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Institut National de la Santé et de la Recherche Médicale (INSERM) CJF 94-03, and INSERM Unité 311, Etablissement de Transfusion Sanguine de Strasbourg, and
Service d’Onco-Hématologie, Hôpital de Hautepierre, Strasbourg, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment, which induces the maturation of
DCs, up-regulates the expression of all v3-, v6-, and v9-containing
isoforms examined. CD44 molecules are involved in the adhesion of DCs
to immobilized hyaluronate (HA), and v3- and v6-containing variants
participate in this function, whereas anti-CD44v9 mAbs were unable
to inhibit DC adhesion to HA. The consequences of ligand binding to
CD44 were examined by culturing DCs on dishes coated with HA or various
anti-CD44 mAbs. HA, the anti-pan CD44 mAb J173, and mAbs
directed against v6- and v9-containing (but not v3-containing) isoforms
provoked DC aggregation, phenotypic and functional maturation, and the
secretion of IL-8, TNF-, IL-1ß, and granulocyte-macrophage CSF. In
addition, IL-6, IL-10, and IL-12 were released by DCs stimulated with
either J173 or HA, although these cytokines were not detected or were
found only at low levels in the culture supernatants of DCs treated
with anti-CD44v6 or anti-CD44v9 mAbs. Our study points to
distinct capacities of the v3-, v6-, and v9-containing isoforms
expressed by human DCs to mediate cell adhesion to HA and/or a signal
inducing DC maturation and the secretion of
cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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haptens, or chemical allergens, DCs can emigrate from the peripheral
tissues to colonize the T cell areas of secondary lymphoid organs
through afferent lymphatics or the blood (1, 2, 3). Phenotypic and
functional changes known as DC maturation underlie this particular
behavior (4, 5, 6). Among these changes, DCs up-regulate the expression of
MHC class I and class II Ags and of accessory molecules involved in
antigenic stimulation and lose their ability to efficiently process new
Ags, while they increase their T cell stimulatory capacity.
Consequently, in lymphoid tissues, mature DCs can sensitize resting T
cells and initiate a primary immune response. Mature DCs are considered
to be the most powerful "professional" APCs and represent a
promising potential tool to improve the immunotherapy of cancer (7). Among the different adhesion molecules expressed by DCs, CD44 comprises a family of polymorphic surface glycoproteins and represents the principal cell surface receptor for hyaluronate (HA), a glycosaminoglycan found in extracellular matrixes (ECM) and in association with cell surfaces (8, 9, 10). A variety of functions have been attributed to CD44 receptors, including leukocyte adhesion, differentiation, homing, activation, and extravasation to inflammatory sites (10). Recent studies have implicated CD44 and HA binding in signal transduction events (11), notably in T lymphocytes and NK cells, where CD44 participates in a tyrosine kinase-dependent signaling pathway (12, 13, 14). CD44 molecules are highly heterogeneous, and extensive glycosylation can modulate their ability to bind HA (15, 16, 17, 18). Moreover, insertion of an additional membrane-proximal region resulting from the alternative splicing of up to 10 exons (termed v1 to v10) gives rise to the so-called CD44 variants (19, 20). Whereas the common ("standard" or "hemopoietic") form of CD44 is widely and often highly expressed, variant isoforms are more restricted. Some carrying the v6 region have been implicated in tumor metastasis and lymphocyte activation, suggesting a possible role in cell homing to the lymph nodes (19), while variants containing v3 represent targets for glycosaminoglycan modification resulting in growth factor binding and presentation (21). Exons v8 to v10 are represented in the so-called epithelial form of CD44 (CD44E) (22), and a variant containing v10 has been shown to mediate a signal inducing endothelial cell proliferation (23). Nevertheless, the functions of CD44 variant isoforms generally remain poorly understood.
Mouse Langerhans cells and DCs isolated from blood and lymph nodes
strongly express CD44 (24). In humans, DCs differentiated from blood
monocytes cultured in the presence of GM-CSF and IL-4 are CD44 positive
(5). However, the role of the CD44 molecules expressed by DCs remains
largely unknown, and the pattern of CD44 variant isoforms has not yet
been determined in this cell type. Accumulating evidence suggests that
regulation of CD44 on the surface of DCs may modulate their immune
function or adhesive phenotype. Thus, recent work has demonstrated the
involvement of CD44 in the emigration of Langerhans cells from the
epidermis and their adhesion to the T cell zones of lymph nodes (25).
TNF- has been shown to increase the overall expression of CD44 in a
variety of cell types, including mouse Langerhans cells and human
monocyte-derived DCs (5, 26, 27). In the latter, expression of a
v9-containing CD44 variant was detected following TNF--induced
maturation (5). Therefore, we attempted in the present work to better
characterize the CD44 isoforms expressed by human monocyte-derived DCs
and to study their regulation during DC differentiation and maturation
induced by TNF-. To gain insight into the function(s) of these
receptors, we sought to determine their implication in DC adhesion to
HA and examined the effects on DC function of the binding of CD44
epitopes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cultures were established in RPMI 1640 medium containing
Glutamax-1 (Life Technologies, Paisley, U.K.), supplemented with 10%
FCS (Life Technologies), 1% nonessential amino acids (Life
Technologies), 1% sodium pyruvate (Sigma, St. Louis, MO), 50 U/ml
penicillin, and 50 U/ml streptomycin (Life Technologies). Hyaluronic
acid (H1751) and BSA (A3350) were purchased from Sigma. Recombinant
human (rh) GM-CSF was a generous gift from Schering-Plough
(Levallois-Perret, France), while rhIL-4 was obtained from PeproTech
(Rocky Hill, NJ), and rhTNF- from Genzyme Corp. (Cambridge, MA).
