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Clinical Research Unit, Department of Dermatology, Johannes Gutenberg University, Mainz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ZAP II containing 18 x 106 independent
clones. This library was screened with freshly isolated Langerhans cell
(fLC)- and cLC-derived probes for cLC-specific cDNAs. The cDNAs
identified were sequenced and analyzed by database searches. Two cDNA
fragments were identified as fragments of fascin, indicating that
fascin is differentially expressed in LC. By competitive RT-PCR, we
confirmed that fascin is highly expressed in cLC cultivated for 1, 2,
and 3 days, while no signals were obtained with fLC. Western blot and
immunofluorescence analysis revealed cLC-specific expression of fascin
on the protein level as well. Fascin is known to be involved in the
organization of the actin cytoskeleton in cytoplasmatic extensions of
nerve growth cones. Its differential expression in maturing LC
coincides with the formation of numerous dendritic projections in LC.
Their formation was inhibited by incubation of LC with fascin antisense
oligonucleotides during cultivation. Therefore, we conclude that fascin
is necessary for the formation of the dendritic processes of maturing
Langerhans cells and may thus influence T cell-LC interaction. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The structural and functional changes that LC undergo during this process are numerous, and some of them pose special demands on the cytoskeleton: before activation, LC reside within the epidermis in contact with surrounding keratinocytes; once activated, LC leave their position and migrate actively to the draining lymph nodes. During movement they retract the few, but very long dendritic processes. Finally, within the lymph nodes they elaborate multiple thin projections. A network of actin filaments associated with actin-binding proteins is important for each of these processes, namely for movement and cell shape as well as for cell attachment (6). The attachment of LC to surrounding cells within the epidermis is mediated by homodimerization of E-cadherin molecules (7). E-cadherin is anchored within the cell to the actin cytoskeleton via actin-binding proteins (8). Activation and emigration of LC is associated with down-regulation of E-cadherin surface expression (9).
The three-dimensional network of actin filaments is structured by interaction of actin filaments with capping proteins and by cross-linking of the filaments via actin-binding proteins. A growing number of actin-binding proteins such as actinin, gelsolin, and villin has been identified, and differential expression of subsets of actin-binding proteins is necessary for specialized functions and structures (10, 11).
Ample evidence exists that the 55-kDa actin-bundling protein fascin organizes actin filaments into bundles (12, 13). It is, furthermore, likely to be involved in formation of dendritic projections, as it was detected in cells with dendritic processes such as neuronal growth cone filopodia and human blood-derived DC (14, 15). Here, we report that fascin is differentially expressed during maturation of murine LC. The formation of pronounced and numerous dendritic projections coincided with strong fascin expression and was impaired by fascin antisense oligonucleotides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c Ann mice were bred in our animal facilities from breeding pairs originally obtained from the Zentralinstitut für Versuchstierkunde (Hannover, Germany). The mice were used at 2 to 5 mo of age.
Monoclonal Abs
Clone 2G9 (anti-I-Ad,b, I-Ed, rat IgG2a) (16) was a kind gift from Dr. M. Mohamadzadeh, Department of Dermatology, University of Mainz, Germany. 55K-2 mAb (anti-human fascin, mouse ascites) (17) was a kind donation by Dr. F. Matsumura, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ. Isotype control mAb were obtained from PharMingen, Hamburg, Germany.
Cell lines
The Th line BK-OVA-1R (18) recognizes OVA in the context of
A
bAßk MHC class II molecules. The
murine B cell lymphoma A20/2J (19), the macrophage cell line P388D1
(20), the fibroblast line WEHI-164 (21), and the melanocyte line B78
(22) were cultured as described before. Cells were harvested and used
directly for mRNA preparation or Western blots, or alternatively, cell
pellets were shock frozen in liquid nitrogen and stored at -70°C for
later analysis.
Preparation of fLC and cLC
Epidermal cell suspensions were prepared from pelts as described previously (23). They were used for LC enrichment either directly or following in vitro cultivation for 1 to 3 days in Iscove’s modified Dulbecco’s medium (IMDM, Life Technologies, Eggenstein, Germany) supplemented with 2 mM L-glutamine, 5 x 10-5 M 2-ME, 100 IU penicillin, 100 µg/ml streptomycin, 12.5 µg/ml amphotericin B, 500 µg/ml gentamicin, and 10% FCS in 175 cm2 tissue culture flasks (Techno Plastic-Products, Trasadingen, Switzerland) (24).
