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IMMUNOBIOLOGY
From the Institute of Cell Biology, ZMBE, and the
Department of Dermatology, University of Münster, Germany;
Harvard Skin Disease Research Center, Division of Dermatology, Brigham
and Women's Hospital, Harvard Institutes of Medicine, Boston, MA;
Department of Pathology, Howard Hughes Medical Institute,
University of Michigan Medical School, Ann Arbor; and Max Planck
Institute of Vascular Biology, Münster, Germany.
Inflammatory processes are associated with the rapid migration of
dendritic cells (DCs) to regional lymph nodes and depletion of these
potent antigen-presenting cells (APCs) from the inflamed tissue. This
study examined whether sites of cutaneous inflammation can be
repopulated with DCs from a pool of immature DCs circulating in the
blood. In adoptive transfer experiments with ex vivo-generated radioactively labeled primary bone marrow-derived DCs injected into
mice challenged by an allergic contact dermatitis reaction, immature
DCs were actively recruited from the blood to sites of cutaneous
inflammation, whereas mature DCs were not. Immature, but not mature,
DCs were able to adhere specifically to immobilized recombinant E- and
P-selectin under static as well as under flow conditions.
P-selectin-dependent adhesion of immature DCs correlates with their
higher level of expression of the carbohydrate epitope cutaneous
lymphocyte-associated antigen (CLA) and is blocked by a novel
inhibitory antibody against mouse P-selectin glycoprotein ligand 1 (PSGL-1). Surprisingly, however, emigration of immature DCs into
inflamed skin is retained in the presence of this anti-PSGL-1 antibody
and is also normal when immature DCs are generated from fucosyltransferase (Fuc-T) Fuc-TVII-deficient mice. By contrast, emigration of wild-type immature DCs is reduced by adhesion-blocking anti-E- and P-selectin antibodies, and immature DCs generated ex vivo
from Fuc-TVII/Fuc-TIV double-deficient mice emigrate poorly. Thus,
fucosylated ligands of the endothelial selectins, determined in part by
Fuc-TIV, and independent of PSGL-1, are required for extravasation of
DCs into sites of cutaneous inflammation.
(Blood. 2002;99:946-956) Dendritic cells (DCs) are bone marrow-derived
leukocytes that are specialized in antigen capture, processing, and
presentation to T lymphocytes and are essential for the initiation and
modulation of antigen-specific immune responses.1 DC
progenitors as well as more mature DCs are present in small numbers in
the blood.2 They seed nonlymphoid tissues and are
primarily localized within epithelia, such as Langerhans cells in the
epidermis.3 On activation these cells undergo phenotypic
changes that allow them to migrate from their site of residence to the
T-cell areas of regional lymph nodes.4 The factors that
mediate trafficking from the periphery to lymphoid organs are well
defined.5-10 However, not much is known about the
immigration of DC precursors to their tissue of residence and only a
little information is available about immigration and turnover of DCs
in inflamed tissues. Because pathogens, allergens, or contact with
CD40L-expressing cells all lead to activation and emigration of
resident DCs toward regional lymph nodes,11 the site of
inflammation is rapidly depleted of resident antigen-presenting cells
(APCs). Thus, for the maintenance of the antigen-specific immune
response, it appears necessary that nonresident APCs be actively
recruited to inflamed tissue. The role of the small population of blood
DCs is not clear, but it is possible that this cell type forms a
"task force" of potent APCs that can rapidly relocate to sites of
inflammation.12 This would result in enhanced local antigen presentation to infiltrating effector T cells and sustained priming of naive T cells after subsequent migration to regional lymph
nodes.2
Selectins and their ligands play a major role for extravasation of
leukocytes from blood into inflamed tissue.13,14 They are
essential for tethering and rolling of leukocytes on vascular endothelium, the initial steps of the adhesion cascade.15
The best analyzed selectin ligand on leukocytes is P-selectin
glycoprotein ligand 1 (PSGL-1),16-18 which is essential
for T-cell as well as for neutrophil emigration.19-22 For
selectin-binding, PSGL-1 and all other known selectin counterreceptors
need to be modified by the minimal ligand structure for all 3 selectins, the tetrasaccharide sialyl Lewis X (sLex)
(NeuAc In humans, it was demonstrated that a DC progenitor population bears a
skin homing molecule known as cutaneous lymphocyte-associated antigen
(CLA) on its surface, defined by the monoclonal antibody (mAb)
HECA452.26 This epitope is a carbohydrate modification whose precise structure has not yet been determined definitively, but
ample evidence suggests that it resembles or is a derivative of
sLex. The mAb HECA452 was shown to block lymphocyte binding
to E-selectin,27,28 suggesting a role of this epitope as a
ligand for E-selectin.
Cutaneous lymphocyte-associated antigen was reported to be prominently
if not exclusively expressed on the sialomucin PSGL-1 on activated
human T cells.29 Although CLA is not well defined in the
mouse, and the HECA452 antibody stains murine skin homing lymphocytes
less reliably than human ones, antigen-specific activation of a mouse
CD8+ T-cell clone also induced expression of CLA, and
PSGL-1 was identified as the major glycoprotein carrier of this
carbohydrate modification on these cells.30 In subsequent
studies, CLA was found to be expressed on human DCs, which prompted
studies to investigate the ability of DCs to interact with the
endothelial selectins. Using intravital microscopy it could indeed be
demonstrated that human DCs were able to roll in postcapillary venules
in the noninflamed mouse skin and these interactions could be inhibited
with antibodies against E- and P-selectin.31
Preliminary, nonquantitative data also suggested that immature
DCs, differentiated from mouse bone marrow cells, could be
recruited into inflamed mouse skin,31 although
neither the potential adhesion mechanisms that mediate this
recruitment nor the capacity of these cells to bind to the endothelial
selectins have been investigated so far.