Ab staining and FACS analysis
Cells were washed twice in PBS (Life Technologies) and incubated
for 30 min with the following primary mAbs: L243 (anti-HLA-DR,
IgG2a, Becton Dickinson, San Jose, CA), W6/32 (anti-HLA-A, -B, and
-C, IgG2a, Dako, Copenhagen, Denmark), BL6 (anti-CD1a, IgG1,
Immunotech, Marseille, France), MAB89 (anti-CD40, IgG1,
Immunotech), MAB104 (anti-CD80, IgG1, Immunotech), IT2.2
(anti-CD86, IgG2b, PharMingen, San Diego, CA), HB-15a
(anti-CD83, IgG2b, Immunotech), J173 (anti-pan CD44, IgG1,
Immunotech), 5F12 (anti-pan CD44, IgG2a, Neomarkers, Fremont, CA),
3G5 (anti-CD44v3, IgG2b, R&D Systems, Abingdon, U.K.), VFF-8
(anti-CD44v5, IgG1, Bender, Vienna, Austria), 2F10
(anti-CD44v6, IgG1, R&D Systems), VFF-9 (anti-CD44v7, IgG1,
Bender), FW11-24-17-36 (anti-CD44v9, IgG1, HB-258, American Type
Culture Collection, Manassas, VA), UCHT-1 (anti-CD3, IgG1,
Immunotech), M
P9 (anti-CD14, IgG2b, Becton Dickinson), 3G8
(anti-CD16, IgG1, Medarex, Annandale, NJ), J4-119 (anti-CD19,
IgG1, Becton Dickinson), or BBIG-I1 (anti-CD54, IgG1, R&D Systems).
Mouse IgG1, IgG2a, and IgG2b (Immunotech) were used as isotype
controls. The cells were then washed twice in Dulbecco’s PBS and
incubated for 30 min with the FITC-conjugated affinity-isolated
F(ab')2 fraction of a sheep anti-mouse IgG Ab (Silenus,
Hawthorn, Australia). Direct staining was performed using PE-conjugated
L243 (anti-HLA-DR, Becton Dickinson), HB-15 (anti-CD83,
Immunotech), or IgG1 (Immunotech). All incubation and washing steps
were conducted at 4°C, and the stained cells were analyzed by flow
cytometry on a FACScan cytometer (Becton Dickinson) using LYSYS II
software (Becton Dickinson).
In vitro differentiation of peripheral blood monocytes into DCs
Human blood monocytes were isolated by continuous flow centrifugation leukopheresis and counterflow centrifugation elutriation as previously described (28). The cells were then cultured in complete medium supplemented with 50 ng/ml rhGM-CSF and 200 U/ml rhIL-4 for 7 days. Cell density was adjusted to 106 cells/ml, and the medium was changed on day 4 of culture. Differentiation of monocytes into DCs was monitored by flow cytometric analysis of cell surface phenotypes. On day 7, DCs expressed high levels of MHC class I and class II (HLA-DR) molecules and the Ags CD1a, CD40, CD80, and CD44, but only small amounts of the CD86 Ag and neither CD83 nor CD3, CD14, CD16, or CD19, in agreement with previous observations (5, 29).
DC stimulation by TNF- or CD44 binding
In TNF- stimulation experiments, day 7 DCs were incubated in
complete medium supplemented with 50 ng/ml rhGM-CSF, 200 U/ml rhIL-4,
and 20 ng/ml rhTNF-. After 24 to 48 h of culture, the cells
were harvested, washed in PBS, and used for phenotypic or functional
analyses. In CD44 stimulation experiments, day 7 DCs were cultured in
60-mm petri dishes (Falcon 1007, Becton Dickinson) coated at 4°C with
10 µg/ml of various anti-CD44 mAbs or a control IgG1 in 3.5 ml of
PBS, or with 50 µg/ml HA in 5 ml of PBS. The dishes were coated
overnight and rinsed twice with PBS before plating the DCs, which were
previously depleted of the few remaining monocytes by treatment with
magnetic beads coated with an anti-CD14 mAb (M450, Dynal, Oslo,
Norway) according to the manufacturer’s instructions. Formation of
cell aggregates was occasionally observed after 24-h culture. The cells
were then harvested for phenotypic or functional analyses, and the
supernatants were recovered for determination of cytokine levels.
All Ab and HA solutions employed for coating dishes were previously tested for their endotoxin content using a Limulus amebocyte assay (Biogenix, Biogenic, Maurin, France), and when necessary, control dishes were prepared using appropriate concentrations of Escherichia coli endotoxin (BioWittaker, Walkersville, MD) dissolved in PBS. Anti-CD44v3, -v6, and -v9 mAb solutions were devoid of detectable contaminating endotoxins. On the contrary, HA isolated from human umbilical cord was found to contain endotoxins at levels of 20 to 30 IU/ml at the concentration of 50 µg/ml HA chosen for coating dishes. Therefore, to ensure that the effect observed with HA was specific and was not due to endotoxin stimulation, control cells were plated on dishes coated overnight with a solution containing 30 IU/ml endotoxin and rinsed twice with PBS, which failed to stimulate DC maturation. This was not the case when DCs were incubated with 30 IU/ml soluble endotoxin added directly to the culture medium or on dishes coated with a highly concentrated (e.g., 900 IU/ml) endotoxin solution; in both cases DC maturation was triggered, as shown by FACS analysis of the cell surface phenotype. In subsequent experiments the low HA concentration chosen for coating dishes (50 µg/ml) could be considered to exclude an effect of contaminating endotoxins on the maturation of DCs.
