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,
,*,
Departments of
*
Microbiology and Immunology,
Pathology,
Medicine, and
Surgery, Dalhousie University, Halifax, Nova Scotia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and increased by
the addition TNF-
to the culture. These cytokines suppress or
enhance DC maturation, respectively. Increased levels of fascin
expression were found to correlate with increased APC activity in a
one-way MLR. Specific inhibition of fascin expression, using antisense
oligonucleotides, markedly reduced this APC allostimulatory activity.
These data demonstrate that fascin expression correlates with DC
maturation into APC, and it plays a significant role in the ability of
DC to function as APC. This observation is the first evidence linking
fascin-mediated dendrite formation with the APC activity of
DC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fascin is a 55-kDa, actin-bundling protein that regulates the rearrangement of cytoskeletal elements, and the interaction between the cytoskeleton and the cell membrane in response to extra- or intracellular signals (15). There is substantial evidence to link fascin expression with dendrite formation. High levels of fascin expression were found to be critical for dendrite development in neurons (16, 17). Recently, fascin expression was found to participate in the development of dendrites in mouse epidermal Langerhans cells (LC) (18). Moreover, when fascin was transfected into a pig epithelial cell line, the cells became dendritic and their motility was markedly increased (19).
Of interest is the fact that DC are the only leukocytes that have been demonstrated to express fascin (20). In the lymphoid organs only IDC, which are involved in APC activity, show high levels of fascin expression. This suggests a direct link between fascin expression and the ability of DC to function as potent APC.
In this study, we evaluated fascin expression and function during DC maturation. The data presented here demonstrate that fascin expression is tightly regulated during maturation of DC. They also demonstrate that fascin expression is critical for the Ag presentation activity of mature DC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult BALB/c and C57BL/6 mice were purchased from Charles River Breeding Laboratories (St. Constant, Quebec, Canada) and housed in the Carleton Animal Care Facility (Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Canada). All animals were housed in compliance with the guidelines established by the Canadian Council on Animal Care and were given standard rodent chow and water ad libitum. The medium used for bone marrow-derived DC (BM-DC) culture was RPMI 1640 (Sigma-Aldrich, Oakville, Ontario, Canada) supplemented with 5% heat-inactivated (30 min, 65°C) FCS (Life Technologies, Grand Island NY), 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 mM 2-ME (British Drug House, Toronto, Ontario, Canada). In this report, we referred to this medium as complete RPMI (cRPMI).
Antibodies
A panel of mAbs was used in the immunocytochemistry and flow cytometry studies. All Abs were titered, and the optimal dilution was used. The anti-fascin mAb (mouse IgG1) was a gift of Dr. Erik Langhoff, Pennsylvania State University (Hershey, PA). Anti-Ia (mouse IgG1), anti-Thy1.2 (CD90; mouse IgG2b), anti-CD11c (N418; hamster IgG), purified mouse IgG1, PE-conjugated rat IgG-2a, FITC-labeled streptavidin, FITC- and PE-labeled rabbit anti-rat IgG2a, and PE-conjugated goat anti-hamster (IgG) Abs were all purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). PE-conjugated anti-I-Ad/I-Ed mAb (rat IgG2a) was purchased from PharMingen (San Diego, CA). Alexa 488 (green) goat anti-mouse IgG conjugate was purchased from Molecular Probes (Eugene, OR). The hybridoma, GL1, producing the anti-B7-2 (rat IgG2a) mAb was purchased from the American Type Culture Collection (ATCC; no. HB-253; Manassas, VA).
Epidermal skin sheet preparation
Epidermal skin sheets were prepared essentially as previously described by Baker et. al (21). Briefly, a mouse ear was dissected and rubbed with the back of a pair of forceps to separate the two halves. The separated skin was then cut into 3- to 4-mm2 squares and incubated in 3.8% NH4SCN (Sigma-Aldrich Canada) for 30 min at 37°C. The skin was then washed in PBS, and the epidermis was separated from the dermis under a dissecting microscope. The epidermis was then fixed in cold (-20°C) acetone (BDH) for 5 min and stored in PBS at 4°C until stained. All staining of the epidermal skin sheet was performed in 1.5-ml microfuge vials (O.H. Johns, Mississauga, Ontario, Canada). The sheets were then mounted on slides, and positive cells were counted under x20 magnification power. The data is presented as the number of positive cells per mm2 of the skin sheet.