In the present study, we have performed a detailed and
quantitative analysis of the migration of immature mouse DCs into
inflamed mouse skin. We found that immature, but not mature, DCs are
recruited in significant numbers into the inflamed tissue. Moreover, we show that immature bone marrow-derived mouse DCs transiently express CLA and a selectin-binding glycoform of PSGL-1, bind to P- and E-selectin, and lose their selectin-binding capacity as well as their
ability to emigrate into inflamed skin on full maturation. Emigration
of immature DCs was dependent on E- and P-selectin and was reduced if
DCs were generated from Fuc-TVII/Fuc-TIV double-deficient mice.
Surprisingly, however, emigration was not reduced if DCs were
preincubated with an adhesion-blocking antibody against PSGL-1 or if
DCs were generated from mice deficient in Fuc-TVII. Our results suggest
that immature DCs require fucosylated ligands of the endothelial
selectins for emigration into inflamed skin and that these ligands are
independent of PSGL-1 and are in part determined by Fuc-TIV.
Mice
Antibodies and selectin-IgG chimeras
Two novel mAbs against mouse PSGL-1 were generated by immunizing Lewis rats with a PSGL-1-human IgG1 chimeric protein. This recombinant protein was constructed by fusing a complementary DNA (cDNA) fragment coding for the extracellular part of mouse PSGL-1 (base pairs 1-918) with a cDNA fragment coding for the hinge region and domains C2 and C3 of human IgG1.33 The cDNA construct was cloned into the pcDNA-3 vector (Invitrogen, Groningen, The Netherlands) and transfected into COS-7 cells; the fusion protein was purified by affinity isolation with protein A Sepharose (Pharmacia, Uppsala, Sweden). Lymphocytes isolated from the spleen and lymph nodes of immunized rats were fused with the mouse myeloma SP2/0. Hybridoma supernatants were tested in enzyme-linked immunosorbent assays (ELISAs) for binding to the fusion protein with human IgG1 as negative control. Two antibodies were obtained that bound to the fusion protein as well as to PSGL-1 on mouse myeloid cells: 4RA10 (rat-IgG1) and 4RB12 (rat-IgG2a). The following mAbs were purified from protein-free hybridoma
supernatants (Nanotools, Teningen, Germany): 2PH1 against mouse PSGL-1,19 RB40.34 against mouse P-selectin,34
4RA10 and 4RB12 against mouse PSGL-1 (described here). Purified rat-IgM
mAb HECA452 was a kind gift from Dr Louis Picker and mAb UZ4 (rat-IgM)
against E-selectin had been kindly provided by Dr Rupert Hallmann.
Negative control antibodies were mAb S7 (rat-IgG) against CD43 and mAb R4-22 (rat-IgM, For sorting with magnetic cell-sorting (MACS) beads (Miltenyi Biotec, Bergisch Gladbach, Germany) the following additional mAbs were used: The mAb Tib164 (anti-CD45R/B220, clone 14.8) was obtained from ATCC. The Fc-block rat-antimouse CD16/CD32 mAb (clone 2.4G2) and the mAbs CD11b/Mac1 (clone M1/70), Gr1 (clone RB6-8C5), CD4 (clone GK1.5), CD8 (clone 53-6.7), and CD24/HSA (clone 30-F1) were obtained from Pharmingen. Cells For the generation of DCs, published methods were used,35,36 with slight modifications. Mouse bone marrow was collected from tibias and femurs of female mice, and passed through a 70-µm nylon mesh after hypotonic lysis of erythrocytes for 3 minutes at room temperature. Cells were resuspended in complete medium (RPMI 1640 containing 5% heat-inactivated fetal calf serum [FCS], 50 µM -mercaptoethanol, 2 mM L-glutamine, 0.1 mM
nonessential amino acids, and 20 µg/mL gentamicin; all from PAA,
Linz, Austria), and seeded into 90-mm tissue culture dishes (Becton
Dickinson, Heidelberg, Germany) for 2 hours at 37°C and 10%
CO2. Nonadherent cells were carefully washed from dishes
and collected and adherent cells were discarded. In some experiments
lineage-positive (Lin+) cells were depleted by MACS after
incubation with mAbs against CD45R/B220, CD11b/Mac1, CD3, GR1,
I-Ab,d,q, I-Ed,k (all rat-IgG) and goat-antirat
IgG microbeads (Miltenyi Biotec) according to the manufacturer's
instructions. The lineage-negative (Lin ) cell fraction
was separated in an automated Magnetic Cell Sorter (Miltenyi Biotec).
Cells were cultured in 80-cm2 bottles (Nunclone, Wiesbaden,
Germany) in the presence of 150 U/mL granulocyte-macrophage
colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) (both from
conditioned cell culture supernatants). Cultures were grown for 5 days
(immature DCs) or 9 to 11 days (mature DCs) with complete change of
medium every second day. In some experiments, mature DCs were incubated
for the final 24 hours of culture with 0.1 µg/mL lipopolysaccharide
(LPS) from Escherichia coli (Sigma).