LC were enriched from epidermal cells by immunomagnetic separation with
Dynabeads M-450 (Dynal, Hamburg, Germany) loaded with anti-MHC
class II mAb 2G9 as described (24). MHC class II-positive, bead-coupled
LC and cells without attached beads were counted using a Neubauer
chamber. LC purity of 
92 to 95% was obtained as determined by the
ratio of bead-rosetted to nonrosetted cells. LC viability, determined
by trypan blue exclusion, was >95%.
Mixed epidermal cell lymphocyte reaction
The mixed epidermal cell lymphocyte reaction was conducted as described (24). Briefly, nylon wool-enriched (25) splenic C57BL/6 T cells were stimulated with titrated numbers of allogeneic fLC or cLC of BALB/c origin. T cell proliferation was measured by incorporation of 28 kBq [3H]thymidine into cellular DNA during the last 12 h of a 96-h incubation period.
cDNA library construction
A cDNA library derived from cLC was constructed in ZAP II
(26). mRNA was isolated from cLC (purity, 95% rosetted cells) using
the QuickPrep mRNA Purification Kit (Pharmacia, Freiburg, Germany).
Double-stranded cDNA was obtained from 5 µg mRNA using the Time Saver
cDNA synthesis kit (Pharmacia) and ligated with
EcoRI/NotI adaptors (Pharmacia) according to the
recommendations of the manufacturer. The adaptor-flanked cDNA fragments
were ligated with dephosphorylated, EcoRI-digested ZAP
II vector arms (Stratagene, Heidelberg, Germany) and were in vitro
packaged using the in vitro packaging kit Gigapack II Gold
(Stratagene).
Preparation of screening probes from fLC and cLC
The cDNA probes were prepared as we described recently (27). Briefly, double-stranded cDNA was obtained from approximately 100 ng mRNA derived from 106 fLC or cLC using the Time Saver cDNA synthesis kit (Pharmacia), ligated with Uni-Amp adaptors (Clontech Laboratories, Palo Alto, CA) and amplified by PCR in 25 cycles with 1 min denaturation at 94°C, 1 min annealing at 60°C, and 2 min extension at 72°C. The radioactive labeling of the double-stranded reamplified cDNAs was performed by random-primed oligo labeling using the Multiprime DNA labeling system (Amersham Buchler, Braunschweig, Germany).
Differential plaque filter screening
Bacteria and recombinant phages were plated on 24.5 x 24.5 cm culture dishes (Nunc, Roskilde, Denmark) in concentrations allowing the distinguishing of single plaques following cultivation overnight. The phages were transferred to nitrocellulose filters, and replicate filters from each plate were treated and hybridized according to Benton and Davis (28). One replicate filter was hybridized with a fLC-derived probe prepared as outlined above, while the corresponding filter was hybridized with a cLC-derived probe. Following autoradiography, the hybridization patterns were compared, and phages yielding cLC-specific signals were picked and replated. Replicate filters were rescreened with fLC- and cLC-derived probes.
Southern hybridization and sequencing
The internal pBluescript SK(-) plasmid containing the
cLC-specific cDNA was recovered from pure, cLC-specific, recombinant
ZAP II phages by in vivo excision (26). The plasmid DNA, purified
using the Plasmid Kit Midi (Qiagen, Hilden, Germany), was restricted,
subjected to Southern hybridization (29), and sequenced using the T7
Sequencing Kit (Pharmacia) with vector-specific primers (T3A: AAT AAC
CCT CAC TAA AGG G; and T7A: AAT ACG ACT CAC TAT AGG G) according to the
recommendations of the manufacturer.
Competitive RT-PCR
mRNA was isolated using the Quick-Prep Micro mRNA Purification Kit (Pharmacia). The RNA concentration and quality were assessed by RT-PCR with primers specific for hypoxanthine-guanine phosphoribosyltransferase (HPRT-1: GGT TGG ATA CAG GCC AGA CTT TGT TG and HPRT-2: GAA GGG TAG GCT GGC CTA TAG GCT). The primers FAS-1 (CCC GCC TCA CTC TGG GAG TA) and FAS-2 (CTC CTA GGC CAC TTC CTG GG) were chosen to amplify a 329 bp fragment of fascin cDNA. For competitor construction, the hybrid primers FASST-1 (CCC GCC TCA CTC TGG GAG TAC CGC TCT AGG CAC CA) and FASST-2 (CTC CTA GGC CAC TTC CTG GGG TAC GAC CAG AGG CA) were used. All primers were generated by MWG-Biotech (Ebersberg, Germany).