Preparation of DC from bone marrow
DC were prepared from BALB/c bone marrow essentially as
previously described (22, 23). Briefly, marrow was flushed
out of mouse femurs and tibias using sterile cRPMI in a laminar flow
hood. The marrow suspension was then passed through nylon mesh to
remove bone marrow particles. The cells were washed, and RBC were lysed
with lysing buffer, containing 155 mM NH4Cl in 10
µM Tris-HCl buffer (Sigma-Aldrich Canada) for 2 min at room
temperature. FcR-positive cells were depleted by incubation (at 37°C
for 1 h) on a Petri dish coated with IgG. After washing,
lymphocytes and Ia-positive cells were removed by incubation with a
mixture of mAbs for 60 min at 4°C followed by Low-Tox rabbit
complement (Cedarlane Laboratories) for 60 min at 37°C. The mAbs used
for depletion were GK1.5 (anti-CD4), HO2.2 (anti-CD8), B21-2
(anti-Ia), and RA3-3A1/6.1 (anti-B220/CD45R) (TIB 207, 150,
229, and 146, respectively; ATCC). Cells (5 x
106) were cultured in 50-ml flasks (Nunc,
Naperville, IL) in 5 ml cRPMI supplemented with either 50 U/ml
recombinant mouse GM-CSF (rmGM-CSF; Cedarlane Laboratories) ± 200
U/ml rmTNF- (Life Technologies), or 50 U/ml rmGM-CSF + 0.5 ng/ml
TGF- (CalBiochem-NovaBiochem, La Jolla, CA). The cultures were fed
every 3 days by aspirating 85% of the medium and adding back fresh
medium with growth factors.
Immunostaining
Immunostaining was performed on lymph node histological sections, epidermal skin sheets, or BM-DC cytospins. For cytospin preparation, 7 x 104 cells were cytocentrifuged onto poly-L-lysine (Sigma)-coated slides. Cells were then fixed in cold (-20°C) acetone for 2 min and stored at -20°C until use. For fascin staining, where mouse anti-fascin mAb was used, slides were fixed in 10% acetate-buffered formalin and incubated in a reagent (Signet Kit; IDlabs, Ontario, Canada) designed to block nonspecific binding of mouse Ab to mouse tissue. For all other staining, slides were fixed in cold (-20°C) acetone (10 min), 4% paraformaldehyde (2 min), dextran-Tris buffer (15 min), and glycine-lysine buffer (15 min). These slides were then incubated (30 min) in 3% H2O2 to block endogenous peroxidase followed by 1 h in horse serum to block nonspecific binding. Titered primary Ab or isotype control (mouse IgG1) was then added to the slides for incubation overnight at room temperature followed by the appropriate biotinylated secondary Ab for 1 h at room temperature. The Ab was localized using streptavidin-HRP (Signet Kit; IDlabs) for 1 h at room temperature and 3-amino-9-ethyl-carbozole (AEC; Sigma) as a chromogen.
Flow cytometric analysis
Cultured DC were harvested on different days and suspended in 100 µl of PBS supplemented with 1% BSA. The cell suspension was then incubated with the appropriate primary Ab. All Abs were incubated for 30 min at 4°C. Permeabilization was required to stain for fascin, which is a cytoplasmic protein. Cells were permeabilized by incubation with 100% methanol (BDH) for 30 min at room temperature. For fascin-MHC class II double staining, cells were stained with anti-fascin mAb followed by FITC-conjugated goat anti-mouse Ab. PE-conjugated anti-MHC class II mAb was then added as a second Ab. For fascin-B7-2 double staining, cells were stained with anti-fascin mAb followed by FITC-conjugated goat anti-mouse Ab. Anti-B7-2 was then added followed by a PE-conjugated rabbit anti-rat Ab. Cells were washed twice with 1% BSA-PBS after each step and fixed with 1% paraformaldehyde-PBS. Fluorescence was analyzed on a total of 10,000 cells per sample using a flow cytometer (FACScan; Becton Dickinson, Mountain View, CA).