The T cells were obtained from the axillary and inguinal lymph nodes of
mice sensitized 7 and 6 days before with 2% (wt/vol) oxazolone
(4-ethoxymethyl-2-phenyl-2-oxazolin-5-one; Sigma) freshly dissolved in
olive oil/acetone (1:4); 50 µL was applied to the shaved abdomen
and 10 µL solution to each footpad of the mice. Lymph nodes were
collected and pressed through a metal sieve with cold
phosphate-buffered saline (PBS; 1% [vol/vol] FCS) to generate single-cell suspensions. T cells were resuspended in RPMI 1640 containing 5% FCS and subjected to a sterile nylon wool column equilibrated in the same medium. After incubation at 10%
CO2 for 45 minutes, nonadherent cells were collected by
washing the column with 40 mL warm media. In a second purification
step, this fraction was incubated with an antibody cocktail including
mAbs against CD45R/B220, CD11b/Mac1, CD16/CD32 (Fc-block), Gr1, and
CD24/HSA for negative depletion. In case of T cells from
Fuc-TVII The mouse neutrophilic progenitor 32Dcl3 and the Chinese hamster ovary (CHO) cell line CHO DUKX B1 and the mouse CD8+ cytotoxic T-cell clone 4G3 were cultivated as previously described.30,37,38 The immature DC cell line XS52 was cultured in the presence of GM-CSF and conditioned media from NS47 cells as described previously.39 Flow cytometry Flow cytometry was done as described.40Immunoprecipitation and Western blot Cell surface biotinylation and immunoprecipitations with mAbs as well as with selectin-IgG chimeras were done as described.20,41 Immunoblots were done as decribed.30Static adhesion assay Static adhesion assays were done in 96-well flat-bottom plates (Maxisorp, Nunc, Wiesbaden, Germany) coated with 5 µg/mL selectin-IgG chimera or human IgG1 in Hanks balanced salt solution (HBSS) overnight at 4°C. Nonspecific binding sites were blocked by incubating with Dulbecco modified Eagle medium (DMEM) containing 10% (vol/vol) FCS for 3 hours.41 To minimize unspecific binding, cells (107 cells/mL) were preincubated with 1 µg CD16/CD32 (Fc-block, Pharmingen) in HBSS per 106 cells. For inhibition studies, purified mAbs were added to the cells prior to the assay at 10 µg/mL and incubated on ice for 10 minutes. To allow adhesion, 5 × 105 cells were added per well and incubated for 20 minutes at 4°C under mild rotation (80 rpm). Plates were kept on ice and washed 4 times with cold HBSS. To check for calcium dependence of the interaction, wells were washed with PBS containing 5 mM EDTA. Adherent cells were fixed with CellFIX in PBS (Becton Dickinson) and numbers were evaluated by computer-aided image analysis with the NIH Image 1.55 software as described.20Cell attachment assay under flow Assays were essentially performed as described.31,42 Glass coverslips were coated overnight at room temperature with 0.75 µg/mL selectin-IgG chimera or human IgG1 in HBSS as negative control and subsequently blocked with 5% (vol/vol) bovine serum albumin (BSA; grade V; Sigma) in HBSS for 3 hours at room temperature in a moist chamber. Cells were used at a density of 5 × 106 cells in 6 mL DMEM containing 10% (vol/vol) FCS and 0.04% (vol/vol) azide. To avoid nonspecific activation of rolling cells via Fc-receptor binding to coated selectin-IgG fusion proteins, cells were preincubated with 1 µg anti-CD16/CD32 (Fc-block, Pharmingen) per 106 cells. Adhesion-blocking antibodies were added at 10 µg/mL final concentration. Immediately before the assay, cells were removed from ice and perfused at room temperature through a rectangular transparent laminar flow perfusion chamber containing the protein-coated cover slip. Evaluation started 60 seconds after starting the pump. The number of rolling cells was counted for 10 areas of 0.125 mm2 each.In vivo contact hypersensitivity model The procedure was performed as described,21,36 with slight modifications. Mice were sensitized twice 7 and 6 days before injection of DCs with 2% (wt/vol) oxazolone (Sigma) in olive oil/acetone (1:4). At 24 hours (in some experiments 6 or 2 hours) before injection of cells mice were challenged at the right ear with 10 µL 0.5% (wt/vol) oxazolone. The untreated left ear served as a control. From this point on mice were caged individually to prevent allergen contamination of the contralateral ear. In some experiments hapten challenge was repeated 4 hours before injection.Cells were counted and labeled at a density of 107 cells/mL
for 1 hour with 51Cr, 100 µCi/107 (3.7 MBq/107) cells (Amersham, Braunschweig, Germany) at
37°C in RPMI 1640, 20% (vol/vol) FCS. After labeling, cells were
washed 3 times in PBS to remove nonincorporated radioactivity. DCs
(5 × 106), 5 × 106 32Dcl3 cells,
or 107 T cells were incubated in 200 µL PBS with 250 µg/mL antibodies against selectin ligands or control antigen,
incubated on ice for 15 minutes, and injected intravenously together
with the antibodies. Antibodies against selectins (400 µg/mouse) were
injected intravenously directly before injection of radiolabeled cells.
In control experiments, cells were fixed with 2% (wt/vol) formaldehyde
for 10 minutes and washed with PBS prior to injection. Immediately
before injection, cells were resuspended to break up clusters. Six
hours after injection mice were killed and organs removed. In some
experiments, a topical steroid (0.1% momethasone-17-[2-furoat]
Ecural cream, Essex, Munich, Germany) was applied to the eczematous ear
lesions 6 and 24 hours after injection of cells, and organs were
removed after 48 hours. Ears, lungs, liver, and spleen were counted in
a
Characterization of DCs differentiated from mouse bone marrow cells To obtain mouse DCs, bone marrow cells were cultured in the presence of GM-CSF and IL-4.35,36 Typically, DCs are characterized by expression of a combination of surface molecules such as major histocompatibility complex class II (MHC-II), B7-2, CD11c, and ICAM-1 that are strongly up-regulated during maturation and that are generally low but not negative on immature DCs. In cultures growing for 5 days we generally observed most cells as DCs of immature morphology and surface phenotype (Figure 1), whereas in cultures growing for 9 to 11 days with LPS added for the last 24 hours, almost all cells uniformly exhibited a mature morphology and surface phenotype (Figure 1). The broader distribution of several markers that we observed on immature DCs at day 5 indicates that the differentiation status of the DC population was still heterogeneous at this time. VLA-4 expression was often found to be stronger than in the particular experiment shown here, especially on mature DCs. In some cultures, Lin+ cells were depleted from bone marrow cells before starting the culture. After 5 days, DCs in these cultures exhibited an almost identical pattern of surface markers as did cells from the nondepleted cultures (Figure 1). All cultures tested were consistently negative for lineage markers such as CD19, CD56, Gr1, and CD3, indicating that the cultures were devoid of significant contaminations by other cell types. Similar results were obtained with cultures from Balb/c as well as from 129Sv mice (not shown).
