (Fig. 3⇓ and data not shown). Consistent with the results of the homotypic aggregation assays of isolated T cells shown in Fig. 1⇑A, DKO T cells infected with a control retrovirus showed an increase in aggregation compared with similarly infected WT cells (Fig. 4⇓A). As shown in Fig. 4⇓B, the reintroduction of PSGL-1FL or CD43FL reversed the hyperaggregation phenotype of the DKO T cells.

FIGURE 3.
FIGURE 3.

Isolation of retrovirus-transduced CD4+ T cells. CD4+ T cells isolated from DKO mice were transduced with the indicated constructs, and GFP+CD4+ T cells in the rectangular gates were sorted by FACSAria. The gates were set so that the sorted cells would express similar levels of the PSGL-1, CD43, or CD8 epitope. The mean fluorescence intensity for CD43, PSGL-1, and CD8 is indicated in each histogram. Results represent one of three similar experiments.

FIGURE 4.
FIGURE 4.

Homotypic aggregation of retrovirus-transduced CD4+ T cells. A, Homotypic aggregation of WT and DKO CD4+ T cells transduced with a control retrovirus. B, Homotypic aggregation of DKO CD4+ T cells transduced with various constructs. The retrovirus-transduced CD4+ T cells were cultured with PMA for 5 h, and the percentage of cells involved in aggregates was determined. Results represent one of three similar experiments. Data are presented as means ± SEM. ∗, p < 0.05.

CD43 may regulate cell adhesion through steric hindrance, via its highly extended extracellular domain, which is extensively decorated with sialylated O-glycans (1). However, it is possible that the intracellular domain of CD43 is critical for its antiadhesive function (28). To determine which domain of PSGL-1 or CD43 mediates its antiadhesive function, we generated chimeric constructs, in which the extracellular and transmembrane domain of PSGL-1 or CD43 was fused to the intracellular domain of human CD8 (PSGL-1/CD8 or CD43/CD8), or the extracellular and transmembrane domain of CD8 was fused to the intracellular domain of PSGL-1 or CD43 (CD8/PSGL-1 or CD8/CD43). DKO CD4+ T cells were infected with vectors encoding each of the chimeric proteins and sorted for GFP expression so that the sorted cells expressed similar levels of chimeric proteins to their full-length counterparts (Fig. 3⇑).

DKO T cells transduced with the PSGL-1/CD8 chimeric construct showed levels of aggregation similar to those of PSGL-1FL-transduced cells (Fig. 4⇑B), indicating that the extracellular/transmembrane domain but not the intracellular domain of PSGL-1 is important for attenuating cell aggregation. Similar, though less dramatic, results were obtained with CD43/CD8 expression (Fig. 4⇑B). In contrast, the increased aggregation of the DKO cells was not reversed by the introduction of CD8/PSGL-1 or CD8/CD43 chimeric constructs or by the full-length CD8 (CD8FL) (Fig. 4⇑B). These results suggest that the intracellular domain of PSGL-1 or CD43 is not required for the antiadhesive effect of these molecules.

The involvement of the extracellular/transmembrane domain but not the intracellular domain in the antiadhesive effect of PSGL-1 and CD43 is consistent with the barrier hypothesis that the extended and negatively charged extracellular domain prevents cell-cell interactions. Because both PSGL-1 and CD43 are highly sialylated and thus negatively charged, we examined the effect of the removal of sialic acids by neuraminidase on T cell aggregation and adhesion. Neuraminidase treatment indeed increased the homotypic aggregation of WT, PSGL-1−/−, CD43−/−, and DKO T cells (Fig. 5⇓A). However, neuraminidase-treated DKO cells still showed an increase in aggregation compared with similarly treated WT cells (Fig. 5⇓A). Similar results were obtained in adhesion assays (Fig. 5⇓B). These results suggest that the antiadhesive role of PSGL-1 and CD43 involves mechanisms independent of the negative charge.

FIGURE 5.
FIGURE 5.

Effect of neuraminidase treatment on T cell aggregation and adhesion. A, Homotypic aggregation of PMA-stimulated T cells treated with neuraminidase. The isolated T cells from WT, PSGL-1−/−, CD43−/−, and DKO mice were incubated with or without neuraminidase for 1 h and cultured with PMA for 5 h. The percentage of cells involved in aggregates was determined. B, Adhesion of PMA-stimulated T cells treated with neuraminidase to fibronectin. The isolated T cells were incubated with or without neuraminidase, stimulated with PMA, added to 96-well plates coated with fibronectin, and incubated for 20 min. Unbound cells were removed, and the number of bound cells was determined. ∗, p < 0.05.