T cell enrichment
T cells were enriched from C57BL/6 spleen by filtration through a warm (37°C for 1 h) nylon wool column as previously described (24). Briefly, spleens were homogenized to achieve a single cell suspension. After RBC were lysed (as above), the cells washed with RPMI 1640, loaded into a nylon wool column, and incubated at 37°C for 1 h. Selection of nonadherent cells was followed by anti-B220 treatment followed by incubation with rabbit Low-Tox complements (as above). T cell purity was routinely between 80 and 86%, as assessed by flow cytometry using Thy1.2 staining.
Mixed lymphocyte reaction
BM-DC were harvested on day 9 and treated with 25 µg/ml mitomycin C (Sigma) for 30 min at 37°C. Treated DC were then applied in graded doses to 2 x 105 T cell-enriched allogeneic spleen cells for 4 days. Cultures were maintained in U-shaped 96-well plates (Nunc), in 200 µl RPMI 1640 supplemented with 10% FCS, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were pulsed with 1 µCi/ml of [3H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA) in the last 18 h of incubation. T cell proliferation was assessed by harvesting the cells on filtermats using a cell harvester (Skatron, Sterling, VA) and measuring the thymidine uptake in a liquid scintillation counter (Beckman Coulter, Fullerton, CA).
Cultivation of bone marrow precursors with antisense oligonucleotides
A fascin antisense oligonucleotide (oligo) and a matched sense control oligo of the same length (17 bp) were purchased from University Core DNA Services, University of Calgary (Calgary, Alberta, Canada). The sequence of the antisense oligo was provided by Dr. J. Bryan of Baylor College of Medicine (Houston, TX; J. Bryan, unpublished observation; Refs. 16, 17). To stabilize the antisense and the control sense oligo, three base pairs at the 3' and the 5' end were phosphorothioated. The sequence of the control oligo was CCGGCACCATGACCGCC and the antisense oligo was GGCGGTCATGGTGCCGG. Bone marrow precursors were cultivated in cRPMI supplemented with GM-CSF alone, GM-CSF plus control oligo (2 µM), or GM-CSF plus antisense oligo (2 µM). The cells were fed with new media supplemented with GM-CSF ± oligo every 3 days. On day 9, DC were harvested and an MLR assay was performed to investigate the effect of inhibiting fascin expression on DC allostimulatory activity.
Image analysis
Five areas on a fascin-immunostained cytospin were randomly selected. Microscopic images on these areas were captured using a JVC model TK-1070U video camera (JVC, Scarborough, Ontario, Canada) and a Nuvista+ frame grabber board (Truevision, Indianapolis, IN) connected to a Macintosh computer. The captured images were analyzed using the software package NIH Image (website: rsb.info.nih.gov/NIH-IMAGE/) on a Power Macintosh 6100/60 computer. The result is reported as percentage of fascin staining in the total DC cell area.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LC in the skin are the classical immature DC (25, 26). In contrast, lymph nodes contain mostly mature DC that have
migrated from different interstitial sites in response to danger
stimuli (1, 2). We chose to examine fascin expression in
lymph node and skin as a first step to evaluate the correlation between
fascin expression and DC maturation. Fascin binding was detected by
immunohistochemistry using a biotinylated anti-mouse Ab as a
secondary Ab and streptavidin-HRP for visualization. The results (Fig. 1
A) reveal strong fascin
staining of DC in the T cell-dependent areas of the lymph node (IDC).
However, in the germinal center (B cell) areas of the lymph node, the
follicular DC were essentially fascin negative. This restriction of
fascin expression to IDC is similar to what has been reported
previously in human lymph nodes (27). Fig. 1B
shows the staining of the epidermal skin sheet for fascin. Very few
fascin-positive cells could be seen. Image analysis revealed that only
10 ± 17 per mm2 (n = 7)
were fascin positive in this skin sheet. However, when these sheets
were stained for MHC class II (image not shown), significant numbers of
positive cells were observed (688 ± 158 cells per
mm2).