Migration of DCs into inflamed skin in the allergic contact dermatitis model We investigated the capacity of the mouse DCs of different maturation stages to migrate into inflamed skin and compared their homing ability with that of other cell types. Recipient mice were sensitized, challenged with oxazolone, and 1 day later, radioactively labeled DCs were injected into the tail veins. As shown in Figure 2, immature DCs cultured for 5 days in the presence of GM-CSF and IL-4 accumulated in the inflamed ear 5 to 12 times better than in the noninflamed ear. This migration was dependent on the immature differentiation state of these cells, because fully mature DCs cultured for 11 days did not specifically accumulate in the inflamed ear. Likewise, mature DCs that had been cultured in the presence of LPS during the last 24 hours of culture did not migrate into inflamed ear skin (Figure 2A). To further control whether the accumulation of DCs required the active participation of these cells we injected paraformaldehyde (PFA)-fixed immature DCs. Again, no specific accumulation of these cells in the inflamed ear was observed (Figure 2A).
Because we had used primary cultures as the source for DCs, we were
concerned that contaminating non-DCs could have contributed to the
selective accumulation of radioactivity at the site of inflammation. To
exclude this possibility, we generated DC cultures that had been
vigorously depleted for Lin+ cells. As shown in Figure 2B,
these Lin To exclude the possibility that the radioactivity measured in the inflamed ear would originate from cells being simply trapped in the dilated microvasculature rather than having emigrated into the surrounding tissue, we topically applied a steroid cream (mometasone-17-[2-furoate]) to the eczematous ears 6 and 24 hours after injection of cells to down-regulate local inflammation and vasodilation. In addition, radioactivity in the ear was analyzed 48 hours after injecting the cells, to exclude transient trapping of labeled cells within the vasculature. At this time point, steroid-treated ears were no longer visibly inflamed and ear swelling had returned to baseline levels of control-treated ears. We found that the accumulated radioactivity measured after 48 hours was in a similar range as after 6 hours (Figure 2C, left panel), indicating that cells had entered the inflamed tissue and had not just been trapped in the dilated vasculature of the ear. Radioactively labeled T cells isolated from the lymph nodes of oxazolone-sensitized mice also accumulated in the inflamed ear skin and were found in comparable numbers at 6 hours and at 48 hours after injection (Figure 2C, right panel). We next investigated whether the emigration of DCs preceded the
allergen-induced skin inflammation, which would suggest a functional
relevance of these cells in elicitation of acute allergic contact
dermatitis. We therefore analyzed the efficiency of DC accumulation in
the inflamed ear at different time points of the inflammation. As shown
in Figure 3, immature DCs homed with
increasing efficiency into the inflamed skin when cells were injected
at 2, 6, and 24 hours after challenging the ear with oxazolone. In addition, this experiment demonstrates that DCs only home to
hapten-challenged ears of sensitized, but not of naive, mice. Thus, the
efficiency of DC emigration into inflamed skin was largely proportional
to the severity of inflammation, although DCs were observed to
extravasate into allergen-challenged skin as early as 2 hours after
hapten application.
Immature, but not mature, mouse DCs bind to P- and E-selectin E- and P-selectin are known to be essential for the emigration of T cells from blood into inflamed skin.20,43 In addition, human DCs bind to E- and P-selectin in vitro and rolling of these cells in mouse venules is dependent on E- and P-selectin.31 Therefore, we first investigated whether immature mouse DCs express ligands for E- and P-selectin. As analyzed by flow cytometry, immature as well as mature DCs expressed PSGL-1 (Figure 1). Whether PSGL-1 on these cells was able to bind to P- or E-selectin (or both) was analyzed in affinity isolation experiments with P- and E-selectin-IgG. As shown in Figure 4, bands at 230 and 130 kd, affinity isolated from immature DCs as well as from 32Dcl3 cells, reacted specifically with the mAb 4RA10 against PSGL-1. As often seen with PSGL-1, not all 230-kd dimers were completely reduced to the 130-kd monomer on the gels. The fact that the E-selectin-binding form of PSGL-1 was slightly larger than the P-selectin-binding form has also been described for a mouse T- cell clone.30 Mature DCs only allowed precipitation of very small amounts of the dimeric form of PSGl-1. Thus, the glycoform of PSGL-1, which can efficiently bind to E- and P-selectin, is preferentially expressed on immature and not on mature DCs. In agreement with this the carbohydrate epitope CLA (resembling sLeX) was clearly expressed on immature mouse DCs, whereas it was strongly reduced on mature DCs (Figure 5). Although CLA is also found on human DCs,31 our finding is surprising because CLA has usually not been described on mouse primary leukocytes.
The expression of CLA and the selectin-binding form of PSGL-1
selectively on immature DCs prompted us to test whether these cells can
bind to the endothelial selectins and whether PSGL-1 would be involved.
Although one adhesion-blocking mAb against mouse PSGL-1 was already
available (2PH1), we included the novel antibody 4RA10 in this analysis
because the antibody blocks PSGL-1-mediated binding to P-selectin more
efficiently (see below). In addition, we raised the nonblocking
anti-PSGL-1 antibody 4RB12. As shown in Figure
6A both antibodies as well as mAb 2PH1
immunoprecipitated PSGL-1 from surface biotinylated 32Dcl3 cells.
Analyzing the epitopes of the 2 new antibodies on PSGL-1 revealed that
the 4RA10 epitope, but not the 4RB12 epitope, was sensitive to
treatment of 32Dcl3 cells with O-sialoglycoprotease (not shown). This
suggests that the 4RB12 epitope is located more proximal to the
transmembrane region of PSGL-1 than the 4RA10 epitope.
In agreement with this, we found that mAb 4RA10 recognizes the 19-amino acid peptide covering the N-terminus of the mature form of PSGL-1 (amino acids 42-60), whereas mAb 4RB12 did not recognize this epitope (not shown). The binding of P-selectin-IgG to the mouse T-cell clone 4G3 as analyzed by flow cytometry could be blocked by 99% (reduction of mean fluorescence intensity) with mAb 4RA10, whereas 2PH1 only reduced the binding of P-selectin-IgG by 80% and no reduction was observed with 4RB12 (Figure 6B). We next performed static and dynamic adhesion assays with immature and
mature DCs. As shown in Figure 7,
immature, but not mature, DCs bound to immobilized P- and
E-selectin-IgG under static conditions. The number of cells
specifically bound was comparable to 32Dcl3 cells. Binding of DC to
P-selectin-IgG was completely blocked by mAb 4RA10. As a negative
control, mAb PS/2 did not block any cell binding to the selectins. We
conclude that immature, but not mature, DCs are able to interact with
each of the 2 endothelial selectins and that PSGL-1 is involved in this
binding. To exclude that the binding of immature DCs to selectins was
due to contaminating non-DC cell populations present in the 5-day
cultures, the cultures were depleted for Lin+ cells by
magnetic bead cell sorting. As shown in Figure
8, removal of potential contaminants
resulted in largely unaffected efficiency of cell binding in the cell
adhesion assays.
