1 University Medical Clinic, Section for Transplantation Immunology and
Immunohematology, Tübingen, Germany
2 Department of Hematology and Oncology, Children's Hospital, University of
Tübingen, Germany
* Author for correspondence (e-mail: gerd.klein{at}uni-tuebingen.de)
Accepted 2 September 2002
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Summary |
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Key words: Thymus, Cell-to-cell interactions, Cellular differentiation, Cell adhesion molecules, Cadherins
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Introduction |
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The cell-cell adhesion molecule family of classical cadherins consists of
more than 15 members (Kemler,
1992; Angst et al.,
2001
). Cadherins are not only critically involved in the
maintenance of tissue architecture and embryonic development but also in
cellular signalling processes (Knudsen et
al., 1998
). However, this gene family has not gained much
attention in thymocyte development, although several cadherins, including
E-cadherin, have been detected on murine thymocytes
(Munro et al., 1996
). The
classical type I cadherins, to which E-, N- and P-cadherin belong, mediate
homophilic adhesive interactions; however, a heterophilic interaction of
E-cadherin with the integrin
E(CD103)ß7 has
been recently determined as an exception to the rule of homophilic cadherin
interactions (Cepek et al.,
1994
; Higgins et al.,
1998
). The integrin
Eß7 is
expressed on intraepithelial T lymphocytes of the skin and gut, but it is also
found on a very small percentage of peripheral blood lymphocytes and
developing T cells in the thymus (Andrew et
al., 1996
; Agace et al.,
2000
; Pauls et al.,
2001
). Analysis of CD103-deficient mice indicated an important
role for
Eß7 in the localisation of mucosal
T lymphocytes (Schön et al.,
1999
). Although the localisation of cutaneous lymphocytes is not
affected in these knockout mice, the
Eß7
deficiency seems to be a risk factor for inflammatory skin diseases
(Schön et al., 2000
).
Whether the development of thymocytes in CD103-deficient mice is altered or
impaired has not been reported so far.
A functional role for E-cadherin in the fetal thymus was determined in
murine fetal thymic organ cultures
(Müller et al., 1997).
E-cadherin has been shown to be expressed by murine thymic epithelial cells as
well as thymocytes at embryonic days 14 to 18, but not on thymocytes of adult
animals (Lee et al., 1994
).
Antibodies against E-cadherin-mediated homophilic interactions blocked
epithelial organisation and early thymocyte development in reaggregate fetal
thymic organ cultures, whereas an antibody selectively inhibiting interactions
of E-cadherin with the integrin
Eß7 did not
interfere with these processes in the embryonic tissue
(Müller et al., 1997
).
The functional significance of the adhesion pair E-cadherinintegrin
Eß7 in the postnatal murine thymus, however,
has not been investigated.
In the present study we have analysed the expression of E-cadherin in human
thymic tissue. Since E-cadherin was not detected on human postnatal thymocyte
subpopulations, the expression pattern of CD103 on human thymocyte
subpopulations was studied in detail. Functional analyses, including cell
adhesion and proliferation studies, were performed to determine the role of
E-cadherin and its ligand Eß7 integrin
during human thymocyte development.
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Materials and Methods |
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Antibodies
Two different monoclonal antibodies against human E-cadherin were used in
this study. The antibody HECD-1 (R&D Systems, Wiesbaden, Germany) was used
for immunoprecipitation, flow cytometric analysis and adhesion blockade,
whereas the antibody 67A4, which was recently characterised as another human
E-cadherin-specific antibody (Bühring
et al., 1996), was used for immunoblotting and
immunohistochemistry. The anti-P-cadherin antibody NCC-CAD 299
(Shimoyama et al., 1989
) was a
kind gift of Dr Hirohashi (National Cancer Center Research Institute, Tokyo,
Japan). Two polyclonal rabbit antisera against human
- und
ß-catenin were purchased from Sigma (Deisenhofen, Germany). Monoclonal
antibodies generated against
-, ß- or
-catenin were
purchased from Transduction Laboratories (
and ß; BD Biosciences,
Heidelberg, Germany) or Sigma (ß and
). The mouse monoclonal
anti-CD103 antibody (clone 2G5.1) was obtained from Serotec (Biozol, Eching,
Germany). Medullary thymic epithelial cells were specifically labelled with
the monoclonal antibody TE4 (IgM) obtained from the American Type Culture
Collection (ATCC; Manassas, VA).
