By
From the * Institut für Zellbiologie, ZMBE, Universität Münster, Germany; Abteilung für
Immunologie, Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany; § Institut für Exp. Medizin u. Bindegewebsforschung, Universität Erlangen-Nürnberg, Germany
We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin-Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin-binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.
The binding of circulating leukocytes to activated endothelium is initiated by transient interactions that are
mediated by the selectins (1). It is well documented in various inflammation models that blocking of the two endothelial selectins, P- and E-selectin, inhibits the entry of neutrophils into inflamed tissue (2). Less is known about the role
of these adhesion molecules for T lymphocyte recruitment
in inflammation. Binding to E-selectin was shown for certain subsets of human CD4+ memory T lymphocytes (3, 4)
and for a large percentage of bovine Binding of activated T cell lines to P-selectin under static
conditions was partially blocked in vitro by high concentrations of an anti-human P-selectin glycoprotein ligand-1
(PSGL-1) antiserum (9, 11). PSGL-1 was originally identified on human neutrophils by affinity isolation with P-selectin
(12, 13) and cloned by expression cloning (14). It was found
to be the major binding site for P-selectin on human leukocytes (11, 15). Rolling of human leukocytes perfused into
rat postcapillary venules was demonstrated to be blocked by
a mAb against human PSGL-1 (16).
Upon activation, T helper lymphocytes polarize into Th1
and Th2 subsets, which are characterized by distinct profiles of secreted cytokines (17, 18). Th1 cells are involved
in cell-mediated inflammatory reactions. Their cytokines
activate cytotoxic and inflammatory functions and Th1 cells
induce delayed-type hypersensitivity (DTH) reactions. Th2
cytokines support antibody production, particularly IgE responses, and in combination with their stimulatory effects on
eosinophil proliferation and function, Th2 cytokines are
commonly found in association with strong antibody and
allergic responses. Although it is well established that Th1 cells
predominate in DTH reactions, it was always unclear whether
their presence is mainly due to polarized differentiation at
these sites or could also be based on preferential immigration of Th1 versus Th2 cells. We have shown recently that
mouse Th1 cells indeed migrate into cutaneous DTH reactions much better than Th2 cells do, and we could demonstrate that this migration is blocked by mAb against P- and
E-selectin (19).
In this study, we have examined which molecules on the
surface of Th1 cells would function as ligands for P-selectin
during migration into cutaneous DTH reactions in the mouse.
We could define the PSGL-1 as the exclusive P-selectin
ligand on Th1 cells by affinity isolation experiments, FACS®
analysis, and cell adhesion assays. Th2 cells carried similar amounts of PSGL-1; however, this form of PSGL-1 was
unable to bind to P-selectin. Antibodies against PSGL-1
could partially block the migration of Th1 cells into cutaneous DTH reactions and showed additive effects with a
mAb against E-selectin.
Cells.
The mouse neutrophilic cell line 32Dcl3 was cultured
as described (20). Th1 and Th2 cells were generated from lymph
node lymphocytes of SPF-reared BALB/c mice. CD4+ T cells
were derived by panning of isolated lymphocytes with mAb
against CD8 (53-672), CD25 (PC/6), Fc-Receptor II/III (2.4G2), Mac-1 (M1/70), and I-Ad (17/227). Of the resulting cells, 98-
99% were positive for CD4 staining. These cells (106/well) were
incubated either in the presence of IL-12 (1,000 U/ml) and IFN- In Vivo Studies.
Th1 cells harvested 6 d after initial activation
were subjected to density gradient centrifugation (17.1% isotonic
Nycodenz; Nyegaard, Oslo, Norway) to remove dead cells. 107
cells per ml were incubated for 1 h at 37°C in RPMI with 10% FCS and 20 µCi 51Cr (Amersham Corp., Arlington, IL). Dead
cells were removed by a second density gradient step, and recovered cells were washed twice with PBS. 106 cells in 400 µl PBS
(with or without antibodies) were injected into the tail vein of
BALB/c mice (7-10 wk; breeding facilities UKE Hamburg)
which had been sensitized to 2.4-dinitrofluorobenzene (DNFB)
by skin painting with a solution of 0.5% DNFB in acetone-olive oil (4:1) at day Flow Cytometric Analysis.
Th1 and Th2 cells, harvested 5 d after initial activation, were separated from dead cells by density
gradient centrifugation, incubated with 25 µg/ml selectin-IgG
chimera or 10 µg/ml affinity-purified rabbit antibodies in HBSS
(Biochrom, Berlin, Germany) with 3% FCS, and stained with PElabeled F(ab Antibodies and Selectin-IgG Chimera.