We also performed dynamic adhesion assays under flow, using a laminar
flow chamber system with the selectin-IgG fusion proteins immobilized
on glass coverslips.44,45 We found that immature DCs could
interact with each of the 2 selectins under shear stress in the
physiologic range (Figure 9A). The number
of interacting cells decreased with increasing shear stress and was
comparable to the number of interacting 32Dcl3 cells. Rolling of DCs
was qualitatively similar to other cell types such as neutrophils. Again, the interaction with P-selectin was completely inhibited by the
mAb 4RA10 (Figure 9B), demonstrating that also under shear stress
PSGL-1 was the predominant P-selectin ligand on immature DCs. Similar
to the results in the static adhesion assays, mature DCs did not
interact with the immobilized selectin fusion proteins under flow
conditions (not shown).
Blocking of the endothelial selectins or lack of fucosylated selectin ligands, but not selective blocking of PSGL-1 or lack of Fuc-TVII, reduces migration of DCs into inflamed skin Because immature, but not mature, DCs clearly and specifically interacted with the endothelial selectins under physiologic shear conditions, we expected that these adhesion mechanisms would also be important for the migration of these cells into inflamed skin in the allergic contact dermatitis model presented above. Therefore, we tried to inhibit the migration of radioactively labeled, immature DCs into the inflamed skin of the ear by intravenous injection of a mixture of the adhesion-blocking antibodies UZ4 and RB40.34 against mouse E- and P-selectin, respectively. These mAbs have been shown to completely block the entry of Th-1 cells into inflamed skin in vivo.43 In control experiments we used in vivo-activated T cells isolated from lymph nodes of inflamed tissues that we labeled radioactively and injected into mice similarly to the immature DCs. As expected, we observed almost complete blockage of T-cell migration into the inflamed ear with antibodies against E- and P-selectin (Figure 10). However, homing of immature DCs was only partially inhibited, suggesting that the endothelial selectins are clearly involved in this process, but are less essential than for T cells (Figure 10).
Next we tested the requirement of selectin ligands on DCs for their
entry into inflamed tissue in the same experimental system. Because
Fuc-TIV and Fuc-TVII are essential for the generation of ligands for
each of the 3 selectins,24,25 we generated T cells and
immature DCs from bone marrow cells of mice that were either deficient
for the FUCTVII gene or for both, the genes of FUCTIV and FUCTVII. Differentiation of
DCs from bone marrow of these mice was largely normal, although in
Fuc-TIV/VII double-deficient mice, fewer DCs could be generated and the
cells had a slightly less differentiated surface phenotype. T cells
isolated from lymph nodes of hapten-sensitized
Fuc-TVII
In agreement with this finding, although surprising in light of the in vitro adhesion results (Figures 7 and 8), we found that preincubation of DCs with the adhesion-blocking mAb 4RA10 against mouse PSGL-1 failed to inhibit the migration of immature DCs into the inflamed ear, whereas it profoundly affected homing of T cells in the same experimental setting (Figure 11B). Increasing the amount of antibody 3-fold gave similar results (not shown). The nonblocking mAb 4RB12 against PSGL-1 had no effect on T cell migration, verifying the specificity of the blocking effect of 4RA10 on T-cell homing. Thus, the endothelial selectins and their ligands are involved in the homing of T cells as well as DCs into inflamed skin, but they vary in their importance for both processes. The endothelial selectins are only partially responsible for DC extravasation. In addition, the selectin ligands that are involved seem to be different from the ones on T cells. PSGL-1, although clearly a major ligand for P-selectin in in vitro adhesion assays, is not essential for DC extravasation in vivo. In agreement with this, Fuc-TVII, which plays a major role in generation of E- and P-selectin ligands in T cells and neutrophils and which is the essential enzyme for the formation of the selectin-binding glycoform of PSGL-1 in neutrophils,46 is dispensable for DC homing into inflamed skin. Instead, the selectin ligands on DCs involved in this process depend on the expression of Fuc-TIV, a minor component in the generation of selectin ligands on T cells and neutrophils.
In this study, we have shown that immature DCs can be recruited
into inflamed areas of murine skin. Because isolation of DCs from the
blood is not feasible in the mouse, we used a well-established culture
system35,36 to generate DCs of different stages of maturity
for our study. By analyzing the migration of radioactively labeled DCs
into sites of allergic contact dermatitis, we could quantitatively
compare the homing efficiency of DCs with that of activated T cells.