RT-PCR was performed as described previously (30) using a model 480 DNA Thermal Cycler (Perkin-Elmer, Ueberlingen, Germany). The competitor fragment (365 bp) was constructed as we described earlier (30). It is flanked by the primer-binding sites for FAS-1 and FAS-2, but has no further sequence homology to fascin. For quantitative analysis of the PCR reactions, equal amounts of the competitor fragments were added to each PCR reaction (30).
Western blotting
Tissues were shock frozen, pulverized under liquid nitrogen in a mortar, and resuspended in SDS sample buffer. Following ultrasonic treatment, protein concentration was determined by Bradford assay (Bio-Rad, München, Germany). Aliquots equivalent to 105 cells or 5 µg protein, respectively, per lane were separated on 15% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Bound primary Ab (anti-human fascin mAb 55K-2) was visualized by horseradish peroxidase-labeled anti-mouse Ig Ab using the BM Chemiluminescence Western Blotting Kit (Boehringer Mannheim, Mannheim, Germany) following the recommendations of the manufacturer.
Immunofluorescence
For fluorescence staining, epidermal cells were grown on coverslides or were transferred onto glass slides by centrifugation (cytospin) for 1 min. Epidermal cells for cytospins were either used directly or following three day cultivation and Ficoll density centrifugation (Histopaque, density 1.077; Sigma, Deisenhofen, Germany). All preparations were fixed for 10 min in 90% aceton at -20°C, washed three times in PBS, and incubated overnight at 4°C or 3 h at 37°C with the primary mAb (55K-2 and 2G9). Following three washes in PBS, cells were incubated for 1 h at 37°C with dichlorotriazinyl aminofluorescein (DTAF)-conjugated goat anti-mouse IgG (0.75 µg) and with phycoerythrin-labeled goat anti-rat IgG (0.5 µg) secondary Abs (both from Dianova, Hamburg, Germany).
Cultivation of LC with antisense oligonucleotides
Epidermal cell suspensions were prepared from pelts (23) and cultivated as described on 8-well or 16-well chamber slides (Nunc). Cells were cultivated either without addition of oligonucleotides or with fluorescein-labeled (0.5 µM) or unlabeled (2 µM) phosphorothioate oligonucleotides. An antisense oligonucleotide (FAS), the reverse complement to the murine and human fascin mRNA, was used as well as a matched control oligonucleotide of the same length (15 bp) and base content, which had similar hybridization characteristics and showed no cross-homology to any known gene. The HPLC-purified oligonucleotides were purchased from Biognostik, Göttingen, Germany. Oligonucleotides were added to epidermal cell suspensions at the beginning of the 3-day cultivation period.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction, validation, and differential screening of a
cLC-derived cDNA library in ZAP II
cLC (5 x 107, purity > 95% rosetted
cells) were subjected to mRNA isolation. The integrity of the mRNA was
analyzed by Northern blotting and hybridization with ß-actin cDNA
probes. A clear, distinct signal was obtained (data not shown). Five
micrograms of mRNA was used for generation of double-stranded cDNA,
subsequent ligation into ZAP II, and in vitro packaging.
Determination of the phage titer of the cDNA library indicated that the
library contained 1.8 x 107 independent clones. Based
on blue/white-screening, we estimated that 90% of the clones were
recombinant. Five randomly picked clones contained cDNA fragments of
300 to 1900 bp. By plaque filter hybridization of 3000 clones with
ß-actin cDNA probes, we obtained eight ß-actin cDNA clones (not
shown).
Approximately 20,000 clones of the cLC-derived cDNA library were plated
onto 24.5 x 24.5-cm culture dishes; replicate filters were taken
and hybridized with fLC- or cLC-derived probes, respectively, that had
been validated before, as described earlier (27). The hybridization
patterns of the replicate filters with fLC- and cLC-derived probes were
compared. Fifty clones yielding strong signals with cLC-derived probes
and weak or no signals with fLC-derived probes were selected, replated,
and rescreened (Fig. 1
). Thirty-eight
clones yielded cLC-derived signals.
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The pBluescript phagemids containing the cLC-specific cDNAs were
recovered by in vivo excision from all 38 isolated clones. Plasmid
DNA was isolated and digested with EcoRI. The restriction
pattern revealed similar patterns for a number of clones. Therefore,
one of them was radioactively labeled and subjected to Southern
hybridization with blots of all 38 clones. Twenty-two clones yielded
strong signals indicating that homologous or identical cDNAs were
cloned. Two of the hybridizing clones and the 16 remaining
nonhybridizing cDNAs were sequenced from both ends with the
vector-specific primers T3A and T7A.