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Fascin expression in in vitro-generated BM-DC
To confirm our in vivo observation regarding the correlation of
fascin expression with DC maturation and to investigate the functional
role of fascin in DC, we generated DC in vitro. These BM-DC mature over
7–9 days in culture, thus allowing enough time to examine fascin
expression during maturation (22). Bone marrow precursor
cells were allowed to grow for 9 days in the presence of cRPMI
supplemented with GM-CSF. This regime pushes DC precursors to mature
into DC (22, 28, 29). On day 9, cells were examined for
fascin expression by immunocytochemistry. We observed a 29-fold
increase in the percentage of cells staining for fascin at day 9 as
compared with day 0. The staining was evenly distributed throughout the
cytoplasm (Fig. 2A). Moreover,
cells that stained positively for fascin were large and displayed long
dendrites, features normally seen in mature DC. We also examined BM-DC
for MHC class II expression, a molecule known to be up-regulated during
DC maturation (8, 30). We observed that BM-DC stain
strongly positive for MHC class II, reflecting their degree of maturity
(Fig. 2B). These data demonstrated that our population of
mature BM-DC expresses both fascin and MHC class II.
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We used double color staining to compare the changes that occur in
fascin expression and MHC class II expression during DC maturation. Day
9 BM-DC were double stained for fascin and MHC class II and examined by
flow cytometry. Nearly all (98.5%) of the fascin-positive cells were
MHC class II positive (Fig. 3A). Furthermore, when we
gated on the fascin-positive cells in Fig. 3A, we found that
67.5% (square in Fig. 3A) of these cells express high
levels of MHC class II, reflecting their maturity. A similar pattern of
fascin and MHC class II double positivity was seen with day 3 and day 6
BM-DC (data not shown). In comparison to day 3, we observed 6.9- and
9.4-fold increases in fascin expression on day 6 and day 9 BM-DC,
respectively. Over the 9 days of examination, there was a direct
correlation between the level of fascin expression and MHC class
IIhigh expression with a correlation coefficient
yield of 0.98 (Fig. 3B). MHC class
IIhigh expression has been used previously as a
marker of mature DC (8, 30). To confirm that the strong
correlation between fascin and MHC class II expression was indeed
correlated with DC maturation, we examined a second marker of DC
maturity, B7-2, during BM-DC maturation. B7-2 is a costimulatory
molecule critical for T cell activation and is known to be up-regulated
during DC maturation (31). We used double color staining
to evaluate the correlation between fascin expression and B7-2
expression in mature DC. Day 9 DM-DC were double stained for fascin and
B7-2 and examined by flow cytometry. Similar to the MHC class II double
staining nearly all (96.0%) of the fascin-positive cells were B7-2
positive (Fig. 4A). B7-2 was
also up-regulated on the BM-DC with time in culture in parallel with
fascin expression (Fig. 4B). The highly significant
correlation between fascin expression and MHC class II and the
up-regulation of B7-2 expression in parallel with fascin strongly links
fascin expression with DC maturation.
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To demonstrate that the increase in fascin expression was due to
BM-DC maturation rather than simply an effect of time in culture, we
suppressed or enhanced BM-DC maturation with additional growth factors
and examined the expression of fascin and MHC class II. BM-DC
precursors were grown in cRPMI supplemented with 1) GM-CSF alone; 2)
GM-CSF plus TGF-, which has been reported to suppress DC maturation
from bone marrow progenitors (23); or 3) GM-CSF plus
TNF-, which has been shown to enhance DC maturation from bone marrow
progenitors (23, 32). Cells were harvested on days 0, 3,
6, and 9 from each group, double stained for fascin and MHC class II,
and analyzed by flow cytometry.