Isolation of thymocyte subpopulations by magnetic cell sorting
Using the MACS CD4 Multisort kit (Miltenyi Biotec, Bergisch Gladbach,
Germany), CD4- CD8- double-negative (DN) thymocytes,
CD4+ CD8+ double-positive (DP) thymocytes, and the
CD4+ and CD8+ single-positive (SP) thymocyte cell
populations could be sorted according to the manufacturers' instructions.
Briefly, thymocytes were collected by density-gradient centrifugation on a
Ficoll® cushion and incubated with CD4 Multisort CD4 microbeads for 30
minutes. After washing with 5 mM EDTA and 0.5% BSA in PBS, the labelled cells
were separated on magnetic columns. Positively selected thymocytes, which were
retained on the magnetic columns, contained the CD4+ SP and the
CD4+ CD8+ DP cell populations, whereas the CD4-depleted
cell population, which ran through the columns, contained the CD8+
SP and the CD4- CD8- DN thymocytes. To remove the CD4
microbeads from the CD4-positively selected cell populations, the cells were
incubated with MACS Multisort release reagent. After 20 minutes, the digestion
was stopped, and the cells were labelled for 30 minutes with CD8 microbeads.
CD4+ CD8+ DP thymocytes were obtained by positive
selection, whereas CD4+ SP cells were found in the depleted cell
population.
The CD4-depleted cell population was incubated for 30 minutes with CD8 microbeads. After applying the labelled cells on magnetic columns, CD8+ SP cells could be separated from the double-negative thymocytes. The purities of the four different thymocyte subpopulations were controlled by flow cytometric analysis.
For the isolation of CD103- CD8+ SP thymocytes, the CD4-depleted thymocyte cell population (which contains CD8+ SP and CD4- CD8- DN thymocytes) was incubated for 1 hour with the anti-CD103 antibody 2G5.1, which is of Ig2a isotype. Rat-anti-murine Ig2a+b microbeads (Miltenyi) were applied as secondary antibodies. After magnetic separation CD103- CD8+ SP and CD103- CD4- CD8- DN thymocytes were found in the run-through of the columns. Following incubation with CD8 microbeads CD103- CD8+ thymocytes were obtained by positive selection.
Isolation of primary thymic epithelial cells
Thymic tissues were cut with a scalpel into small pieces of about 1
cm3 and digested at 37°C with 1 mg/ml collagenase A and 50
µg/ml DNase I in RPMI 1640 medium for 45 minutes (Roche, Mannheim,
Germany). After this digestion step, the tissue fragments were collected by
gravity sedimentation. The supernatants contained mainly thymocytes, which
were discarded. The tissue fragments were digested a second time with
collagenase A and DNase I for 30 minutes. The cell suspension obtained after
the second digestion was washed with RPMI 1640 medium. Then, by repeated 5
minutes gravity sedimentation, the thymic epithelial cells could be isolated
from thymocytes, which were found in the supernatants. The combined thymic
epithelial cells were washed with PBS and cultured in serum-free
OptiMemTM medium (GIBCO-BRL, Eggenstein, Germany) on Petri dishes coated
with collagen type I (Roche). The serum-free medium was changed every third
day. After one week of culture, the first clusters of adherent thymic
epithelial cells could be observed, which could be expanded to almost
confluent monolayers by extended culture periods of up to eight weeks.
Immunohistochemistry
Specimens of human thymuses were frozen in Tissue Tek embedding medium
(Vogel, Gießen, Germany) and stored at -70°C until use. 5 µm
thymic cryostat sections, as well as primary thymic epithelial cells grown on
eight-well chamber CultureSlides coated with collagen type I (Falcon,
Heidelberg, Germany), were fixed with methanol or acetone at -20°C for 5
minutes and washed with PBS. For indirect immunofluorescence staining, the
tissue sections and the adherent thymic epithelial cells were incubated for 1
hour with the primary antibodies diluted 1:100 in PBS containing 0.1% BSA.