Comparison of the sequences obtained with the Geall databases
(Deutsches Krebs-Forschungszentrum, Heidelberg, Germany) revealed that
the cross-hybridizing clones contained identical cDNAs of a
so far unknown gene. Two of the remaining cDNAs were identical with the
murine fascin cDNA (accession No. L33726). The clone pcLC12 contained a
nearly complete cDNA of 2587 bp, while clone pcLC5 contained 1221 bp of
the 3' end (Fig. 2A).
Fascin was selected for further investigation, as a mAb was available
(17), its ability to organize actin into bundles was known (12, 13),
and its functional role in organizing the actin cytoskeleton promised
to be of importance.
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To analyze at what time point during maturation of LC fascin
expression is induced, we performed competitive RT-PCR using mRNA
isolated from equal numbers of fLC and from cLC cultivated for 1, 2,
and 3 days. Equal amounts of mRNA were subjected to reverse
transcription, and aliquots were used for PCR with specific primers for
the housekeeping gene, HPRT. Signals of equal intensity were obtained
indicating comparable amounts of mRNA (Fig. 2
C).
Aliquots of the same RT reaction were used for PCR with fascin-specific
primers. Serial dilutions of the RT reaction were mixed with a constant
amount of competitor molecules. The competitor molecules were generated
as we described recently (27). They are flanked by the primer-binding
sites for the fascin primers but have no further homology to fascin to
minimize cross-hybridizations. Following gel electrophoresis,
competitor-derived (upper bands; 365 bp) and
cDNA-derived (lower bands; 329 bp) fragments were
distinguishable by size. As shown in Figure 2B, no fascin
signals were obtained with fLC, while strong signals were obtained with
cLC cultivated for 1, 2, and 3 days. The signal intensities on days 1
through 3 were similar.
Detection of fascin protein by immunofluorescence analysis in cLC, but not in fLC
Freshly isolated or 3-day-cultured epidermal cells were
transferred onto glass slides by cytospin. Double fluorescence labeling
for MHC class II (red) and fascin (green) revealed that fLC, identified
by bright MHC class II staining, were negative for fascin staining,
while cLC were clearly positive (Fig. 3).
MHC class II-negative cells, either freshly isolated or cultivated,
were negative for fascin staining. With isotype-matched control Ab, no
staining was observed.
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a). The entire cells,
including all dendritic processes, stained clearly positive for fascin
(Fig. 4b).
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Extracts of equal numbers of fLC and cLC separated by paramagnetic
beads coupled with anti-MHC class II mAb, as well as equal amounts
of protein derived from brain, liver, spleen, and kidney, were
subjected to Western blotting. Fascin was detected by mAb 55K-2 only in
extracts of cLC, but not of fLC (Fig. 5).
The estimated molecular mass (approximately 55 kDa) of the
corresponding protein is identical with that of fascin. In addition, a
prominent fascin signal was obtained with brain extract (Fig. 5), which
is compatible with the notion that fascin is highly expressed in
neurons and glial cells (31). A strong signal was obtained with splenic
cells as well. Weak signals were obtained with kidney and liver cells.
Among a panel of cell lines including B cells, T cells, macrophages,
fibroblasts, and melanocytes, only the fibroblast cell line yielded a
fascin signal (Fig. 5). No signals were obtained with isotype control
Abs (not shown).
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Epidermal cells were cultivated in 8- or 16-well chamber slides,
with or without addition of phosphorothioat oligonucleotides, for 3
days. The class II-positive cLC were then immunostained with mAb 2G9.
Incubation with 2 µM FAS oligonucleotides (reverse complement to
murine and human fascin mRNA) clearly inhibited the formation of
dendritic projections (Fig. 6c) compared with
untreated cells (Fig. 6a). In parallel experiments, 2
µM control oligonucleotides were added to rule out cytotoxic or
nonspecific effects of the antisense oligonucleotides (Fig. 6b). Cellular uptake was monitored by addition of 0.5
µM fluorescein-labeled oligonucleotides (Fig. 6d).