Semiquantitative RT-PCR
Total RNA was isolated from highly purified DCs using the
guanidium isothiocyanate method (30). cDNA synthesis was conducted in a
100-µl reaction mix containing 2 µg of total cellular RNA, 0.4
U/µl RNase inhibitor (Eurogentec, Seraing, Belgium), 1 mM dNTP
(Boehringer Mannheim, Mannheim, Germany), 1x concentrated
hexanucleotide mixture (Boehringer Mannheim), and 0.4 U/µl AMV
reverse transcriptase (Finnzymes Oy, Espoo, Finland) in the
reaction buffer provided by the manufacturer. After 45-min incubation
at 42°C, the mixture was heated to 95°C for 5 min. The whole
variable region of CD44 was amplified using oligonucleotides
hybridizing with sequences present in the flanking exons 5 and 15
(Table I
), which code for the common or
standard region of CD44 (19). cDNA samples (5 µl) were added to a
50-µl reaction mix containing 200 nM of each oligonucleotide
(Eurogentec), 0.5 mM dNTP (Boehringer Mannheim), 1.5 mM
MgCl2, and 0.05 U/µl Taq DNA polymerase (Goldstar,
Eurogentec) in 1x reaction buffer. Amplification was performed in a
DNA thermal cycler (Hybaid, OmniGene, Teddington, U.K.) as follows:
30 s at 95°C, 15 s at 55°C, and 2 min at 72°C for 35
cycles, followed by 10 min at 72°C. PCR products were separated on a
2% agarose gel and transferred onto Hybond-N+ membranes
(Amersham France, Les Ulis, France), and Southern blots were hybridized
with a panel of oligonucleotide probes corresponding to the CD44
variable exons v3 to v10 (Table I). The probes were labeled with
digoxigenin-dideoxyUTP (DIG oligonucleotide 3' end labeling kit,
Boehringer Mannheim). Homologous DNA fragments hybridized on Southern
blots were revealed by chemiluminescence using a DIG luminescent
detection kit (Boehringer Mannheim). After a series of short exposures
to Hyperfilm-MP (Amersham), band intensities were quantified with the
program Visiolab 2000 (Biocom, Les Ulis, France). All results were
normalized relative to an actin sequence amplified from each cDNA
sample. Actin PCR was conducted using the oligonucleotides given in
Table I, with 25 cycles of 30 s at 95°C, 15 s at 56°C,
and 60 s at 72°C. The PCR product was run on a 1.5% agarose
gel, the actin band intensity was quantified by gel scanning as
described above, and the result was taken as a standard indicative of
the total amount of cDNA added to the amplification mixture.
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, MIP-1, GM-CSF, and IL-1ß amplification were provided by
Dr. Eric Tartour (Institut Curie, Paris, France), while primers for
IL-15 amplification were synthesized by Eurogentec (Table I
). Serial
1/10 dilutions of each cDNA sample were subjected to PCR according to
the protocol described above, using amplification cycles of 1 min at
95°C, 1 min at 55°C, and 1.5 min at 72°C. The number of cycles
(n = 25–35) was adjusted for each cytokine so as to
remain in the linear range. PCR products were separated on a 2%
agarose gel in the presence of ethidium bromide (Appligene, Illkirch,
France) and quantified with the program Visiolab 2000. Again, results
were normalized relative to an actin sequence amplified from each cDNA
sample. Cytokine titration
Cytokine concentrations in DC culture supernatants were
determined using two-site sandwich ELISA kits (human IL-6, IL-8, IL-10,
IL-12p70, GM-CSF, IL-1ß, and TNF-, Quantikine, R&D Systems)
according to the manufacturer’s recommendations. Absorbances (450–540
nm) were read in an ELISA microplate reader (Molecular Devices, Palo
Alto, CA).
DC adhesion to HA
Flat-bottom 96-well plates (Maxisorp, Nunc, Rockilde, Denmark)
were coated overnight with 5 mg/ml HA (Sigma) in PBS, rinsed with PBS,
and saturated with 0.5% BSA (Sigma) for 2 h at 4°C. Control
wells were coated either with BSA or with an endotoxin concentration
equivalent to that found in 5 mg/ml HA solutions (
3000 IU/ml) to
measure unspecific cell adhesion, which was negligible. Anti-CD44 or
control mAbs (100 µl of a 40 µg/ml solution in serum-free medium)
were added to a first well, 50 µl of serum-free medium was added to
the following well, and serial 1/2 dilutions were performed to
obtain a final volume of 50 µl of diluted mAb solution in all wells.
Cells (105 in 50 µl) were added to each well and left to
sediment on ice for 1 h. The wells were then filled with cold PBS
to obtain a positive meniscus, the plates were wrapped in Parafilm and
centrifuged in an inverted position for 5 min at 400 rpm (22 g), and
medium and unbound cells were eliminated by flicking. Adherent cells
were stained using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye
reduction assay (MTT; Sigma) as described by Mossmann (31). Aliquots
(200 µl) of culture medium containing 10% MTT were added to all
wells, while a series of calibration wells contained graded quantities
of DCs added directly to the MTT medium as standards indicative of
total cell number. After 3-h incubation at 37°C, the plates were
briefly centrifuged, and the supernatant medium (150 µl) was
carefully removed from each well. Newly formed blue formazan crystals
were dissolved by adding 100 µl of 10% Triton-100 (Sigma) in
acidified isopropanol, and absorbances (540–650 nm) were read in an
ELISA microplate reader.