The mAb UZ4 (rat IgM)
against mouse E-selectin (23) and the mAb RB40.34 (rat IgG 1)
against mouse P-selectin (24) were produced in and purified from
protein-free hybridoma medium (Gibco/BRL, Karlsruhe, Germany). In analogy to Yang et al. (25), we raised the rabbit anti-
mouse PSGL-1 antiserum 124 against a peptide covering amino
acids 42-60 of the sequence of murine PSGL-1. This sequence
starts after the cleavage site of the propeptide, and the analogous
region of human PSGL-1 is known to be necessary for P-selectin
binding (26) and to harbor the binding epitope of the adhesion
blocking mAb PL-1 (27). Specific antibodies were purified by affinity isolation on the BSA-conjugated PSGL-1 peptide linked to
CNBr-Sepharose as described (28). The FACS® signal which was
obtained with these antibodies on 32Dcl3 cells was abolished after
pretreatment of the cells with O-sialoglycoprotease (treatment done
as described in 29). For the preparation of Fab-fragments, 2.5 mg
of affinity-purified anti-PSGL-1 antibodies were incubated in 500 µl of 20 mM sodium phosphate, pH 7.0, 10 mM EDTA, 10 mM cysteine with 100 µl of washed papain Sepharose beads (Pierce Chemical Co., Rockford, IL) for 4 h at 37°C. The supernatant
was dialyzed against PBS, and Fc fragments and undigested antibodies were removed with protein A-Sepharose (Pharmacia LKB,
Piscataway, NJ). The P-selectin-IgG fusion protein was produced
as described (28).
Immunoprecipitation.
Th1 and Th2 cells harvested 5 d after
initial activation were subjected to density gradient centrifugation, washed three times in PBS, and surface biotinylated in 1 ml
of PBS containing 0.5 mg/ml Sulfo-NHS-biotin (Pierce) for 30 min on ice. The reaction was blocked by incubating the cells for
10 min in 1 ml of DMEM (Gibco/BRL) without FCS on ice.
Cells were washed with PBS twice and subjected to immunoprecipitations performed as described (29). Immunoprecipitated proteins were separated by electrophoresis on 6% SDS-PAGE and
transferred to nitrocellulose (Schleicher & Schuell, Keene, NH).
Filters were analyzed for biotinylated proteins with peroxidase-conjugated streptavidin (Dianova, Hamburg, Germany) and the ECLsystem (Amersham Corp., Arlington Heights, IL).
Cell Adhesion Assay.
Adhesion assays in rotating 96-well microtiter plates coated with selectin-Ig fusion proteins were performed as described (29), except for evaluating the number of
bound cells, which was done by computer-aided image analysis
using the NIH Image 1.55 software. For each value, three wells
were analyzed, counting three randomly chosen areas per well.
We have recently shown that Th1 cells but not Th2 cells
efficiently enter sites of acute inflammation in the skin (19). This migration was blocked by a combination of antibodies
against P- and E-selectin (19). Based on these findings, we
have now examined which molecules on Th1 cells would
function as ligands for P-selectin in this process. We first
analyzed which glycoproteins on the surface of Th1 cells
bind to P-selectin. To this end, equal numbers of Th1 and
Th2 cells were surface biotinylated and subjected to affinity
isolation experiments with a P-selectin-Ig fusion protein.
As shown in Fig. 1 (lane 5), two proteins of 230 and 130 kD
were isolated with P-selectin-Ig that could be reprecipitated with affinity-purified antibodies of the rabbit antiserum 124 against an amino-terminal peptide of mouse
PSGL-1 (lane 6). The 230-kD form is the not completely
reduced dimeric form of the 130-kD PSGL-1. No selectin
ligands could be isolated with the P-selectin probe from
Th2 cells (lane 2), although the PSGL-1 protein was detectable with antibodies (lane 3) at similar levels as on Th1
cells (lanes 3 and 4). Interestingly, the 130-kD monomeric form of PSGL-1 on Th2 cells was slightly smaller than the
one on Th1 cells (compare lane 3 with 5). Whereas antibodies against PSGL-1 precipitated a broader band from
Th1 cells (lane 4), including a slightly smaller molecular
species of the size as in Th2 cells, P-selectin-Ig only precipitated the higher molecular weight species. We conclude
that only Th1 cells express a modified form of PSGL-1 that
is capable of binding to P-selectin. Furthermore, PSGL-1 was the only glycoprotein ligand on Th1 cells that could be
detected by this technique.
In line with the immunoprecipitation results, a comparison of Th1 and Th2 cells by FACS® analysis revealed that
both cell types expressed similar levels of PSGL-1 (Fig. 2, A
and B). However, 45% of the Th1 cells, but none of the Th2
cells, could be stained with P-selectin-Ig (Fig. 2, C and D).