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, category 1 cLC outnumbered
category 2 cLC by a factor of 4, when the epidermal cells had been
cultured without addition of oligonucleotides. In cultures to which
control oligonucleotides had been added, a factor of 3.2 resulted. In
contrast, when FAS antisense oligonucleotides were present during
cultivation of epidermal cells, category 1 cLC were outnumbered by
category 2 cLC (factor of 0.2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results obtained by differential screening were confirmed by investigations on the mRNA and protein level, indicating that the screening procedure and the probes generated for screening were reliable. While fascin was not detectable on the mRNA level in fLC, we detected significant expression of mRNA by quantitative RT-PCR already on day 1 and comparable transcript levels on day 2 and 3 of culture. For detection of fascin expression on the protein level, mAb 55K-2 was used (17). Although generated against human fascin, this mAb cross-reacts with the homologous proteins of mouse and Xenopus, as the gene is highly conserved (32). Differential expression of fascin mRNA was paralleled by differential expression of fascin protein. The data indicate that fascin expression is regulated on the mRNA level and that fascin may be required early after activation as well as at later time points. Expression of fascin in mouse cLC is compatible with results published recently by Mosialos et al. (15) and Pinkus et al. (33) showing that fascin is highly expressed in human DC. Furthermore, we observed high levels of fascin in spleen, known to harbor elevated numbers of mature DC. Weak fascin signals with cells derived from kidney and liver may be due to DC resident in these organs. Whether DC contribute to fascin expression in these organs has to be shown in further studies.
Many blood-derived or skin-derived cell types do not express fascin. Thus, fascin was not expressed in fLC and in freshly isolated and in cultured MHC class II-negative epidermal cells, as indicated by immunofluorescence, and was not detectable by Western blotting in several murine cell lines including B cells, T cells, macrophages, and melanocytes.
Fascin expression is, however, not restricted to DC. It was reported to be expressed in fibroblasts, neurons, glial cells, and in EBV-transfected B cells, but not in untransfected T cells and B cells (17, 31). Accordingly, we obtained strong fascin signals with murine brain cells and with the murine fibroblast cell line WEHI-164. Nevertheless, lacking deal markers for DC, fascin may serve as a marker molecule for DC, taking into account its strong expression in cLC and its absence in many other blood- and skin-derived cell types. Furthermore, fascin may serve as a marker for maturing LC and possibly other DC.
The actin cytoskeleton is essential for a number of important cellular functions including motility (34, 35), exocytosis (36), phagocytosis (37), formation of stress fibers (34), activity of ion channels and transport proteins (38), cell surface receptor perturbation (39), cell polarity, and contact to extracellular matrix components (40). It is not yet clear whether fascin is involved in these processes. Fascin organizes actin into bundles in vitro, and Drosophila singed mutants deficient in the Drosophila fascin homologue show that fascin is required for proper bristle formation as well as formation of actin filaments in nurse cells during oogenesis (12). The limited expression of fascin and the differential expression in LC eliminate the possibility that it might be of crucial importance for most of the processes listed above, e.g., fLC show phagocygotic activity despite lack of detectable fascin expression. Fascin may be involved in formation and/or motility of the newly shaped dendritic processes. It might not be merely coincidence that the cells with the most elaborated projections, namely DC and neurons, express fascin. In neurons, fascin protein is not detectable in axons; it is restricted to the cytoplasm of the cell body and expressed particularly in the dendritic processes of the nerve growth cone (14). The latter strikingly resemble dendritic projections of cLC and other DC in shape and are mobile as well. In neurons, this movement might be necessary in the course of the pathfinding and growth of the axon to its destination. In DC, this active movement allows DC to attach to T cells and to interact with them closely. Fascin is expressed within the dendritic projections of cLC. As these cover the entire surface of the cell, there are no regions that might be devoid of fascin.
The fact that anti-fascin oligonucleotides impair the dendritic morphology of cLC strongly supports the view that fascin is pivotal for the formation of numerous dendritic projections of maturing LC. It is unlikely that the impaired dendritic morphology is due to unspecific effects of the antisense oligonucleotides, as the number of cells with dendritic morphology is only slightly reduced by the same concentration of control oligonucleotides. As the dendritic projections are likely to support and enhance the LC-T cell contact, fascin could be involved indirectly in the process of Ag presentation and T cell stimulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. B. Reske-Kunz, Klinische Forschergruppe Allergie, Universitäts-Hautklinik/Verfügungs-gebäude, Obere Zahlbacher Strasse 63, D-55131 Mainz.
3 Abbreviations used in this paper: DC, dendritic cell(s); fLC, freshly isolated Langerhans cell(s); cLC, cultured Langerhans cell(s); DTAF, dichlorotriazinyl aminofluorescein; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
Received for publication September 17, 1997. Accepted for publication December 19, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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X174 x HaeIII. C, The concentration and
quality of the mRNA used in B, derived from fLC (d0) and cLC
cultivated for 1 (d1), 2 (d2), or 3 (d3) days, was assessed by RT-PCR.
Aliquots of the same RT reaction as described in B were
amplified with specific primers for the housekeeping gene, HPRT. c,
Indicates negative control without mRNA; m, molecular weight marker