Regardless of the treatment group, there was an increase in fascin
expression over time and, again, most of the fascin-positive cells
(94 ± 6%) were MHC class II positive. However, the number of
fascin-positive DC was significantly (p <
0.001) reduced at day 6 (by 80%) and at day 9 (68% reduction;
p < 0.001) in the group treated with GM-CSF plus
TGF- compared with those treated with GM-CSF alone (Fig. 5). The cells in the group treated with
TGF- were small and had few mature DC-like cells when examined in
culture by light microscopy. In contrast, the number of fascin-positive
DC was significantly (p < 0.001) increased at
day 6 (by 12%) and at day 9 (26% increase; p <
0.001) in the group treated with GM-CSF plus TNF- when compared with
those treated with GM-CSF alone (Fig. 5). The number of CD11c-positive
cells in the TGF- (32%), GM-CSF alone (33%), and TNF- (34%)
groups were not statistically different (ns; p >
0.05), indicating that the overall numbers of DC were similar. Light
microscopy examination demonstrated more mature DC-like cells in
the group treated with TNF-. This clearly demonstrates that
enhancement of BM-DC maturation with TNF- increased fascin
expression. In contrast, suppression of BM-DC maturation with TGF-
markedly reduced fascin expression.
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Increased levels of fascin correlates with enhanced Ag presentation activity by BM-DC
Upon maturation, DC increase their potency at stimulating naive T cell proliferation in a MLR assay (1, 4). Because our data correlated fascin expression with DC maturation, we examined whether the increase in fascin expression would be associated with enhanced allostimulatory activity.
BM-DC were generated in the presence of different growth factor
combinations that we have shown above to alter fascin expression and
BM-DC maturation. At day 9, BM-DC were harvested from the different
groups and added in graded doses to a fixed number of naive allogeneic
T cells. T cell proliferation was then assessed in a 4-day MLR assay.
In all groups, there was an increase in T cell proliferation when DC
numbers increased (Fig. 6). DC that were
treated with GM-CSF were potent APC. However, the allostimulatory
activity of BM-DC treated with GM-CSF plus TGF-, which express a
lower level of fascin, was markedly reduced. This reduction become more
evident at a higher DC/T cell ratio, with a 91%
(p < 0.001) reduction at 1:25 ratio. In
contrast, the allostimulatory activity of BM-DC treated with GM-CSF
plus TNF-, which express a higher level of fascin, was increased by
37% (p < 0.001) at the same DC/T cell ratio
as compared with those treated with GM-CSF alone. In addition, when we
plotted the correlation between fascin-positive cells and the levels of
T cell alloactivation in MLR, we found a strong correlation between
fascin levels and T cell alloactivation with a correlation coefficient
yield of 0.97. These data demonstrate a strong correlation between the
level of fascin expression and T cell allostimulation.
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The correlation between fascin expression and allostimulatory
activity does not prove a role for fascin in Ag presentation because DC
maturation is also associated with the up-regulation of MHC class II
and costimulatory molecules. To isolate the effects of fascin on BM-DC
allostimulatory activity, we used antisense oligonucleotides to inhibit
fascin expression during maturation. Bone marrow precursor cells were
seeded in cRPMI supplemented with GM-CSF alone, GM-CSF plus antisense
oligo, or GM-CSF plus control oligo for 9 days, and then evaluated by
MLR. BM-DC viability in the no oligo (97%), control oligo (95%), and
antisense oligo (95%) treatment groups was not significantly different
(ns; p > 0.05). The change in DC morphology following
antisense oligo or control oligo treatment was assessed using double
staining with anti-fascin and anti-CD11c Abs. Consistent with
previous studies that demonstrated the role of fascin expression in
dendrite formation, antisense oligo-treated BM-DC became smaller and
had fewer dendrites, whereas control-treated DC had a normal morphology
(Fig. 7). In the control oligo-treated
group, 37 ± 1% of the CD11c+ cells were
dendritic as compared with 6 ± 0.1% in the antisense
oligo-treated group (p < 0.001). Fascin
expression in the antisense oligo-treated groups was suppressed by
70%, as judged by image analysis, when compared with control
oligo-treated group (Fig. 8A).
However, MHC class II and B7-2 expression on BM-DC were not
significantly different (ns; p > 0.05) between control
oligo- and antisense oligo-treated groups (Fig. 8, B and
C).