Although recruitment of DCs was less efficient than that of activated T
cells, several controls demonstrated that the accumulation of DCs was
specific. First, about 10 times more DCs were recruited to the inflamed
than to the noninflamed ear. Second, only immature, but not mature DCs
(resulting from the same DC cultures) homed to inflamed skin. Third,
PFA-fixed immature DCs did not accumulate in the inflamed ear,
demonstrating that this process required the active participation of
the recruited cells. Fourth, accumulated radioactivity persisted for at
least 48 hours and was not affected by steroid-induced resolution of cutaneous inflammation, arguing against the possibility that the DCs
were simply trapped within inflamed blood vessels without actually
transmigrating into tissue. In addition, transmigration of
calcein-labeled DCs from blood into inflamed skin was demonstrated by
confocal microscopy in a previous study.31 Fifth, by flow cytometry, all DC cultures were virtually devoid of T cells, B cells,
and granulocytes, suggesting that contaminating non-DCs were not
responsible for the selective accumulation of radioactively labeled
cells at the inflamed tissue site. To further exclude this possibility,
we depleted the bone marrow cultures of differentiated T cells, B
cells, granulocytes, and macrophages. These Lin In earlier studies, we found that human DCs can bind to E- and P-selectin.31 Moreover, by intravital microscopy, we observed that human DCs roll in postcapillary venules of mouse ears.31 This prompted us to now analyze whether this is also the case for mouse DCs and whether binding was dependent on the differentiation state of these cells. We now demonstrate binding of immature, but not of mature, DCs to immobilized recombinant forms of the selectins in vitro under static assay conditions and under physiologic conditions of flow. This confirms that differentiation of mouse bone marrow cells in the presence of GM-CSF and IL-4 leads to the induction of E- and P-selectin ligands on the differentiating DCs. Expression of these ligands, but not of PSGL-1, is transient and vanishes when cells reach the mature phenotype. It has been shown previously that human DCs can roll along the surface of mouse blood vessels and that this rolling depends on E- and P-selectin.31 However, it was not analyzed whether this interaction was indeed responsible for the extravasation of DCs. Here we show that the combined administration of adhesion-blocking antibodies against E- and P-selectin inhibited the entry of immature mouse DCs into inflamed skin, although inhibition was only partial. The same batches of antibodies were able to completely abolish the recruitment of hapten-specific T cells into inflamed skin, which rules out that the amount or quality of the antibodies might not have been sufficient to block the function of the endothelial selectins. We conclude that the endothelial selectins are indeed involved in the migration of immature mouse DCs into inflamed skin, but that additional mechanisms enable these cells to extravasate independently of the endothelial selectins. One obvious alternative candidate would be the integrin VLA-4 that is expressed on these cells (Figure 1) and that has been reported to also support leukocyte extravasation47 and rolling.48 Furthermore, it is conceivable that DCs, due to their large size, might already be in sufficiently tight contact with the blood vessel wall when they exit from the capillary system so that they are less dependent on selectin-mediated rolling to overcome the endothelial barrier. Yet another possibility is that DCs make use of adhesion mechanisms that are not found on other leukocytes. Recently, a novel lectin called DC-SIGN was identified on human DCs that mediates the binding of these cells to ICAM-2 and -3 on T cells and that is essential for DC-induced T-cell proliferation.49 Thus, novel adhesion mechanisms could exist on DCs, which might assist
or even dominate some of the known ones. In this context it is
interesting that we recently found that monocytes and DCs are still
able to extravasate into tissues in CD18 ( Further differences between the molecular mechanisms involved in the
extravasation of DCs on the one hand and T cells on the other hand were
found on the level of the selectin ligands. Blocking of PSGL-1 with
antibodies did not inhibit extravasation of DCs although it blocked
extravasation of T cells almost completely. This was especially
surprising because our in vitro studies showed that PSGL-1 was the
major P-selectin ligand on immature DCs in static as well as in
adhesion assays under flow (1 dyne/cm2). A possible
explanation for this apparent discrepancy would be that, as stated
above, additional adhesion mechanisms or the large size of immature DCs
might enable them to be in sufficiently tight contact with the blood
vessel wall when they exit from the capillary system that they are less
dependent on a highly effective capturing molecule such as PSGL-1 and
that other normally less effective selectin ligand molecules might
compensate for a lack of PSGL-1. In agreement with this interpretation,
we found that DC generated from Fuc-TVII The generation of carbohydrate epitopes on mouse DCs related to selectin functions seems to differ from such epitopes on other mouse leukocytes in yet another aspect. The carbohydrate epitope CLA is clearly transiently induced on the surface of DC during the course of differentiation. This is remarkable because neither primary isolated leukocytes from mouse bone marrow nor any major lineage population of leukocytes from mouse peripheral blood express the carbohydrate epitope CLA, which is known to be expressed on E-selectin-binding human leukocytes. The only other mouse leukocytes known to express CLA on their surface are the myeloid cell line 32Dcl3 (this report) and the mouse CD8+ T-cell clone 4G3 on antigen-specific activation.30 The structure of this carbohydrate epitope is not known in all detail; however, it is likely that it is a derivative of the tetrasaccharide sLex. It is generally assumed that mouse leukocytes express carbohydrate epitopes that somehow resemble sLex and bind to the selectins, but that are derived in a way that prevents recognition by the known anti-sLex antibodies.14 Our results suggest that enzymes that affect antibody recognition of sLex-like structures on these cells seem to be regulated during the course of DC differentiation in the mouse. We demonstrate here that immature, but not mature, DCs are actively recruited from blood to inflamed tissues, which might constitute a possible mechanism to provide potent APCs for presentation of antigen to effector T cells within inflamed tissues. This recruitment of APCs to inflamed tissue would allow for a sustained inflammatory response in the case of persisting antigenic challenge. Moreover, this mechanism would ensure a continuous flow of antigen-loaded DCs to regional lymph nodes, resulting in prolonged priming of naive T cells during chronic inflammation. Although much less efficient, the time course of DC recruitment is comparable to that of T cells,43 starting as early as 2 hours after hapten application, which is before dermatitis becomes visible. It will be interesting to test whether recruitment of APCs is not only involved in the maintenance but also in the initiation of the inflammatory tissue response. In addition it will be important to further elucidate the molecular mechanisms that allow immature DCs to enter sites of inflammation.
We thank Dr Louis Picker for donating the mAb HECA452 and Dr Rupert Hallmann for the mAB UZ4.
Submitted May 22, 2001; accepted August 10, 2001.
Supported in part by grants to D.V. (SFB 293/A1) and S.G. (SFB293/B1, Gr1022/3-3, Gr1022/7-1) from the Deutsche Forschungsgemeinschaft. J.B.L. is an Investigator of the Howard Hughes Medical Institute.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dietmar Vestweber, Institute of Cell Biology, ZMBE, and Stephan Grabbe, Department of Dermatology, University of Münster, Von-Esmarch Str 56, D-48149 Münster, Germany; e-mail: vestweb{at}uni-muenster.de and grabbe{at}uni-muenster.de.
1. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245-252[CrossRef][Medline] [Order article via Infotrieve]. 2. O'Doherty U, Peng M, Gezelter S, et al. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology. 1994;82:487-493[Medline] [Order article via Infotrieve]. 3. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1991;9:271-296[CrossRef][Medline] [Order article via Infotrieve]. 4. Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol. 1997;9:10-16[CrossRef][Medline] [Order article via Infotrieve].
5.
De Smedt T, Pajak B, Muraille E, et al.
Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo.
J Exp Med.
1996;184:1413-1424
6.
Roake JA, Rao AS, Morris PJ, Larsen CP, Hankins DF, Austyn JM.
Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1.
J Exp Med.
1995;181:2237-2247 7. MacPherson GG, Jenkins CD, Stein MJ, Edwards C. Endotoxin-mediated dendritic cell release from the intestine: characterization of released dendritic cells and TNF dependence. J Immunol. 1995;154:1317-1322[Abstract].
8.
McWilliam AS, Nelson D, Thomas JA, Holt PG.
Rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces.
J Exp Med.
1994;179:1331-1336
9.
Austyn JM.
New insights into the mobilization and phagocytic activity of dendritic cells.
J Exp Med.
1996;183:1287-1292
10.
Price AA, Cumberbatch M, Kimber I, Ager A.
Alpha 6 integrins are required for Langerhans cell migration from the epidermis.
J Exp Med.
1997;186:1725-1735
11.
Sallusto F, Lanzavecchia A.
Mobilizing dendritic cells for tolerance, priming, and chronic inflammation.
J Exp Med.
1999;189:611-614
12.
Hart DN.
Dendritic cells: unique leukocyte populations which control the primary immune response.
Blood.
1997;90:3245-3287 13. Lasky LA. Selectin-carbohydrate interactions and the initiation of the inflammatory response. Ann Rev Biochem. 1995;64:113-139[CrossRef][Medline] [Order article via Infotrieve].
14.
Vestweber D, Blanks JE.
Mechanisms that regulate the function of the selectins and their ligands.
Physiol Rev.
1999;79:181-213 15. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994;76:301-314[CrossRef][Medline] [Order article via Infotrieve]. 16. McEver RP, Cummings RD. Role of PSGL-1 binding to selectins in leukocyte recruitment. J Clin Invest. 1997;100:485-491[Medline] [Order article via Infotrieve].
17.
Moore KL, Stults NL, Diaz S, et al.
Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.
J Cell Biol.
1992;118:445-456 18. Sako D, Chang X-J, Barone KM, et al. Expression cloning of a functional glycoprotein ligand for P-selectin. Cell. 1993;75:1179-1186[CrossRef][Medline] [Order article via Infotrieve].
19.
Borges E, Eytner R, Moll T, et al.
The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum.
Blood.
1997;90:1934-1942
20.
Borges E, Tietz W, Steegmaier M, et al.
P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin.
J Exp Med.
1997;185:573-578
21.
Yang J, Hirata T, Croce K, et al.
Targeted gene disruption demonstrates that P-selectin glycoprotein ligand-1 (PSGL-1) is required for P-selectin-mediated but not for E-selectin-mediated neutrophil rolling and migration.
J Exp Med.
1999;190:1769-1782
22.
Hirata T, Merrill-Skoloff G, Aab M, Yang J, Furie BC, Furie B.
P-selectin glycoprotein ligand-1 (PSGL-1) is a physiological ligand for E-selectin in mediating T-helper 1 lymphocyte migration.
J Exp Med.
2000;192:1669-1676 23. Lowe JB. Selectin ligands, leukocyte trafficking, and fucosyl transferase genes. Kidney Int. 1997;51:1418-1426[Medline] [Order article via Infotrieve]. 24. Maly P, Thall AD, Petryniak B, et al. The a-(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell. 1996;86:643-653[CrossRef][Medline] [Order article via Infotrieve]. 25. Weninger W, Ulfman LH, Cheng G, et al. Specialized contributions by alpha(1,3)-fucosyltransferase-IV and FucT-VII during leukocyte rolling in dermal microvessels. Immunity. 2000;6:665-676.
26.
Strunk D, Egger C, Leitner G, Hanau D, Stingl G.
A skin homing molecule defines the Langerhans cell progenitor in human peripheral blood.
J Exp Med.
1997;185:1131-1136 27. De Boer OJ, Horst E, Pals ST, Bos JD, Das PK. Functional evidence that the HECA-452 antigen is involved in the adhesion of human neutrophils and lymphocytes to tumour necrosis factor-alpha-stimulated endothelial cells. Immunology. 1994;81:359-365[Medline] [Order article via Infotrieve].
28.
Berg EL, Yoshino T, Rott LS, et al.
The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1.
J Exp Med.
1991;174:1461-1466 29. Fuhlbrigge RC, Kieffer JD, Armerding D, Kupper TS. Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells. Nature. 1997;389:978-981[CrossRef][Medline] [Order article via Infotrieve].
30.
Borges E, Pendl G, Eytner R, Steegmaier M, Zöllner O, Vestweber D.
The binding of T cell-expressed P-selectin glycoprotein ligand-1 to E- and P-selectin is differentially regulated.
J Biol Chem.
1997;272:28786-28792
31.
Robert C, Fuhlbrigge RC, Kieffer JD, et al.
Interaction of dendritic cells with skin endothelium: a new perspective on immunosurveillance.
J Exp Med.
1999;189:627-635 32. Homeister JW, Thall AD, Petryniak B, et al. The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing. Immunity. 2001;15:115-126[CrossRef][Medline] [Order article via Infotrieve].
33.
Hahne M, Jäger U, Isenmann S, Hallmann R, Vestweber D.
Five TNF-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.
J Cell Biol.
1993;121:655-664 34. Bosse R, Vestweber D. Only simultaneous blocking of L- and P-selectin completely inhibits neutrophil migration into mouse petritoneum. Eur J Immunol. 1994;24:3019-3024[Medline] [Order article via Infotrieve].
35.
Labeur MS, Roters B, Pers B, et al.
Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.
J Immunol.
1999;162:168-175 36. Lutz MB, Kukutsch N, Ogilvie AL, et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods. 1999;223:77-92[CrossRef][Medline] [Order article via Infotrieve].