After washing with PBS, bound antibodies could be detected by
Cy3TM-conjugated goat anti-mouse or Cy3TM-conjugated goat
anti-rabbit antibodies (Dianova) diluted 1:500 or 1:1000, respectively. Cell
nuclei could be identified by counterstaining with
4',6-diamino-2-phenylindol-dihydrochloride (DAPI; 1 µg/ml). Control
staining was performed by omitting the first antibody. Photographs were taken
on a Zeiss axiophot microscope.
For double-immunofluorescence staining of E-cadherin with epithelial cell markers, the labelled sections were also visualised by epifluorescence light microscopy. Digital pictures from every fluorescence channel were taken and superimposed for the specific antibody stains as well as for each negative control labelling using the software DOKU® from Soft Imaging Systems (Leinfelden-Echterdingen, Germany).
For enzymatic horseradish peroxidase staining, the EnVisionTM system of Dako Diagnostics (Hamburg, Germany) was applied. After blocking of endogenous peroxidase activity the cryostat sections were first incubated with normal rabbit antiserum for 30 minutes and then with the first antibodies for another 30 minutes. For control staining, the sections were labelled with the antibody W6/32.HL, which recognises the heavy chain of MHC class I antigens (positive control) or the antibody W6/32.HK, an inactive variant of W6/32.HL (negative control). After extensive washing, the sections were incubated with EnVisionTM-horseradish peroxidase-coupled goat anti-mouse antibodies for 30 minutes. Incubation with the chromogenic substrate DAB (3,3'-diaminobenzidine tetrahydrochloride) revealed specific signals. The sections were counterstained with Mayer's hemalaun.
Flow cytometric analysis
Unfractionated thymocytes were labelled with the individual cadherin
antibodies as described recently (Armeanu
et al., 1995). Expression of the integrin receptor
E (CD103) on the different thymocyte subpopulations isolated
by magnetic cell sorting was studied by single-fluorescence analysis. Briefly,
for each staining 1-5x105 cells were incubated with 20 µl
polyglobin® (Bayer, Leverkusen, Germany) for 30 minutes to block
unspecific binding of antibodies, washed with PBS containing 0.1% BSA and
0.05% NaN3 and incubated for 45 minutes with the primary anti-CD
103 antibody 2G5.1. After intensive washing, a FITC-conjugated
anti-mouse-IgG2a (Caltag Laboratories, Hamburg, Germany) was applied for 30
minutes as secondary antibody.
For dual, triple and four colour flow cytometric analysis, DN and
CD8+ SP thymocyte subpopulations were analysed using FITC- or
PE-conjugated antibodies against /ß TCR and
/
TCR, a
PE-conjugated antibody against CD24, APC-conjugated antibodies against CD25
and CD69, and a Cy-Chrome-conjugated antibody against CD62L. All these
fluorochrome-conjugated antibodies were purchased from BD Biosciences
(Heidelberg, Germany). In the multiple immunofluorescence analyses, the CD103
antibody was detected with secondary FITC- or PE-conjugated anti-IgG2a
antibodies (Caltag). The purity of the DN and CD8+ SP thymocyte
subpopulations were assessed with the directly labelled CD4-PE (clone B-F5)
and CD8-FITC (clone MCD8) antibodies, which were obtained from DPC-Biermann
(Bad Nauheim, Germany). Isotype control antibodies, obtained from
DPC-Biermann, were used as negative controls. After labelling and extensive
washing, 10,000 thymocytes were analysed for cell-surface antigen expression
in the dual colour analysis using a FACSort flow cytometer (Becton Dickinson,
Heidelberg, Germany) and the WinMDI 2.8 software. For the triple and four
colour flow cytometric analysis, at least 20,000 events were analysed on an
FACScalibur (Becton Bickinson).