Staining with P-selectin-Ig was completely blocked by preincubating the cells with affinity-purified antibodies against PSGL-1, indicating that P-selectin binding to Th1 cells is
mediated exclusively by PSGL-1 (Fig. 2 E).
Using nonstatic (rotation) adhesion assays, we analyzed
the binding of Th1 and Th2 cells to P-selectin-Ig coated
onto plastic. In agreement with the results obtained by
FACS® analysis, Th1 cells bound efficiently to P-selectin-
Ig and, this binding was blocked by 89% with affinity-purified antibodies against PSGL-1 (Fig. 3). Binding of Th2
cells to P-selectin was low and comparable to the residual
binding of anti-PSGL-1-blocked Th1 cells. Thus, PSGL-1
almost exclusively mediates P-selectin binding to Th1 cells
in FACS® analysis as well as in adhesion assays and is the
only glycoprotein ligand that can be detected by affinity
isolation with a P-selectin probe from these cells.
To determine whether PSGL-1 would be involved in the
P-selectin-mediated step during migration of Th1 cells into
inflamed sites of the skin, we examined the effect of affinitypurified antibodies against PSGL-1 on Th1 cell homing in
a DNFB-elicited cutaneous DTH reaction. 51Cr-labeled
cells were preincubated with the antibodies for 15 min and
injected into mice intravenously. Organs were taken 1 h later
and analyzed for the presence of infiltrated lymphocytes by
measuring radioactivity. Affinity-purified, complete rabbit antibodies against PSGL-1 blocked T cell migration into
inflamed skin by 43% but also caused some trapping of
these cells in the lung (not shown). Therefore, Fab fragments were generated. As shown in Fig. 4, anti-PSGL-1
Fab fragments reduced the entry of Th1 cells into the inflamed skin area by 26% (± 5.2%), while no effect was seen
with Fab fragments from preimmune serum. Migration of
Th1 cells into noninflamed control skin of the same mice
was low. No effect of the Fab fragments on Th1 cell accumulation in other organs was observed (not shown). The
anti-P-selectin antibody RB40.34 and the anti-E-selectin
mAb UZ4 blocked Th1 cell entry into inflamed skin by
42% (± 9.8%) or 48% (± 14%), respectively, when injected together with the cells. The effect of anti-PSGL-1 Fab fragments and the anti-E-selectin mAb were additive,
resulting in 83% (± 5.2%) reduction of Th1 cell entry into
inflamed skin. This is in agreement with the additive inhibitory effect of 92% that we observed when the anti-P- and
anti-E-selectin mAbs were simultaneously injected (19).
Our data demonstrate that PSGL-1 is the major P-selectin ligand on Th1 cells and that it is relevant for the entry
of these cells into inflamed skin. The fact that anti-PSGL-1
Fab fragments inhibited Th1 cell migration less efficiently
than the anti-P-selectin mAb is probably due to the lower
blocking efficiency of the Fab fragments as compared with
complete antibodies. In in vitro adhesion assays, 50 µg/ml
anti-PSGL-1 Fab inhibited adhesion of mouse 32Dcl3 neutrophilic cells to P-selectin-Ig by only 35%, while 20 µg/ ml complete anti-PSGL-1 antibodies blocked adhesion by
70% (not shown).
Although the cell surface expression level of PSGL-1 on
Th1 and Th2 cells is similar, only PSGL-1 on Th1 cells is
able to mediate binding to P-selectin. It is well known that
PSGL-1 requires certain posttranslational modifications for
binding to P-selectin, such as fucosylation (14), tyrosinesulfation (30), and branched carbohydrate side chains
generated by the core-2 enzyme (33). It is likely that
one or several of these modifications are upregulated in the
course of the differentiation process that leads to the acquisition of the Th1 cell phenotype. Indeed, careful analysis of
the apparent molecular weight of PSGL-1 on Th1 and Th2
cells revealed that the P-selectin-binding form of PSGL-1
on Th1 cells has a slightly larger apparent molecular weight
than the nonfunctional form on Th2 cells (Fig. 1, lanes 3 and 5). Besides the different repertoire of cytokines, the
functional form of PSGL-1 is a cell surface molecule that
distinguishes Th1 cells from Th2 cells. The expression of
active PSGL-1 is relevant for the migration of Th1 cells
into inflamed skin.
/
T cells (5). A small
percentage of CD4+ T cells from peripheral blood (6) and
also chronically activated CD4+ T cells (7) was found to
bind to P-selectin. Human CD4+ T cell clones were described to bind to E- and P-selectin in static (8) as well as
flow adhesion assays (9), and T cell recruitment into inflamed skin was blocked with polyclonal antibodies against
P-selectin in vivo in the rat (10).