Intracellular domain of PSGL-1 or CD43 is responsible for the reversal of increased T cell proliferation

We next examined which domain of PSGL-1 or CD43 is involved in regulating T cell proliferation. DKO T cells transduced with each construct were stimulated with anti-CD3ε and anti-CD28 in the presence of [3H]thymidine for 24 h, and proliferative responses were determined by [3H]thymidine incorporation. The increased proliferation of DKO T cells was reversed by the reexpression of either PSGL-1FL or CD43FL (Fig. 6⇓), indicating that this hyperproliferative phenotype was indeed due to the loss of PSGL-1 and/or CD43. In contrast, expression of PSGL-1/CD8 or CD43/CD8 had little effect (Fig. 6⇓). These results suggest that the extracellular/transmembrane domain of PSGL-1 or CD43 does not play a major role in regulating T cell proliferation, and suggest that the intracellular domain of PSGL-1 or CD43 is involved in this process. Indeed, the introduction of CD8/PSGL-1 or CD8/CD43 decreased the proliferative response compared with CD8FL (Fig. 6⇓). These results suggest that the intracellular domain of PSGL-1 and CD43 is required for the negative regulation of T cell proliferation.

FIGURE 6.
FIGURE 6.

Proliferative responses of retrovirus-transduced DKO CD4+ T cells. The retrovirus-transduced DKO CD4+ T cells were stimulated with plate-immobilized anti-CD3ε and anti-CD28 in the presence of [3H]thymidine for 24 h, and proliferative responses were determined by [3H]thymidine incorporation. Results represent one of three similar experiments. Data are presented as means ± SEM. ∗, p < 0.05; ∗∗, p < 0.01.

PSGL-1 and CD43 regulate the development of CD4+ T cell-mediated colitis in vivo

The above results suggested that PSGL-1 and CD43 both regulate T cell adhesion and proliferation. To determine whether they regulate T cell-mediated immune responses, which require optimal T cell adhesion and proliferation in vivo, we used a mouse model of inflammatory bowel disease, which is caused by adoptively transferring naive T cells into SCID or RAG-1−/− recipient mice (24, 25). For our experiments, we used CD4+CD25−CD45RBhigh T cells from WT, PSGL-1−/−, CD43−/−, and DKO mice for the adoptive transfer into RAG-1−/− recipient mice. The recipient mice were analyzed 6 wk after cell transfer. RAG-1−/− mice that received WT cells developed colitis, with clinical signs such as diarrhea and wasting. RAG-1−/− mice that received PSGL-1−/−, CD43−/−, or DKO cells also developed colitis. The body weight of the RAG-1−/− mice with colitis at 6 wk after cell transfer was reduced compared with untreated RAG-1−/− mice, but was comparable regardless of the T cell genotype that was transferred (data not shown). To quantify the severity of the colitis, we determined the disease activity as the sum of four parameters: hunching, wasting, colon thickening, and stool consistency. The disease activity score of the mice that received WT cells was 3.6 (n = 8), whereas that of the mice receiving PSGL-1−/−, CD43−/−, or DKO cells was 5.6 (n = 7), 5.0 (n = 10), or 5.7 (n = 10), respectively (Fig. 7⇓A). Thus, the single and double knockout phenotypes all exacerbated the induced colitis.

FIGURE 7.
FIGURE 7.

Loss of PSGL-1 exacerbates T cell-mediated colitis. Naive CD4+ T cells were isolated from WT, PSGL-1−/−, CD43−/−, and DKO mice by FACSAria, and transferred into RAG-1−/− mice. The recipient mice developed colitis and were analyzed at 6 wk after T cell transfer. A, Disease activity score. The mice were scored for disease activity as described in Materials and Methods. B, Histological analysis of colon cross-sections. Tissue samples from the middle colon were prepared for histological staining with H&E. Crypt abscesses are indicated by arrowheads. C, Histological colitis score. Individual sections were scored blind as described in Materials and Methods. D, The number of CD4+ T cells in the large intestine lamina propria. Cells were isolated from the large intestine lamina propria, enriched for leukocytes by percoll, and stained with anti-CD4-PE. The numbers of CD4+ T cells were determined. Values are means ± SEM for seven to ten mice in each experimental group. ∗, p < 0.05; ∗∗, p < 0.01.