Mixed leukocyte reaction
The stimulator cells were DCs that had been cultured for 24 h in the presence of anti-CD44 or isotype control mAbs, HA, or control endotoxin as described above. After resuspension, these cells were washed in PBS and irradiated (3000 rad from a 137Cs source). Various numbers of stimulator cells (ranging from 104-625) were added to allogeneic responder cells (1.5 x 105) in the wells of round-bottom 96-well plates (Nunc) in a final volume of 200 µl of culture medium containing 10% human serum (ETS, Strasbourg, France). The responder cells were PBLs or allogeneic CD4+ naive T cells obtained from lymphocyte suspensions depleted of CD45R0+ and CD8+ cells. All cultures were set up in triplicate. Controls included DCs alone, responder cells alone, and responder cells cultured in the presence of 10 U/ml IL-2 (Chiron France, Suresnes, France) and 100 ng/ml Con A (Sigma). After 5 days of culture, 1 µCi of [3H]thymidine (Amersham) was added to each well, and thymidine incorporation was measured 18 h later using a Skatron cell harvester and a Betaplate counter (LKB Wallac, EG&G Instruments, Evry, France).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD44 expression is known to be modulated on the surface of DCs (5, 24, 26). In particular, the appearance of a v9-containing CD44 isoform
has been reported in monocyte-derived DCs following maturation induced
by TNF- (5). Given the diversity of CD44 isoforms already
characterized in various cell types, we wished to know whether other
variants were expressed by monocyte-derived DCs. Hence we chose to
examine the profile of transcripts encoding CD44 isoforms and to
compare variant CD44 mRNAs in monocyte-derived DCs to those expressed
by fresh blood monocytes. To this aim, the entire variable region of
CD44, spanning exons v1 to v10, was PCR amplified (Fig. 1a), and the PCR products were
hybridized with oligonucleotide probes corresponding to all exons of
the variable region from v3 to v10. Since v1 is known to be
nonfunctional in human cells (20), while v2 has been found only in
large and rarely expressed CD44 variants (10), probes for these two
exons were not tested. Some variable exons were barely detectable in
either monocytes or DCs, for instance v4, v5, and v7 (data not shown),
which may have been due either to the absence of such variants or to
the large size of their PCR products, as long fragments are known to be
less efficiently amplified by the PCR technique. In contrast, other
exons were readily amplified, and their relative abundances were
modified upon DC differentiation from monocytes (Fig. 1b).
The relative abundances of two v3-containing mRNAs and a v6-containing
transcript did not vary significantly between monocytes and day 7 DCs
(Fig. 1b, d0–d7). The size of the v6-containing variable
domain indicated that it must contain only the v6 exon and therefore
correspond to the CD44v6 form known to be induced in activated
lymphocytes (19). Two main v9-containing CD44 variant transcripts,
giving bands of approximately 380 and 520 bp, were significantly
decreased (about fourfold by densitometry scanning) in DCs compared
with those in monocytes (Fig. 1b). Hybridization with the
v10 probe gave two doublets of 300 to 400 and 520 to 640 bp, and the
second doublet diminished about twofold in DCs relative to monocytes
(Fig. 1b). Using probes for v8, a 520-bp band was also found
to decrease in intensity following DC differentiation from monocytes
(data not shown). In view of its size and hybridization pattern, this
520-bp PCR product may well correspond to the CD44E-type isoform
(CD44v8–v10), while the overall results obtained using probes for v8,
v9, and v10 probably reflect a decrease in the abundance of this
CD44E-type transcript upon DC differentiation. It is noteworthy that
although quantitative regulation was observed, no change in the general
splicing pattern of the variable domain could be detected following DC
differentiation from monocytes, nor was this splicing pattern modified
when DCs were treated with TNF- on day 7 of culture. However, using
semiquantitative PCR analysis, mature DCs stimulated with TNF- for
48 h showed elevated levels of v3-, v6-, v9-, and v10-containing
transcripts compared with unstimulated DCs on day 7 or 9 of culture
(data not shown). This led us to study more closely the regulation of
CD44 isoforms on the surface of DCs during their maturation induced by
TNF-.
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We therefore examined whether the CD44 isoforms characterized at
the transcript level were in fact expressed on the surface of DCs and
regulated upon TNF- stimulation. In view of the preceding results,
the surface expression of v3-, v6-, and v9-containing isoforms and that
of total CD44 were studied by immunocytochemistry and flow cytometry.
After 7-day differentiation in the presence of rhGM-CSF and rhIL-4, DCs
showed intense and relatively homogeneous staining with the
anti-pan CD44 mAb J173. Although staining with anti-CD44v5 or
anti-CD44v7 mAbs was almost negative (data not shown), the three
variant epitopes, v3, v6, and v9, were indeed found to be expressed on
the surface of DCs (Fig. 2), in agreement
with the PCR results. However, their levels of expression were somewhat
weak on unstimulated, immature DCs, with mean fluorescence intensities
about 100-fold lower than that of total CD44 stained with J173.