Immunoblotting and immunoprecipitation
Thymic protein extracts were obtained by homogenisation and sonication of
the tissue in Tris-buffered saline (TBS) containing 1% NP-40, 1% Triton X-100,
1 mM CaCl2, 1 mM MgCl2, 1 mM PMSF and 1 mM aprotinin and
subsequent incubation on ice for 30 minutes. After centrifugation at 12,500
g, the resulting protein extracts were separated on 10%
polyacrylamide gels and transferred to nitrocellulose filters. Non-specific
protein-binding sites were blocked with a TBS solution containing 0.1%
Tween-20 (TTBS) and 5% skimmed milk powder. The filters were probed for 1 hour
with the primary monoclonal or polyclonal antibodies diluted in blocking
solution. After washing with TTBS, bound antibodies were detected either by
alkaline-phosphatase-conjugated rabbit -mouse or goat
-rabbit
immunoglobulins (Dako Diagnostics) followed by colorimetric reaction with the
Fast BCIP/NBT system (Sigma).
For non-radioactive immunoprecipitations, the thymic cell lysates were incubated with protein-A-sepharose (Amersham Pharmacia Biotech, Freiburg, Germany) for 1 hour for preclearing the lysates. After centrifugation, specific immune complexes were formed by the addition of anti-cadherin or anti-catenin antibodies to the supernatants followed by the addition of protein-G-sepharose. After rotation for 1 hour at 4°C, the precipitated antigens were washed several times and dissolved by boiling in SDS-PAGE sample buffer. After separation in 10% polyacrylamide gels, the precipitated antigens were detected by immunoblotting using antibodies against ß-catenin or E-cadherin.
Cell adhesion assay
Isolated thymic epithelial cells were cultured in serum-free medium in
24-well plates coated with collagen type I (Roche). The non-thymic human
epithelial carcinoma cell line A431, which expresses high levels of
E-cadherin, was used in parallel experiments. The adherent epithelial cells
were washed twice with pre-warmed PBS and incubated with 2.5% BSA/PBS for 1
hour at 37°C to block unspecific cell binding sites of the wells. After
blocking, the wells were washed again with medium. Isolated thymocyte
subpopulations were labelled with 2 µl BCECF-AM
[2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxylmethyl
ester; Sigma], dissolved in DMSO at 37°C for 15 minutes. After washing,
3x105 thymocytes were added to each well and allowed to
adhere to the confluent monolayers of thymic epithelial cells for 1 hour at
37°C. To inhibit cell-cell interactions, either the thymocytes were
preincubated under constant rotation for 30 minutes with anti-integrin
antibodies or the thymic epithelial cells were preincubated with
anti-E-cadherin antibodies. The murine antibody W6/32. HL, which recognises
HLA-A, -B and -C molecules on the surface of the cells was used as an
irrelevant control antibody. Then the cell adhesion assays were performed in
the presence of the respective antibodies. After 1 hour of incubation, the
non-adherent thymocytes were removed by gently washing with prewarmed PBS.
Finally, 500 µl PBS were added to each well, and the BCECF-AM-fluorescence
was quantified with a fluorometer (Fluoroskan Ascent; Thermo Biosciences,
Egelsbach, Germany) at excitation wavelength 485 nm and emission wavelength
538 nm. The ratio (%) of the adhering thymocytes was calculated as follows:
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The assays were carried out in triplicate. After quantification, attached thymocytes were fixed with 2% formaldehyde and stained with 0.5% crystal violet to visualise cell attachment also under the light microscope.
Cell proliferation assay
CD8+ SP or CD103- CD8+ thymocytes isolated
by magnetic cell sorting were incubated with confluent monolayers of primary
thymic epithelial cells cultured in 96-well plates. 1x105
thymocytes in MEM D-valine medium containing 10% human AB serum were added to
each well. The combined cell populations were cultured for 48 hours without
addition of any cytokine. During the last 16 hours, 1 µCi
[3H]thymidine was added to each well. To analyse the influence of
various antibodies on cell proliferation, the antibodies were present during
the whole culture period. After incubation, the thymocytes together with the
thymic epithelial cells were harvested on filter papers.
[3H]thymidine incorporation was measured in a liquid scintillation
counter. All experiments were carried out in triplicate.