We also performed histological analyses of colon cross-sections from untreated or RAG-1−/− mice or RAG-1−/− mice that received the transferred naive CD4+ T cells as described above. Six weeks after the transfer, typical pathologic changes of colitis, such as cell infiltration, crypt elongation, and crypt abscesses, were observed in mice that received the naive CD4+ T cells (Fig. 7⇑B). For the histological colitis score, individual sections were scored for cell infiltration, crypt elongation, and the number of crypt abscesses. The mice that received PSGL-1−/−, CD43−/−, or DKO cells showed a higher colitis score than those that received WT cells (Fig. 7⇑C).

Because marked cell infiltration was observed in the colon of the T cell recipient mice, we examined the number of CD4+ T cells in the colon of the mice that received the WT, PSGL-1−/−, CD43−/−, or DKO cells. In the colon of the mice that received DKO cells, the number of infiltrated CD4+ T cells was ∼2-fold greater than in the mice that received WT cells (Fig. 7⇑D). These results suggest that PSGL-1 and CD43 both negatively regulate the development of CD4+ T cell-mediated colitis in vivo.

Discussion

Although it is well established that PSGL-1 serves as a selectin ligand to mediate the migration of T cells and neutrophils into inflamed sites, none of its other roles are well understood. In this study, we provide evidence that PSGL-1 is a negative regulator of T cell adhesion and proliferation in vitro and of T cell immune responses in vivo.

Sialomucins often act as antiadhesive molecules and the sialomucin CD43 has both antiadhesive and proadhesive functions (7). Previous studies demonstrated that the targeted disruption of CD43 in mice leads to enhanced T cell adhesion (3). We speculated that PSGL-1, a sialomucin that shares many features with CD43, such as an extended extracellular structure, abundant expression on T cells, and microvillar localization, might also function as an antiadhesive molecule. Our data showed that a deficiency of PSGL-1 enhanced T cell adhesion and that the loss of both PSGL-1 and CD43 had an additive effect on T cell adhesion. Retroviral gene transfer experiments suggested that the antiadhesive function of PSGL-1 and CD43 was mediated by their extracellular/transmembrane domain. A similar requirement for the extracellular/transmembrane domain was reported for the antiadhesive function of CD34 on mast cells (29). These results are consistent with the barrier hypothesis that the highly extended and negatively charged extracellular domain of sialomucins provides a physical and electrostatic barrier around a cell. The observation that a PSGL-1 and/or CD43 deficiency enhanced T cell adhesion even after removal of sialic acids suggests that the antiadhesive role of their extracellular/transmembrane domain involves mechanisms independent of the electrostatic barrier. In contrast, Walker et al. (28) previously reported that the expression of a mutant CD43 that lacks the intracellular domain in a CD43−/− T cell line did not reverse its hyperaggregation phenotype. The reason for this inconsistency is unknown, although it is possible that the lack of the intracellular domain may have affected the configuration of the extracellular domain in the cell line used in their study.

Our study also demonstrated that a deficiency of PSGL-1 enhances the proliferative responses of T cells. Consistent with previous studies (3, 27), we also demonstrated that CD43−/− T cells show an increase in proliferative responses, and the loss of both PSGL-1 and CD43 additively increased proliferation. These results are in contrast to the report by Carlow et al. (30), which showed that a CD43 deficiency had no effect on T cell responsiveness. In fact, we did not observe the enhanced proliferation of CD43−/− T cells when the cells were stimulated with higher or lower concentrations of anti-CD3ε plus anti-CD28 (data not shown). In addition, when the proliferative response was assessed by [3H]thymidine incorporation, the role of PSGL-1 and CD43 as antiproliferative molecules was most prominent within 1 day after stimulation (data not shown), suggesting that PSGL-1 and CD43 regulate T cell proliferation during the early phase.