Moreover, despite a relative homogeneity in variant CD44 expression
among unstimulated DC cultures (Fig. 2), a minor population of cells
stained negatively for the v3, v6, and v9 epitopes. TNF- induced the
maturation of DCs, as assessed by staining for HLA-DR, CD40, CD80, and
CD86, all of which were up-regulated on the cell surface following
TNF- treatment (5), and for CD83, a marker of mature DCs (29) (data
not shown). After 48-h stimulation with TNF-, total CD44 expression
was slightly, but significantly, up-regulated (1.5- to 2-fold; Fig. 2, upper left panel). On the other hand, expression of the v3,
v6, and v9 isoforms was more strongly induced (Fig. 2); the mean
fluorescence intensities corresponding to these epitopes increased up
to 5-fold in DCs stimulated by TNF-. This would suggest that
variant CD44 isoforms may play a more important role in DCs that have
undergone maturation in the presence of TNF-.
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Some members of the CD44 family have been implicated in cell
adhesion to the extracellular matrix component HA (9), but the
contributions of variant isoforms to this process remain a matter of
debate. As a first step to investigate the function(s) of the CD44
molecules expressed by DCs, we examined their involvement in cell
attachment to HA. DCs were indeed capable of adhering to a plastic
surface coated with a highly concentrated HA solution (5 mg/ml) within
1 h of incubation at 4°C (Fig. 3).
Although the percentage of adherent cells varied between experiments
(40–80%; data not shown), possibly due to donor-specific parameters
or to slight variations in experimental conditions, Ab inhibition
clearly demonstrated that CD44 molecules were involved in DC adhesion
to HA. Cell adhesion was measured in the presence of graded
concentrations of the mAbs J173 and 5F12, which recognize common CD44
epitopes and are known to block HA binding mediated by CD44 (32, 33).
At saturating concentrations, 5F12 and J173 were able to partially
inhibit the adhesive interaction between DCs and HA (by 50%
compared with a control IgG). Unexpectedly, DCs stimulated with
TNF-, which expressed higher levels of CD44, showed no significant
modulation of their overall capacity to bind to immobilized HA (data
not shown). Regulatory events occurring at the post-translational level
may therefore further modulate HA binding regardless of the total
amount of surface CD44.
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). It should be noted that the blocking
effect observed in the presence of both anti-CD44v3 and
anti-CD44v6 mAbs was stronger than that obtained using anti-pan
CD44 mAbs despite the lower level of surface expression of the variant
epitopes compared with total CD44 (Fig. 2). In contrast, an
anti-CD44v9 mAb was totally ineffective at inhibiting DC adhesion
to HA. These results indicate that CD44 variants containing v3 and v6
play an important role in the binding of DCs to HA, whereas
v9-containing variants do not seem to contribute to this function. DC aggregation induced by ligation of CD44 isoforms
Since a role for surface CD44 in triggering cell aggregation has
been described in lymphoid and transfected cell lines (9, 34, 35), we
decided to investigate the effects on DCs of the ligation of various
CD44 epitopes, including variant region domains. To this aim, day 7 DCs
were incubated in the presence of immobilized Abs directed against all
forms of CD44 (J173, 5F12), against v3-, v6-, or v9-containing
isoforms, or in the presence of control IgG. In parallel, DCs were
cultured in dishes coated with a dilute solution of HA (50 µg/ml).
Large cell aggregates were observed following 24-h incubation with
either the mAb J173 (Fig. 4) or
immobilized HA (data not shown), while aggregation was also obtained by
culture of DCs in the presence of anti-CD44v6 (Fig. 4) and
anti-CD44v9 (data not shown) mAbs. The aggregates formed by
stimulation with J173 were often larger than those induced by treatment
with anti-CD44v6 or anti-CD44v9, which could be due to the
greater number of common CD44 epitopes on the cell surface or to the
existence of a small DC population negative for variant CD44 expression
(Fig. 2). The mAbs 5F12 and anti-CD44v3 did not provoke any
detectable cell aggregation compared with control IgG (Fig. 4). To
ensure that aggregation of DCs was not induced by contaminating
endotoxin in HA or mAb samples, we regularly titrated endotoxin levels
in our reagents and performed control cultures on dishes coated with
solutions containing equivalent concentrations of endotoxin (see
Materials and Methods). Furthermore, the anti-variant
CD44 mAb solutions used for coating dishes were devoid of detectable
endotoxin. It is noteworthy that anti-v6 and anti-v9, but not
anti-v3, Abs induced cell aggregation, even though all three
variants showed comparable levels of surface expression (Fig. 2). Our
experiments therefore point to distinct capacities of CD44 variant
isoforms to trigger DC aggregation.
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DC maturation is accompanied by the modulation of adhesion and
costimulatory molecule expression and by the appearance of CD83 on the
cell surface (5, 6, 29). In view of the aggregation observed in
cultures stimulated through ligation of CD44 epitopes, the surface
phenotype of DCs was analyzed by flow cytometry after 24-h incubation
in the presence of immobilized anti-CD44 mAbs, control IgG, HA (50
µg/ml), or control endotoxin. Results from six independent
experiments are summarized in Figure 5a. DCs from cultures that had
aggregated in the presence of the anti-pan CD44 mAb J173,
anti-CD44v6, or anti-CD44v9 mAbs or HA showed enhanced surface
expression of MHC class II, CD40, and CD86 (B7-2) Ags (Fig. 5a) and of CD54 and CD80 (data not shown) compared with DCs
cultured on dishes treated with control IgG or endotoxin. In contrast,
no significant variation in the expression of these five maturation
markers was found on cells from the nonaggregated cultures stimulated
with 5F12 or anti-CD44v3. Figure 5b (left
panel) presents superimposed FACS profiles of DCs treated with
5F12, J173, or anti-CD44v9 and subsequently stained for class II
(DR), CD40, and CD86 expression. Induction of these Ags on the surface
of DCs treated with HA is shown in Fig. 5b (right
panel) relative to the endotoxin control. Together, these results
indicate that ligation of CD44 by HA or anti-CD44 Abs that induce
DC aggregation leads to a concomitant up-regulation of the expression
of surface markers, reflecting enhanced cell maturation and potentially
implicated in T cell interactions.