37.
Levinovitz A, Mühlhoff J, Isenmann S, Vestweber D.
Identification of a glycoprotein ligand for E-selectin on mouse myeloid cells.
J Cell Biol.
1993;121:449-459
38.
Zöllner O, Vestweber D.
The E-selectin ligand-1 is selectively activated in Chinese hamster ovary cells by the alpha(1,3)-fucosyltransferases IV and VII.
J Biol Chem.
1996;271:33002-33008 39. Kitajima T, Ariizumi K, Bergstresser PR, Takashima A. T cell-dependent loss of proliferative responsiveness to colony-stimulating factor-1 by a murine epidermal-derived dendritic cell line, XS52. J Immunol. 1995;155:5190-5197[Abstract].
40.
Marquardt T, Luhn K, Srikrishna G, Freeze HH, Harms E, Vestweber D.
Correction of leukocyte adhesion deficiency type II with oral fucose.
Blood.
1999;94:3976-3985
41.
Lenter M, Levinovitz A, Isenmann S, Vestweber D.
Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells.
J Cell Biol.
1994;125:471-481 42. Grabbe S, Steinert M, Mahnke K, Schwarz A, Luger TA, Schwarz T. Dissection of antigenic and irritative effects of epicutaneously applied haptens in mice: evidence that not the antigenic component but nonspecific proinflammatory effects of haptens determine the concentration-dependent elicitation of allergic contact dermatitis. J Clin Invest. 1996;98:1158-1164[Medline] [Order article via Infotrieve]. 43. Austrup F, Vestweber D, Borges E, et al. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. Nature. 1997;385:81-83[CrossRef][Medline] [Order article via Infotrieve].
44.
Kuijper PH, Gallardo Torres HI, van der Linden JA, et al.
Platelet-dependent primary hemostasis promotes selectin- and integrin-mediated neutrophil adhesion to damaged endothelium under flow conditions.
Blood.
1996;87:3271-3281
45.
Zöllner O, Lenter MC, Blanks JE, et al.
L-Selectin from human, but not from mouse neutrophils binds directly to E-selectin.
J Cell Biol.
1997;136:707-716
46.
Huang MC, Zollner O, Moll T, et al.
P-selectin glycoprotein ligand-1 and E-selectin ligand-1 are differentially modified by fucosyltransferases Fuc-TIV and Fuc-TVII in mouse neutrophils.
J Biol Chem.
2000;275:31353-31360 47. Hourihan H, Allen TD, Ager A. Lymphocyte migration across high endothelium is associated with increases in alpha 4 beta 1 integrin (VLA-4) affinity. J Cell Sci. 1993;104:1049-1059[Abstract]. 48. Berlin C, Bargatze RF, Campbell JJ, et al. a4-Integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell. 1995;80:413-422[CrossRef][Medline] [Order article via Infotrieve]. 49. Geijtenbeek TB, Torensma R, van Vliet SJ, et al. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell. 2000;100:575-585[CrossRef][Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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|
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 |
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|
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 S. Massberg, I. Konrad, K. Schurzinger, M. Lorenz, S. Schneider, D. Zohlnhoefer, K. Hoppe, M. Schiemann, E. Kennerknecht, S. Sauer, et al. Platelets secrete stromal cell-derived factor 1{alpha} and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo J. Exp. Med., May 15, 2006; 203(5): 1221 - 1233. [Abstract] [Full Text] [PDF]
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 H. Langer, A. E. May, K. Daub, U. Heinzmann, P. Lang, M. Schumm, D. Vestweber, S. Massberg, T. Schonberger, I. Pfisterer, et al. Adherent Platelets Recruit and Induce Differentiation of Murine Embryonic Endothelial Progenitor Cells to Mature Endothelial Cells In Vitro Circ. Res., February 3, 2006; 98(2): e2 - e10. [Abstract] [Full Text] [PDF]
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M. Gunzer, H. Riemann, Y. Basoglu, A. Hillmer, C. Weishaupt, S. Balkow, B. Benninghoff, B. Ernst, M. Steinert, T. Scholzen, et al. Systemic administration of a TLR7 ligand leads to transient immune incompetence due to peripheral-blood leukocyte depletion Blood, October 1, 2005; 106(7): 2424 - 2432. [Abstract] [Full Text] [PDF]
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 M.-C. Huang, A. Laskowska, D. Vestweber, and M. K. Wild The alpha (1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids J. Biol. Chem., November 27, 2002; 277(49): 47786 - 47795. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
2,3Gal
isotype) (Pharmingen). The P-selectin-IgG and E-selectin-IgG chimeras were produced as described
before.
counter for 2 minutes per probe (LKB Systems, Freiburg, Germany).
The percentage of radioactivity in each ear relative to the sum counted
in all organs was calculated.
). The y-axis (percent of cpm) refers to radioactivity in the ear
with respect to radioactivity of all harvested organs set as 100%. (A)
Immature DCs cultured for 5 days, mature DCs cultured for 11 days
without LPS (mature DC) or with LPS for the last 24 hours (mature DC,
LPS activated), immature DCs fixed in PFA (immature DC, PFA fixed), and
the immature DC line XS52 were compared. The means and SDs for each
value were calculated from 3 independent animals. Immigration of DCs
was statistically significant for immature DCs and XS52, respectively
(P < .001), but not for mature DCs. (B) Bulk cultures of
immature DCs were not depleted for Lin+ cells, whereas
Lin
) or E-selectin-IgG (
) immobilized on glass coverslips
that were inserted into a planar flow chamber with a variable flow rate
of 1.0 to 3.2 dynes/cm2. (B) At a constant flow rate of 1 dyne/cm2, cells were either used directly (no block) or
preincubated with 10 µg/mL of the blocking antibody 4RA10 against
mouse PSGL-1 (
, 
, +)
was calculated as percent of total radioactivity measured in all
harvested organs (percent of cpm). Each value represents groups of 4 mice. Inhibition of homing due to blockade of selectins was
statistically significant (DCs, P = .001; T cells,
P < .0001).