We also showed that the intracellular domain of PSGL-1 and CD43 is required for their antiproliferative effect. In support of this finding, two groups have published data demonstrating that a CD43 mutant lacking the intracellular domain is ineffective in reverting the hyperproliferative phenotype of CD43−/− T cells (28, 31). Although the intracellular domains of PSGL-1 and CD43 have no obvious homology, this domain is known to be involved in signal transduction. In addition, the intracellular domain interacts with the adapter proteins ezrin and moesin (32, 33), which function together to regulate T cell activation (34). The cytoplasmic domain of CD43 is also phosphorylated upon PMA stimulation (35). However, it remains unknown whether PSGL-1 and CD43 under physiological conditions bind to yet-undefined receptors to deliver an inhibitory signal for proliferation. Additional experiments will be required to clarify the mechanism of the antiproliferative effect mediated by the intracellular domains of PSGL-1 and CD43.

Besides PSGL-1 and CD43, several sialomucins, including CD34, podocalyxin, and endomucin, which can serve as L-selectin ligands when modified with selectin-binding glycans, are reported to have similar dual functions (36, 37, 38). We propose that these sialomucins generally function as antiadhesive molecules to prevent inappropriate cell adhesion and activation, but when they are modified to bind selectins in specific cells or tissues, they switch roles and become proadhesive, promoting cell-cell interactions.

We found that, in vivo, a deficiency of PSGL-1 as well as CD43 exacerbated the development of inflammation in a mouse colitis model induced by the adoptive transfer of naive CD4+ T cells into RAG−/− hosts. This colitis model is reported to be mediated by Th1 cells that migrate into the colon in a selectin-independent manner (24). Importantly, we showed that the numbers of CD4+ T cells in the colon of DKO cell-recipient mice were significantly higher than in mice that received WT or singly deficient cells. These results are consistent with a role for PSGL-1 and CD43 in negatively regulating T cell adhesion and proliferation.

In conclusion, our study shows for the first time that PSGL-1 functions as a negative regulator of T cell adhesion and proliferation, adding this molecule to the expanding category of dual-functioning sialomucins. Together with CD43, PSGL-1 constitutes an additional layer of regulation for T cell adhesion and proliferation, and possibly for T cell-mediated immune responses.

Acknowledgments

We thank Dr. Bruce Furie for the PSGL-1-deficient mice, Dr. Kazunori Kato for the anti-ICAM-1 mAb, Dr. Toshio Kitamura for the pMXs-IG vector and PLAT-E cells, and Yuko Furukawa and Norie Yoshizumi for technical assistance. We also thank Dr. Takeshi Watanabe for valuable comments.

Disclosures

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 a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan; a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science; and a Grant of Long-Range Research Initiative (LRI) by the Japan Chemical Industry Association (JCIA).

  • ↵2 Address correspondence and reprint requests to Dr. Takako Hirata, Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan. E-mail address: thirata{at}ak.med.kyoto-u.ac.jp

  • ↵3 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand-1; WT, wild type; DKO, PSGL-1 and CD43 double-knockout; FL, full length.

  • ↵4 The online version of this article contains supplementary material.

  • Received July 6, 2009.
  • Accepted September 29, 2009.