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, a and
b), thus providing further evidence of a mature DC
phenotype. In addition, the expression of v3-, v6-, and v9-containing
CD44 variants was higher on aggregated DCs incubated in the presence of
HA (data not shown), similar to the up-regulation of these isoforms
observed during DC maturation induced by TNF- (Fig. 2). Finally,
cultures stimulated with J173, anti-CD44v9, or HA showed a marked
increase (about twofold) in the expression of the ICAM-1 adhesion
molecule compared with DCs treated with the mAb 5F12 or control IgG
(data not shown).
Modification of the immunostimulatory potential of DCs following their
in vitro maturation has been described by several authors (4, 5, 36),
and mature DCs, in particular, are more potent inducers of T cell
proliferation in MLR. To evaluate the consequences of the phenotypic
changes triggered through binding of CD44 epitopes, DCs cultured for
24 h in the presence of anti-CD44 mAbs or HA were used to
stimulate allogeneic PBLs or naive T cells. Induction of T cell
proliferation over the control level could be measured following DC
stimulation by CD44 ligands inducing aggregation, namely the J173,
anti-CD44v6, and anti-CD44v9 mAbs or HA (Fig. 6). Conversely, culture of DCs in the
presence of 5F12 or anti-CD44v3 mAbs, which did not induce DC
maturation, did not augment their stimulatory capacity compared with
that of cells cultured in the presence of control IgG (Fig. 6). Thus,
the up-regulation of DC maturation markers was accompanied by an
increase in T cell proliferation in MLR. This result together with
those presented in Figure 3 show that the binding of common or variant
CD44 domains, whether these are involved in adhesion to HA (like the
epitopes of the J173 and anti-CD44v6 mAbs) or do not bind HA (like
the CD44v9 epitope), can trigger the maturation of DCs in vitro.
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Lastly, we addressed the question of whether the binding of
surface CD44 isoforms, in particular those involved in triggering DC
maturation, could lead to the production of cytokines by DCs. This
property has previously been described for the CD44 molecules expressed
on monocytes (37), T cells (38), NK cells (12), and macrophages (39).
The pattern of cytokine production was examined in DCs cultured for
24 h in the presence of HA (50 µg/ml) or immobilized
anti-CD44 mAbs or on control plates. Cytokine transcripts were
amplified using a semiquantitative RT-PCR method (Fig. 7a). mRNAs encoding GM-CSF,
IL-1ß, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, MIP-1, and
TNF- were readily detected in all samples, including controls (Fig. 7a and data not shown), thus demonstrating the presence of
basal levels of cytokine transcripts in both aggregated and
nonaggregated DC cultures. There were no significant variations in the
levels of transcripts encoding GM-CSF, IL-1ß, IL-12p35, IL-12p40,
IL-15, MIP-1, or TNF- between different DC cultures (data not
shown). In contrast, IL-8 mRNA was clearly up-regulated in DCs
incubated with J173 or anti-CD44v6, but not in DCs incubated with
5F12 or a control IgG (Fig. 7a). Treatment with the mAb J173
also increased levels of IL-6 and IL-10 mRNAs, whereas DC stimulation
with anti-CD44v6 did not significantly modulate levels of these
transcripts (Fig. 7a and data not shown).
|
b shows the results of one experiment, representative of
eight. IL-6 and IL-10 production was strongly enhanced in DCs
stimulated with mAb J173 compared with that in DCs stimulated with a
control IgG or HA (50 µg/ml) or with control endotoxin (Fig. 7b, upper panel). In addition, IL-8 production
was up-regulated not only by incubation with J173 or HA but also by
treatment with anti-CD44v6 or anti-CD44v9 (Fig. 7b, lower panel). Whereas the up-regulation of
IL-6, IL-8, and IL-10 mRNAs in DC populations paralleled the increases
in cytokine levels in the culture medium, no such variations were
detected in IL-1ß and TNF- mRNAs. Since the production of these
latter cytokines is known to be regulated at both the transcriptional
and the post-transcriptional level (40), this may account for their
increased secretion by DCs in the absence of mRNA up-regulation.
TNF- and low concentrations of IL-1ß and GM-CSF were likewise
detected in culture supernatants of DCs stimulated by J173 or HA or by
ligation of v6- or v9-containing CD44 isoforms. IL-6 and IL-10
secretion was sometimes detectable after stimulation with
anti-CD44v6 or anti-CD44v9, but these responses were generally
weak relative to the cytokine levels obtained using J173 or HA.
However, stimulation of DCs with a combination of anti-CD44v6 and
anti-CD44v9 could increase the secretion of IL-6 and IL-10, whereas
addition of anti-CD44v3 to either anti-CD44v6 or
anti-CD44v9 did not enhance the secretion of these cytokines (data
not shown). IL-12 (p70) was secreted by DCs treated with J173 or HA,
but this cytokine was never detected in cultures stimulated with
anti-variant CD44 mAbs even when these were used in combination.