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Results |
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The expression of E- and P-cadherin on stromal cells of the human thymus was also analysed on isolated thymic epithelial cells grown in culture for several weeks. These primary cells express both E- and P-cadherin mainly on their cell borders (Fig. 3A,B).
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For functional interactions, cadherins have to be associated with
cytoplasmic catenins. Therefore the distribution of -, ß- and
-catenin in human thymus was determined by immunofluorescence staining.
ß-catenin was found to be equally distributed on cortical and medullary
thymic epithelial cells, whereas a prevalence of medullary epithelial cells
for
-catenin could be observed (Fig.
1C,D).
-catenin was evenly expressed on cortical and
medullary epithelial cells (data not shown). On isolated thymic epithelial
cells, ß-catenin and
-catenin were found to be strongly enriched
at the sites of cell-cell contact (Fig.
3C,D).
The expression of both cadherins and catenins in thymic tissue as well as
in cultured primary thymic epithelial cells was confirmed by western blotting.
The specific antibodies detected the 120 kDa bands of E- and P-cadherin and
also the 102, 95 and 86 kDa bands of the three different catenins
(Fig. 4A,C). Several additional
immunoreactive bands were detected with the antibodies against ß- and
-catenin, which most probably represent proteolytically degraded
products. The existence of intact E-cadherincatenin complexes in the
thymus was investigated by co-immunoprecipitation. In these experiments, the
cadherin-catenin complexes could be precipitated by antibodies against
E-cadherin,
-, ß- or
-catenin, respectively
(Fig. 4B), which suggested
direct functional interactions in thymic epithelial cells.
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By immunofluorescence staining of thymic tissue, no staining of thymocytes for E- or P-cadherin could be detected. However, this type of analysis is not sensitive enough to exclude the possibility that no thymocyte subpopulation could express cadherins on their cell surface. Therefore, flow cytometric analyses of isolated thymocytes were performed (Fig. 5). The results confirmed that no thymocyte subpopulation in the postnatal human thymus expressed E- or P-cadherin, indicating that no cadherin-mediated homophilic interaction of thymocytes with thymic epithelial cells is likely to occur.
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Expression of CD103 on subpopulations of human thymocytes
The integrin E(CD103)ß7 is the only
heterophilic ligand for E-cadherin known so far. Since thymocytes did not
express E-cadherin, the expression pattern of CD103 on human thymocyte
subpopulations was determined by flow cytometric analysis
(Fig. 6A). About 12% (normal
range: 10-17%) of the early CD4- CD8- DN thymocyte
subpopulation expressed CD103, whereas the more mature CD4+
CD8+ DP thymocytes did not express CD103 at all. On CD4+
SP thymocytes, CD103 was also hardly detectable (<2%). However, a strong
expression was observed on CD8+ SP thymocytes where more than one
third of the cells (normal range: 30-50%) expressed CD103. An
immunohistochemical staining of human thymus with the anti-CD103 antibody
revealed strong surface labelling of thymocytes in the medullary region where
CD8+ SP thymocytes are expected to be located
(Fig. 7).
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To determine whether the CD103+ CD8+ SP cells
represent a specific subpopulation of CD8+ SP thymocytes,
CD8+ SP cells were isolated by MACS technology and stained with
antibodies against the T-cell receptors (TCR) ß and
(Fig. 6C). A more than 90%
enrichment of the CD8+ SP cells was achieved using MACS separation.
Only 4.3% of these cells expressed the
TCR, whereas 89%
expressed the
ß TCR. Double labelling of the CD8+ SP
cells with the anti-CD103 antibody and the anti-
ß and
TCR antibodies clearly showed that the overwhelming majority of
CD103+ CD8+ SP thymocytes expressed the
ß
TCR. A further characterisation of the CD103+ CD8+ SP
thymocyte subpopulation was performed with antibodies against CD24, CD62L and
CD69 in a four colour flow cytometric analysis
(Fig. 6D). Whereas CD24 was
hardly detected on the CD103+ CD8+ SP thymocyte
subpopulation, a strong expression of CD62L and CD69 was detected on these
cells. These results show that a prominent subpopulation of
TCR
ß+ CD62L+ CD69+
CD8+ SP thymocytes is characterised by CD103 expression.