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). We
observed no significant difference (ns; p > 0.05) in
the allostimulatory activity between DC that had been treated with
GM-CSF alone and those treated with GM-CSF plus control oligo. However,
the allostimulatory activity of BM-DC that were treated with the GM-CSF
plus antisense oligo was markedly suppressed (p
< 0.001) compared with those treated with the GM-CSF plus control
oligo or GM-CSF alone. Similar results were observed for the fascin
antisense experiments when we used a population that contained 90%
BM-DC generated by a different protocol (33). These data
demonstrate for the first time a direct role for fascin in the
interaction between DC and T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although DC have been described as the most potent professional APC, their function in this respect is critically dependent on their degree of maturity (1, 4, 5). When DC-precursors leave the bone marrow they circulate in the blood and eventually reside in nonlymphoid tissues as immature DC. At these sites, especially at interfaces with the environment, immature DC are highly efficient in Ag uptake and processing but are poor in Ag presentation and T cell activation (7, 34). Upon maturation, DC migrate via the lymphatics to the secondary lymphoid organs and become extremely potent in Ag presentation, but poor in Ag uptake and processing (9, 10, 11). The increase in the Ag presentation ability of mature DC is associated with up-regulation of a variety of molecules, including MHC class II and B7-2, the major triggers for the initiation of T cell activation (35, 36, 37, 38, 39). DC express higher levels of MHC class II and costimulatory molecules than other professional APC. However, this alone does not account for their greater potency in activating Ag-dependent immune responses (8, 31).
It has recently been recognized that other features unique to DC enhance their ability to present Ag to T cells. All of these functions are dependent on the state of maturation of the DC. Mature DC retain MHC class II peptide complexes on their surface for prolonged periods in culture, whereas other APC have a turnover measured in hours (40, 41). Migration of DC from the periphery into the lymph node is closely regulated during DC maturation through changes in chemokine receptor expression (42, 43, 44). In the lymphoid organs only mature DC produce high levels of the DC-CK1 chemokine, which preferentially attracts naive T cells (12). These findings indicate that the changes that occur during DC maturation play a significant role in their ability to act as potent APC.
DC also undergo a variety of morphological changes during maturation, including the development of numerous long dendrites (13, 14). Dendrites are a feature common to both neurons and DC, and both cell types express a cytoskeletal protein known as fascin (16, 20). Indeed, several studies have clearly demonstrated that fascin is important for the development of dendrites (16, 18, 19). If dendrite formation is linked to APC activity then one would expect a correlation between fascin expression, dendrite formation, and APC activity. We first confirmed that the DC in the IDC compartment of the lymph node, where APC activity is highest, were fascin positive. This has been previously shown in human IDC (20), and we have here confirmed it in mice. We then confirmed that LC in the skin, which are the classic immature DC, do not stain for fascin.
The in vivo demonstration of fascin expression in mature DC, and not in immature DC, suggests a link between maturation and fascin expression. To confirm this association we examined DC maturation, in vitro, from bone marrow precursors. We found that fascin was expressed in mature DC as evaluated by morphology. Fascin expression was also correlated with the up-regulation of MHC class II and B7-2. These data establish an association between fascin expression and maturation and a tentative link with APC activity. To directly relate this to APC activity of mature DC we performed MLR to assess APC activity under different conditions of reduced fascin expression. We found a strong correlation between the level of fascin and the ability of DC to activate T cells in MLR. More specifically, fascin antisense oligonucleotides effectively reduced fascin expression by 70%, and the cells treated with these oligonucleotides showed reduced alloactivation. This reduced allostimulatory effect was not due to changes in MHC class II or B7-2 expression or reduced DC viability. Therefore, fascin is another DC protein regulated during maturation that is critical for APC activity. These data provide the first evidence suggesting that dendrite formation plays a functional role in the interaction between DC and T cells.
Although we have shown that fascin expression is directly involved in enhancing the ability of DC to activate T cells, the exact mechanism underlying this process is not fully understood. There are a number of explanations that might account for this observation. Fascin expression resulting in dendrite formation may increase the DC surface area, and this may favor interaction with a greater number of T cells. However, it may also be a much more active process. Fascin expression in epithelial cells results in active extension of lamellipodia (19). Likewise, fascin might permit coordinated extension of dendrites maximizing the surface contact area between DC and T cells (15).