Significant induction of cytokine secretion was not observed following
treatment of DCs with 5F12 or with anti-CD44v3. Our results
therefore demonstrated that DC maturation induced by the ligation of
common or variant CD44 epitopes on the cell surface was accompanied by
cytokine secretion (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; 2) v3- and v6-containing isoforms play an important
role in DC adhesion to the ECM component HA, whereas variants carrying
v9 do not appear to contribute to this function; and 3) signaling
through CD44 can be achieved not only by binding of HA or of the
anti-pan CD44 mAb J173 to this receptor, but also by selective
stimulation of v6- or v9-containing CD44 variants. This signaling
induces DC maturation, as reflected by phenotypic changes and increased
T cell stimulatory capacity in MLR, cell aggregation in culture, and
cytokine secretion. The effects of CD44 ligation on DC phenotype and
function are summarized in Table II.
Transcripts encoding v3-, v6-, and v9-containing CD44 variants are
differentially regulated during monocyte differentiation into DCs.
Although levels of v3- and v6-containing mRNAs were relatively stable,
two transcripts containing v9 were markedly reduced in DCs compared
with fresh monocytes. One of these v9-containing transcripts was
identified as CD44v8-v10, the epithelial form of CD44 (CD44E) (22). No
changes were apparent in the general splicing pattern of CD44, as we
failed to detect DC-specific mRNA species. Maturation of DCs in the
presence of TNF- increased the surface expression of v3-, v6-, and
v9-containing isoforms compared with immature DCs, as did incubation of
DCs with LPS, another inducer of DC maturation (41) (unpublished
results). These findings are in agreement with other studies
demonstrating the modulation of CD44 expression on DCs by TNF- (5, 26). An increase in the CD44v9 epitope on the surface of mature DCs has
previously been reported by Sallusto and Lanzavecchia (5).
Interestingly, v9-containing variants are also known to be expressed by
tissue macrophages at inflammation sites (42), suggesting that these
variants may play a role in local immune responses.
Anti-CD44v3 and anti-CD44v6 mAbs were able to partially block the binding of DCs to immobilized HA, while a combination of these two mAbs almost totally inhibited DC adhesion, a blocking effect even stronger than that observed with either of the anti-pan CD44 mAbs, J173 and 5F12. Our data therefore suggest that v3- and v6-containing isoforms play a major role in the adhesion of DCs to immobilized HA. On the contrary, an anti-CD44v9 mAb was unable to block the binding of DCs to HA in our adhesion assays. The capacity of v9-containing CD44 isoforms to bind HA has long been a matter of debate (9, 10, 22), and more recently, the CD44E (v8–v10) and other v9-containing isoforms have been demonstrated to poorly bind immobilized HA (17). Moreover, other authors have reported an anti-CD44v9 mAb to be ineffective in blocking the adhesion of human DCs to frozen sections of paracortical lymph nodes, whereas anti-CD44v6 and anti-pan CD44 mAbs could block this binding (25). These results and those of our study support the hypothesis that DC adhesion to the lymph nodes could be mediated by the binding of v3- or v6-containing isoforms to HA present in this organ, but through a mechanism independent of CD44v9.
The engagement of CD44 molecules expressed on the surface of DCs, either by specific mAbs or by the ECM ligand HA, can induce their phenotypic and functional maturation. Thus, HLA class II molecules, CD40, ICAM-1, CD80, and CD86 were up-regulated and CD83 was induced on the surface of DCs stimulated by CD44 ligation, and these cells showed increased allogeneic T cell stimulatory capacity. Interestingly, only one of the two anti-pan CD44 mAbs, J173 but not 5F12, was able to stimulate DC maturation, whereas both these Abs could inhibit cell attachment to HA. Since other studies have demonstrated that CD44 undergoes conformational changes in the course of ligand binding (10), it is possible that the difference between the effects of these mAbs results from the blockage by 5F12 of a conformational change necessary for the signal transduction event. A number of investigations have implicated CD44 in signal transduction events leading to the activation of diverse cell types, and in the case of lymphocytes, CD44 bound by HA was found to behave as a costimulatory molecule (11, 13, 14, 33, 43, 44). Our study indicates that HA can in addition induce the maturation of DCs. Control experiments were performed to check that contamination of HA preparations with endotoxin was not responsible for this effect. Thus, culture of DCs on plastic dishes coated with a concentration of endotoxin equivalent to that found in HA solutions failed to trigger their maturation. Nevertheless, it is still possible that HA and endotoxin could bind to each other, resulting in increased endotoxin deposition on culture dishes in the presence of HA, or that the DC stimulatory activity of HA may be seen only in the presence of low levels of endotoxin. It should also be noted that another ECM component, type 1 collagen, has been reported to induce the maturation of DCs derived from mouse liver progenitors (45). Interestingly, CD44 modified by chondroitin sulfate has been shown to bind to type 1 collagen (10, 46), suggesting that DC maturation might also be triggered by interaction of CD44 with collagen.
Stimulation through CD44 has been shown to produce homotypic cell aggregation in a variety of hemopoietic cells (34, 35). Cell clustering, which is likewise a characteristic feature of our mature DC cultures, probably results from the up-regulation of several surface molecules mediating cell-cell interactions, and preliminary results suggest, for instance, that ICAM-1 (CD54) is one receptor involved in the formation of DC aggregates (H. Haegel-Kronenberger, unpublished observations). Previous studies have also implicated mAb binding to CD44 in the formation of clusters between blood DCs and T cells (47). In vivo, the binding of CD44 molecules present on the surface of DCs may favor both direct interactions and cooperation among DCs or between DCs and T cells. Thus, CD44v6 has been implicated in the onset of a primary immune response in the mouse (48). In addition, the standard form of CD44 has been shown to stimulate T cell proliferation when expressed by APC (49), suggesting that increased CD44 expression on the surface of DCs may modulate T cell stimulation, either through a direct effect on T cells or by inducing cytokine secretion in DCs.