References

  1. ↵
    Fukuda, M., S. Tsuboi. 1999. Mucin-type O-glycans and leukosialin. Biochim. Biophys. Acta 1455: 205-217.
    OpenUrlPubMed
  2. ↵
    Cyster, J. G., D. M. Shotton, A. F. Williams. 1991. The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation. EMBO J. 10: 893-902.
    OpenUrlPubMed
  3. ↵
    Manjunath, N., M. Correa, M. Ardman, B. Ardman. 1995. Negative regulation of T-cell adhesion and activation by CD43. Nature 377: 535-538.
    OpenUrlCrossRefPubMed
  4. ↵
    Stockton, B. M., G. Cheng, N. Manjunath, B. Ardman, U. H. von Andrian. 1998. Negative regulation of T cell homing by CD43. Immunity 8: 373-381.
    OpenUrlCrossRefPubMed
  5. ↵
    Woodman, R. C., B. Johnston, M. J. Hickey, D. Teoh, P. Reinhardt, B. Y. Poon, P. Kubes. 1998. The functional paradox of CD43 in leukocyte recruitment: a study using CD43-deficient mice. J. Exp. Med. 188: 2181-2186.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Matsumoto, M., A. Shigeta, M. Miyasaka, T. Hirata. 2008. CD43 plays both antiadhesive and proadhesive roles in neutrophil rolling in a context-dependent manner. J. Immunol. 181: 3628-3635.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Ostberg, J. R., R. K. Barth, J. G. Frelinger. 1998. The Roman god Janus: a paradigm for the function of CD43. Immunol. Today 19: 546-550.
    OpenUrlCrossRefPubMed
  8. ↵
    Rosenstein, Y., J. K. Park, W. C. Hahn, F. S. Rosen, B. E. Bierer, S. J. Burakoff. 1991. CD43, a molecule defective in Wiskott-Aldrich syndrome, binds ICAM-1. Nature 354: 233-235.
    OpenUrlCrossRefPubMed
  9. ↵
    Baum, L. G., M. Pang, N. L. Perillo, T. Wu, A. Delegeane, C. H. Uittenbogaart, M. Fukuda, J. J. Seilhamer. 1995. Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells. J. Exp. Med. 181: 877-887.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    van den Berg, T. K., D. Nath, H. J. Ziltener, D. Vestweber, M. Fukuda, I. van Die, P. R. Crocker. 2001. Cutting edge: CD43 functions as a T cell counterreceptor for the macrophage adhesion receptor sialoadhesin (Siglec-1). J. Immunol. 166: 3637-3640.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Matsumoto, M., K. Atarashi, E. Umemoto, Y. Furukawa, A. Shigeta, M. Miyasaka, T. Hirata. 2005. CD43 functions as a ligand for E-selectin on activated T cells. J. Immunol. 175: 8042-8050.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Fuhlbrigge, R. C., S. L. King, R. Sackstein, T. S. Kupper. 2006. CD43 is a ligand for E-selectin on CLA+ human T cells. Blood 107: 1421-1426.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Matsumoto, M., A. Shigeta, Y. Furukawa, T. Tanaka, M. Miyasaka, T. Hirata. 2007. CD43 collaborates with P-selectin glycoprotein ligand-1 to mediate E-selectin-dependent T cell migration into inflamed skin. J. Immunol. 178: 2499-2506.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Alcaide, P., S. L. King, C. J. Dimitroff, Y. C. Lim, R. C. Fuhlbrigge, F. W. Luscinskas. 2007. The 130-kDa glycoform of CD43 functions as an E-selectin ligand for activated Th1 cells in vitro and in delayed-type hypersensitivity reactions in vivo. J. Invest. Dermatol. 127: 1964-1972.
    OpenUrlCrossRefPubMed
  15. ↵
    McEver, R. P., R. D. Cummings. 1997. Role of PSGL-1 binding to selectins in leukocyte recruitment. J. Clin. Invest. 100: S97-103.
    OpenUrlPubMed
  16. ↵
    Vestweber, D., J. E. Blanks. 1999. Mechanisms that regulate the function of the selectins and their ligands. Physiol. Rev. 79: 181-213.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Patel, K. D., M. U. Nollert, R. P. McEver. 1995. P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils. J. Cell Biol. 131: 1893-1902.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Hirata, T., G. Merrill-Skoloff, M. Aab, J. Yang, B. C. Furie, B. Furie. 2000. P-Selectin glycoprotein ligand 1 (PSGL-1) is a physiological ligand for E-selectin in mediating T helper 1 lymphocyte migration. J. Exp. Med. 192: 1669-1676.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Hirata, T., B. C. Furie, B. Furie. 2002. P-, E-, and L-selectin mediate migration of activated CD8+ T lymphocytes into inflamed skin. J. Immunol. 169: 4307-4313.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Yang, J., T. Hirata, K. Croce, G. Merrill-Skoloff, B. Tchernychev, E. Williams, R. Flaumenhaft, B. C. Furie, B. Furie. 1999. Targeted gene disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectin-mediated neutrophil rolling and migration. J. Exp. Med. 190: 1769-1782.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Xia, L., M. Sperandio, T. Yago, J. M. McDaniel, R. D. Cummings, S. Pearson-White, K. Ley, R. P. McEver. 2002. P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow. J. Clin. Invest. 109: 939-950.