Cell polarity may be another mechanism by which cytoskeletal proteins influence APC-T cell interactions. Recently, it has been demonstrated that rearrangements of the T cell actin cytoskeleton result in clustering of TCR molecules, thereby enhancing TCR cross-linking (45). These changes result in sustained T cell signaling, which is an important step in T cell activation (46). Through its actin-bundling function fascin may induce a similar rearrangement of MHC molecules on the DC contributing to the immunological synapse that develops between APC and T cells. Cell polarity may also result in the directional secretion of cytokines by DC as has been demonstrated for T cells (47, 48). Finally, fascin is important in the motility and migration of cells (15, 19). Migration of DC to lymph nodes during their maturation is critical for the generation of the immune response, and this may be influenced by fascin expression.
In summary, this study clearly demonstrates that fascin is expressed in DC upon maturation. More importantly, it indicates that fascin expression in mature DC is critical for their generation of dendrites and their ability to activate T cells. This observation is the first evidence linking dendrite formation with the ability of DC to activate T cells. Although the exact nature of this interaction remains to be elucidated, further studies of the mechanisms that control fascin expression in DC may improve our understanding of the interaction between DC and T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kenneth A. West, Department of Medicine, Suite 5087, Dickson Building, 5820 University Avenue, Halifax, Nova Scotia, B3H 2Y9 Canada.
3 Abbreviations used in this paper: DC, dendritic cell(s); BM-DC, bone marrow-derived DC; IDC, interdigitating DC; LC, Langerhans cell(s); cRPMI, complete RPMI; rm, recombinant mouse.
Received for publication July 6, 1999. Accepted for publication October 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and TNF- on the development of dendritic cells from progenitors in mouse bone marrow. Stem Cells 15:144. cooperate in the generation of dendritic Langerhans cells. Nature 360:258.[Medline]
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 N. Setterblad, V. Blancheteau, A. Delaguillaumie, F. Michel, S. Becart, G. Lombardi, O. Acuto, D. Charron, and N. Mooney Cognate MHC-TCR interaction leads to apoptosis of antigen-presenting cells J. Leukoc. Biol., June 1, 2004; 75(6): 1036 - 1044. [Abstract] [Full Text] [PDF]
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M. M. Al-Alwan, R. S. Liwski, S. M. M. Haeryfar, W. H. Baldridge, D. W. Hoskin, G. Rowden, and K. A. West Cutting Edge: Dendritic Cell Actin Cytoskeletal Polarization during Immunological Synapse Formation Is Highly Antigen-Dependent J. Immunol., November 1, 2003; 171(9): 4479 - 4483. [Abstract] [Full Text] [PDF]
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M. Bros, X.-L. Ross, A. Pautz, A. B. Reske-Kunz, and R. Ross The Human Fascin Gene Promoter Is Highly Active in Mature Dendritic Cells Due to a Stage-Specific Enhancer J. Immunol., August 15, 2003; 171(4): 1825 - 1834. [Abstract] [Full Text] [PDF]
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N. Setterblad, C. Roucard, C. Bocaccio, J.-P. Abastado, D. Charron, and N. Mooney Composition of MHC class II-enriched lipid microdomains is modified during maturation of primary dendritic cells J. Leukoc. Biol., July 1, 2003; 74(1): 40 - 48. [Abstract] [Full Text] [PDF]
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M. I. Zimmer, A. T. Larregina, C. M. Castillo |
), GM-CSF plus TNF-
). The cells were harvested on days 0
(without growth factors), 3, 6, and 9, permeabilized, and double
stained for fascin and MHC class II. This figure was obtained from flow
cytometric analysis and represents the number of fascin-positive cells
of 104 collected. The results are expressed as mean of
positive cells ± SE and are representative of three independent
experiments. Statistical significance was determined using one-way
ANOVA where ** indicate a p value of <0.01 and
*** indicate a p value of <0.001.
), or antisense oligonucleotide (