The CD103+ DN thymocyte subpopulation was also characterised in
more detail (Fig. 6B). Flow
cytometric analysis revealed a more than 97% enrichment of these cells by MACS
separation. Triple staining with antibodies against CD103, CD25 and
ß TCR or
TCR showed that the CD103+ DN
thymocyte subpopulation do not express CD25 or
ß TCR
significantly. However, about 20% of the CD103+ DN thymocytes
showed an expression of
TCR, whereas the remaining 80% of these
cells do not. These results strongly indicate that the majority of the
CD103+ DN thymocytes are immature precursors.
Adhesion of CD8+ CD103+ thymocytes to thymic
epithelial cells
CD103+ thymocytes were evaluated for adhesive interactions with
E-cadherin+ thymic epithelial cells. After incubation of
CD8+ SP thymocytes for 1 hour with a confluent layer of primary
thymic epithelial cells, strong adhesive interactions between these two cell
types could be observed (Fig.
8A). The adhesive interactions were strongly inhibited by
pre-incubation of the CD8+ SP thymocytes with anti-CD103 antibody
(Fig. 8B), indicating a direct
involvement of the E-cadherin-integrin Eß7 ligand pair
in the observed attachment of the cells. To quantify the adhesive
interactions, the isolated CD8+ SP thymocytes were labelled with a
fluorescent dye that does not interfere with the cell-binding process. About
40% of the labelled CD8+ SP thymocytes attached to the thymic
epithelial cell layer (Fig.
8C), corresponding to the amount of CD103+ thymocytes
in the CD8+ SP cell population. Addition of the control antibody
W6/32. HL, which binds to the surface of the thymocytes, did not influence the
adhesive interactions (Fig.
8C). However, addition of either anti-CD103 antibody or
anti-E-cadherin antibody to the interacting cells drastically diminished
cellular interactions (Fig.
8D). No synergistic effect was observed when both antibodies were
simultaneously added. Although cell binding of CD103+
CD8+ SP cells to isolated thymic epithelial cells could be
drastically inhibited by anti-CD103 and anti-E-cadherin antibody treatment, a
100% inhibition could never be observed, indicating that other adhesive
mechanisms might still play a role in the binding of thymocytes to thymic
epithelial cells.
|
Expression of E-cadherin on the cell surface was sufficient for binding of CD103+ CD8+ SP thymocytes. This was shown using the non-thymic epithelial cell line A431 instead of primary thymic epithelial cells. The isolated thymocytes attached well to A431 cells, and again the attachment could be blocked by antibodies against E-cadherin or CD103 (data not shown).
The DN thymocytes also contain a subpopulation (>10%) that express CD103. Therefore, DN thymocytes were also evaluated for cellular interactions with primary thymic epithelial cells and the non-thymic epithelial carcinoma cell line A431. DN thymocytes attached to both E-cadherin+ epithelial cell types, and antibodies against E-cadherin or CD103 showed a strong inhibitory effect (data not shown).
Co-culturing CD8+ CD103+ thymocytes with thymic
epithelial cells induces cell proliferation
The functional significance of the observed adhesive interactions between
CD103+ thymocytes and an adherent thymic epithelial cell layer was
determined in cell proliferation assays. Incubation of confluent epithelial
cell layers with CD8+ SP thymocytes consistently increased cell
proliferation, which could not be blocked by incubation with the control
antibody W6/32. HL (Fig. 9A). Addition of antibodies against E-cadherin resulted in a modest blockade of
cell proliferation. However, antibodies against CD103 drastically diminished
cell proliferation in the co-culture assay (n=5;
Fig. 9A). Incubation of
CD8+ SP thymocytes with the CD103 antibody without epithelial cells
did not show an influence on cell proliferation (data not shown).
|
Since CD8+ SP thymocytes contain CD103+ and CD103- subpopulations, the CD103- CD8+ SP cells were also analysed for cell proliferation with primary thymic epithelial cells. CD103+ CD8+ SP cells could not be directly analysed since the anti-CD103 antibody used for cell separation was shown to be strongly inhibitory in cell proliferation studies. CD103- CD8+ SP thymocytes did not show a proliferative response when co-cultivated with thymic epithelial cells (n=3), whereas unseparated CD8+ SP thymocytes did show a proliferative response (Fig. 9B). Again this mitogenic effect could be efficiently blocked by antibodies against CD103. CD103+ CD8+ SP thymocytes attached well to E-cadherin+ A431 cells. However, this adhesive interaction did not lead to an induction of cell proliferation (n=2; data not shown).