(200 U/ml) (for generation of Th1 cells) or in the presence of IL-2
(50 U/ml) and IL-4 (10 ng/ml) (for the generation of Th2 cells)
in 24-well plates coated with mAb 145-2C11 against CD3. After
2 d, cells were transferred to noncoated plates without changing
medium and cultured for another 3 or 4 d as described (21). Under these conditions, the differentiated effector cells have returned into a resting state (21). Purity of Th1/Th2 subsets were
verified for cytokine production by intracellular FACS® analysis
(22). The two generated T cell populations contained routinely
>50% cells of the desired phenotype and were contaminated by
<1% with cells of the opposite phenotype.
21,
20, and challenged with 0.3% DNFB at
day
1. After 1 h, mice were killed, and distribution of radioactivity in different organs and the remaining body was measured in
a
-scintillation counter.
)2 donkey anti-human IgG (Dianova, Hamburg, Germany) or FITC labeled goat anti-rabbit IgG (Sigma). Analysis was
performed on a Becton Dickinson FACSCalibur® and CellQuest
analysis software.
Fig. 1.
PSGL-1 from Th1 cells but not from Th2 cells can be affinity
isolated by P-selectin-IgG. Equal numbers of Th1 or Th2 cells were surface biotinylated, and detergent extracts were incubated with either immobilized human Ig as control (Co), P-selectin-IgG (P), or affinity-purified antibodies against PSGL-1 (). Specifically bound proteins were
either directly electrophoresed (lanes 1-5 and 8) or subjected to a reprecipitation (as indicated) with affinity-purified anti-PSGL-1 rabbit antibodies (lane 6) or immobilized nonimmune rabbit antibodies (lane 7).
Isolated proteins were separated on 6% polyacrylamide gels under reducing conditions. The material in lanes 6 and 7 corresponds to twice as
many cells as used for lanes 1-5 and 8. The front of the gel is marked by an
arrow on the left. Molecular mass markers (in kD) are indicated on the left.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
FACS® analysis of
Th1 and Th2 cells with P-selectin-Ig and antibodies against
PSGL-1. Th1 and Th2 cells (as
indicated) were analyzed by flow
cytometry either with affinitypurified rabbit antibodies against
mouse PSGL-1 (A and B, solid
lines) or with P-selectin-Ig (C and
D, solid lines). Dotted lines show
negative control staining either
with nonimmune rabbit IgG (A
and B) or with human IgG (C
and D). E shows the staining of
Th1 cells with P-selectin-IgG
after preincubation of the cells
either with nonimmune rabbit
IgG (faint line) or with affinitypurified rabbit anti-PSGL-1 antibodies (bold line). P-selectin-Ig
was detected with PE-conjugated F(ab)2 donkey anti-human IgG,
and rabbit antibodies were detected with FITC-conjugated
goat anti-rabbit IgG.
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
Adhesion of Th1 and Th2 cells to immobilized P-selectin-Ig.
Cell adhesion assays were performed with Th2 or Th1 cells (as indicated)
in 96-well microtiter plates coated with human IgG (hIgG) or P-selectin-
IgG (P-Sel). Before the assay, cells were incubated with HBSS(--), HBSS
with 50 µg/ml rabbit nonimmune antibodies (Co), or HBSS with 50 µg/
ml affinity-purified antibodies against PSGL-1 ( PSGL). Bound cells
were counted by computer-aided image analysis in three randomly chosen areas of defined size (per well) in three different wells for each determination. The depicted experiment represents one of three similar experiments.
[View Larger Version of this Image (22K GIF file)]
Fig. 4.
Partial inhibition of Th1 cell immigration into inflamed skin
by antibodies against PSGL-1. Radiolabeled Th1 cells were injected together with PBS (no Ab) or the same buffer containing 100 µg of nonimmune rabbit IgG Fab fragments (Co Fab), 100 µg of affinity-purified anti-PSGL-1 Fab fragments (PSGL-1), 200 µg of mAb RB40 against mouse P-selectin (P-Sel), 200 µg of mAb UZ4 against mouse E-selectin (E-Sel). Immigration of cells into the noninflamed control skin region of
the same mice is depicted as solid bars. For each determination, four mice
were analyzed. Experiments shown by the left graph were performed
with a different preparation of Th1 cells than the experiments depicted by
the right graph. Numbers on the left refer to the percentage of injected
cells that were found in the analyzed skin area of 2.5 cm2.
[View Larger Version of this Image (25K GIF file)]
Address correspondence to Dietmar Vestweber, Institute of Cell Biology, ZMBE, Technologiehof, Mendelstr. 11, D-48149 Münster, Germany.
Received for publication 18 October 1996
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