    OpenUrlCrossRefPubMed
  22. ↵
    Kitamura, T., Y. Koshino, F. Shibata, T. Oki, H. Nakajima, T. Nosaka, H. Kumagai. 2003. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Exp. Hematol. 31: 1007-1014.
    OpenUrlCrossRefPubMed
  23. ↵
    Morita, S., T. Kojima, T. Kitamura. 2000. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7: 1063-1066.
    OpenUrlCrossRefPubMed
  24. ↵
    Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. B. Caddle, R. L. Coffman. 1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1: 553-562.
    OpenUrlCrossRefPubMed
  25. ↵
    Powrie, F.. 1995. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity 3: 171-174.
    OpenUrlCrossRefPubMed
  26. ↵
    de Jong, Y. P., A. C. Abadia-Molina, A. R. Satoskar, K. Clarke, S. T. Rietdijk, W. A. Faubion, E. Mizoguchi, C. N. Metz, M. Alsahli, T. ten Hove, et al 2001. Development of chronic colitis is dependent on the cytokine MIF. Nat. Immunol. 2: 1061-1066.
    OpenUrlCrossRefPubMed
  27. ↵
    Thurman, E. C., J. Walker, S. Jayaraman, N. Manjunath, B. Ardman, J. M. Green. 1998. Regulation of in vitro and in vivo T cell activation by CD43. Int. Immunol. 10: 691-701.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Walker, J., J. M. Green. 1999. Structural requirements for CD43 function. J. Immunol. 162: 4109-4114.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Drew, E., J. S. Merzaban, W. Seo, H. J. Ziltener, K. M. McNagny. 2005. CD34 and CD43 inhibit mast cell adhesion and are required for optimal mast cell reconstitution. Immunity 22: 43-57.
    OpenUrlCrossRefPubMed
  30. ↵
    Carlow, D. A., S. Y. Corbel, H. J. Ziltener. 2001. Absence of CD43 fails to alter T cell development and responsiveness. J. Immunol. 166: 256-261.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Tong, J., E. J. Allenspach, S. M. Takahashi, P. D. Mody, C. Park, J. K. Burkhardt, A. I. Sperling. 2004. CD43 regulation of T cell activation is not through steric inhibition of T cell-APC interactions but through an intracellular mechanism. J. Exp. Med. 199: 1277-1283.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Serrador, J. M., M. Nieto, J. L. Alonso-Lebrero, M. A. del Pozo, J. Calvo, H. Furthmayr, R. Schwartz-Albiez, F. Lozano, R. Gonzalez-Amaro, P. Sanchez-Mateos, F. Sanchez-Madrid. 1998. CD43 interacts with moesin and ezrin and regulates its redistribution to the uropods of T lymphocytes at the cell-cell contacts. Blood 91: 4632-4644.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Serrador, J. M., A. Urzainqui, J. L. Alonso-Lebrero, J. R. Cabrero, M. C. Montoya, M. Vicente-Manzanares, M. Yanez-Mo, F. Sanchez-Madrid. 2002. A juxta-membrane amino acid sequence of P-selectin glycoprotein ligand-1 is involved in moesin binding and ezrin/radixin/moesin-directed targeting at the trailing edge of migrating lymphocytes. Eur. J. Immunol. 32: 1560-1566.
    OpenUrlCrossRefPubMed
  34. ↵
    Shaffer, M. H., R. S. Dupree, P. Zhu, I. Saotome, R. F. Schmidt, A. I. McClatchey, B. D. Freedman, J. K. Burkhardt. 2009. Ezrin and moesin function together to promote T cell activation. J. Immunol. 182: 1021-1032.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Mody, P. D., J. L. Cannon, H. S. Bandukwala, K. M. Blaine, A. B. Schilling, K. Swier, A. I. Sperling. 2007. Signaling through CD43 regulates CD4 T-cell trafficking. Blood 110: 2974-2982.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Baumheter, S., M. S. Singer, W. Henzel, S. Hemmerich, M. Renz, S. D. Rosen, L. A. Lasky. 1993. Binding of L-selectin to the vascular sialomucin CD34. Science 262: 436-438.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Sassetti, C., K. Tangemann, M. S. Singer, D. B. Kershaw, S. D. Rosen. 1998. Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34. J. Exp. Med. 187: 1965-1975.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Kanda, H., T. Tanaka, M. Matsumoto, E. Umemoto, Y. Ebisuno, M. Kinoshita, M. Noda, R. Kannagi, T. Hirata, T. Murai, M. Fukuda, M. Miyasaka. 2004. Endomucin, a sialomucin expressed in high endothelial venules, supports L-selectin-mediated rolling. Int. Immunol. 16: 1265-1274.
    OpenUrlAbstract/FREE Full Text
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