These results showed that the E-cadherin-integrin
Eß7 ligand pair is necessary but not
sufficient to induce cell proliferation, suggesting that primary thymic
epithelial cells can provide additional signals for cell proliferation not
found on non-thymic epithelial cells.
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Discussion |
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The cadherin family shows a very restricted expression pattern during
hematopoietic cell development. In the human bone marrow, N-cadherin is
present on very early CD34+ CD19+ hematopoietic
progenitor cells but is downregulated on more mature progenitor cells
(Puch et al., 2001).
N-cadherin can also be detected on T cell leukaemia and lymphoma cells, but
not on normal leukocytes (Tsutsui et al.,
1996
; Kawamura-Kodama et al.,
1999
). E-cadherin expression is found on bonemarrow-derived
dendritic Langerhans cells of the epidermis
(Tang et al., 1993
). On
myeloid blood cells E-cadherin expression is restricted to distinct
developmental stages of erythropoiesis, whereas other myeloid cells do not
seem to express E-cadherin (Armeanu et al.,
1995
; Armeanu et al.,
2000
; Bühring et al.,
1996
; Lammers et al.,
2002
). On developing T-lymphocytes, Munro and colleagues detected
E-cadherin as well as the classical type II cadherin-6, -8 and -11 by RT-PCR.
These results were obtained with murine adult CD4+ CD8+
thymocytes, but they were not corroborated by any immunological technique
(Munro et al., 1996
). On the
other hand, by flow cytometry analyses, Lee and co-workers only detected
E-cadherin on murine fetal thymocytes and on a very small percentage of
postnatal thymocytes, but not on adult thymocytes
(Lee et al., 1994
). The
expression of E-cadherin on the cell surface of murine fetal thymocytes was
confirmed in subsequent studies with fetal thymic organ cultures. Antibodies
that block homophilic E-cadherin interactions inhibited epithelial
organization and thymocyte development in reaggregate fetal thymic organ
cultures (Müller et al.,
1997
). However, antibodies that were able to block heterophilic
interactions between
Eß7 integrin and
E-cadherin did not interfere with early thymocyte development, indicating an
important role for homophilic E-cadherin interactions during murine fetal
thymocyte development. Homophilic E-cadherin-mediated interactions between
thymocytes and thymic epithelial cells did not seem to play a role in the
postnatal human thymus, since no human postnatal thymocyte subpopulation
seemed to express E-cadherin, as shown in the present study. Whether human
fetal thymocytes, like their murine counterparts, also express E-cadherin has
not been analysed so far.
Although no E-cadherin expression was found on postnatal human thymocytes, cortical and medullary thymic epithelial cells still express E-cadherin with the highest expression level found on medullary thymic epithelial cells. Using suitable culture conditions, primary thymic epithelial cells strongly expressing functional E-cadherin-catenin complexes could be grown in vitro. However, the expression of E-cadherin on the primary thymic epithelial cells was critically dependent on the culture conditions used, as slight changes in these conditions led to a downregulation of E-cadherin as well as an upregulation of N-cadherin (S.K. and G.K., unpublished). Therefore, all primary thymic epithelial cells used in the present study for functional analyses had to be tested for E-cadherin expression.
The integrin E(CD103)ß7 is the
heterophilic counter-receptor for E-cadherin and is mainly expressed on
intraepithelial lymphocytes (Cepek et al.,
1993
; Higgins et al.,
1998
). In the skin,
Eß7 seems to
contribute to tissue-specific epidermal localization of CD8+
lymphocytes (Pauls et al.,
2001
).