The maturation of DCs could be induced by stimulation with an anti-CD44v6 or an anti-CD44v9 mAb, but not by treatment with an anti-CD44v3 mAb. This is the first report showing that variant isoforms of CD44 carrying the v6 or v9 region possess signaling activity. Variants containing v6 are therefore potentially involved in both cell attachment to HA and the triggering of DC maturation. Whether signaling through CD44v6 occurs in vivo after the binding of HA by DCs, particularly in the lymph nodes (19, 25), remains to be determined and may be of importance for the physiologic role of human DCs. In the case of v9-containing CD44 isoforms, while our results indicate that they can trigger a maturation signal in DCs, there is still no positive evidence that these variants bind HA. Thus, in contrast to the v6-containing variants implicated in both adhesion to HA and signal transduction, v9-containing isoforms may contribute only to this latter function. The possible existence of a specific ligand for CD44v9 other than HA that is capable of triggering a maturation signal in vivo remains a subject for further investigation. Finally, v3-containing CD44 isoforms seem to mediate DC attachment to HA without inducing signal transduction. This lack of a stimulatory effect might be related to the anti-CD44v3 mAb used, which could block adhesion but be unable to trigger a signal, in a manner similar to that of the anti-pan CD44 mAb 5F12. Alternatively, it is possible that the lack of signaling activity is an intrinsic feature of v3-containing CD44 isoforms. In summary, concerning the adhesive and signaling functions of CD44 variants, the results of this study point to distinct capacities of the v3-, v6-, and v9-containing isoforms expressed by human DCs to mediate either one or both of these properties.
Secretion of IL-8, IL-1ß, TNF-, and GM-CSF increased during the
maturation of DCs in response to all CD44 stimuli, namely HA and the
mAbs J173, anti-CD44v6, and anti-CD44v9. The DC maturation
observed under these conditions might well result from secretion into
the culture medium of TNF- and IL-1ß, acting in an autocrine
fashion. It is noteworthy that monocytes have been shown to secrete
IL-1ß and TNF- and that alveolar macrophages produce IL-8 in
response to CD44 triggering (37, 39). This would, in fact, appear to be
a common feature of myeloid cell lineages stimulated through CD44.
IL-6, IL-10, and IL-12 production were also triggered following CD44
ligation by HA or J173. In contrast, when v6- or v9-containing variants
were bound by mAbs, secretion of IL-6 and IL-10 was either not induced
or was induced only at low levels compared with those obtained with
anti-pan CD44 stimuli. Simultaneous stimulation with
anti-CD44v6 and anti-CD44v9 nevertheless triggered the release
of larger amounts of these cytokines. In the case of IL-12, secretion
was not observed following treatment with anti-CD44v6 or CD44v9
mAbs, either alone or in combination. Rather than a specific property
of variant CD44 isoforms, this may reflect the requirement for binding
of a greater number of CD44 molecules (and hence of the standard or
common form) on the cell surface, to trigger IL-6, IL-10, or IL-12
secretion. The pattern of cytokine release induced in our DC cultures
is characteristic of mature DC lineage cells (41, 50) and therefore
provides additional evidence for the mature phenotype of DCs stimulated
through CD44.
Our observations suggest that the in vivo engagement of CD44 epitopes
on DCs at sites of inflammation could contribute to several aspects of
the immune response. Firstly, T cell responses to antigenic stimulation
may be optimized notably through the action of cytokines involved in T
cell activation or differentiation. Thus, IL-6 secreted from skin and
lymph node DCs behaves as a T cell costimulatory signal (51). IL-10 can
skew the Th1/Th2 balance to a Th2-type response (52), while IL-12 has
been shown to act mainly as a stimulator of Th1-type responses (53),
suggesting that the balance between the IL-10 and IL-12 produced by DCs
may regulate Th responses. Secondly, the recruitment and/or
differentiation of other immune cell types, including DCs or DC
precursors, may be facilitated through the secretion of other
cytokines. In this respect, the proinflammatory chemokine IL-8 is
notably involved in the transendothelial migration of neutrophils,
monocytes, and lymphocytes (54, 55). TNF- and IL-1ß participate in
inflammatory responses and regulate the maturation of DCs (56, 57).
Moreover, TNF- can in combination with GM-CSF induce the
differentiation of DCs from CD34+ precursors (58), while
GM-CSF also plays a role in the differentiation of DCs from blood
monocytes. Thus, the triggering of surface CD44 molecules on DCs may
regulate not only the functional maturation of DCs but, more generally,
the efficiency of the immune response.
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Acknowledgments |
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Footnotes |
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2 This paper is dedicated to the memory of Professor Francis Oberling, who died on January 2, 1998.
3 Address correspondence and reprint requests to Dr. Hélène Haegel-Kronenberger, ETS, 10 rue Spielmann, BP 36, 67065 Strasbourg Cedex, France. E-mail address:
4 Abbreviations used in this paper: DC, dendritic cell; HA, hyaluronate; ECM, extracellular matrix; GM-CSF, granulocyte-macrophage CSF; rh, recombinant human; MIP-1, macrophage inflammatory protein-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Received for publication November 24, 1997. Accepted for publication June 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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