Eß7 is also expressed on
developing T-cells as shown for murine and human thymocytes. Whereas Andrew
and co-workers found that
Eß7 was expressed
on comparable subsets of both CD4+ SP and CD8+ SP murine
thymocytes, the study of Lefrancois and colleagues showed that the majority of
murine CD103+ thymocytes were medullary CD8+ SP
thymocytes (Andrew et al.,
1996
; Lefrancois et al.,
1994
). In the human thymus, an average of 26% of CD8+
SP thymocytes were quantified as CD103-expressing thymocytes
(McFarland et al., 2000
).
These cells were predominantly
ß TCR+
TCR-, which is in agreement with our present study. An analysis of
T-cell-receptor rearrangement excision circles (TREC) showed that the
CD103+ CD8+ cells within the human thymus represent an
actual stage of thymopoiesis and not a subset of mature peripheral memory T
cells recirculating in the thymus
(McFarland et al., 2000
).
Our present functional studies showed that the expression of CD103 on human
thymocytes led to adhesive interactions with thymic epithelial cells. This was
shown for CD4- CD8- DN and CD8+ SP
thymocytes. Quantification of the adhering thymocytes revealed that only the
fraction of thymocytes expressing CD103 attached to thymic epithelial cells.
However, adhesion of CD103+ thymocytes was not restricted to thymic
epithelial cells, since the CD103+ thymocytes could also adhere to
epithelial carcinoma cells expressing E-cadherin. The specificity of these
interactions was verified by inhibition studies using antibodies against
either CD103 and/or against E-cadherin. Recently, it has been shown that the
integrin Eß7 can also mediate cell adhesion
in an E-cadherin-independent way (Strauch
et al., 2001
). However, the antibodies against CD103 and
E-cadherin caused similar inhibitory effects on thymocyte adhesion to
epithelial cells, suggesting that an E-cadherin-independent binding mechanism
of CD103+ thymocytes does not seem to play a role.
Cell proliferation of thymocytes can be directly influenced by adhesive
interactions with cellular and extracellular matrix components. Fibronectin
and laminins as prominent representatives of extracellular matrix molecules
have been shown to either enhance or downregulate cell proliferation of
developing T cells (Halvorson et al.,
1998; Vivinus-Nebot et al.,
1999
). Cellular adhesion of human CD103+
CD8+ SP thymocytes to epithelial carcinoma cells did not lead to
enhanced thymocyte proliferation, whereas adhesive interactions of these
thymocytes with cultivated thymic epithelial cells did. The proliferative
response seemed to be directly linked to CD103 expression since (1) antibodies
against CD103 inhibited cell proliferation in the co-culture system and (2)
CD103- CD8+ thymocytes did not proliferate on thymic
epithelial cells. Although the expression of CD103 on CD8+ SP
thymocytes seemed to be necessary, it was not sufficient to induce
CD8+ thymocyte cell proliferation, since only the interaction with
E-cadherin+ thymic epithelial cells, but not with
E-cadherin+ epithelial carcinoma cells, led to an enhanced
proliferative response. The nature of the additional signal(s) present on
thymic epithelial cells is, however, not known.
There is increasing evidence that integrins play a pivotal role in
regulating T-cell maturation (Mojcik et
al., 1995; Andrew et al.,
1996
; Salomon et al.,
1997
; Halvarson et al., 1998;
Vivinus-Nebot et al., 1999
;
Savino et al., 2000
;
Schmeissner et al., 2001
).
Integrins mainly interact with extracellular matrix molecules but also with
cellular adhesion molecules like VCAM-1 and E-cadherin. The expression pattern
of the integrin ligands in the thymus as well as the integrin expression on
thymocytes seems to be finely tuned, being responsible for their influence at
defined stages of T cell maturation (Crisa
et al., 1996
; Kutle
a
et al., 2002
). Developing T cells have to undergo various cycles
of proliferation, migration, adhesion and arrest during their differentiation
process. Therefore, a coordination of adhesive interactions and induction of
proliferation as shown here for the intergrin
Eß7 and its ligand E-cadherin is most likely
to be of high significance for human T cell development.
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Acknowledgments |
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References |
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