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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3745-3755
Thymocyte Contact or Monoclonal Antibody-Mediated Clustering of
 3 1 or 64 Integrins Activate Interleukin-6 (IL-6)
Transcription Factors (NF- B and NF-IL6) and IL-6 Production in Human
Thymic Epithelial Cells
By
Dunia Ramarli,
Maria Teresa Scupoli,
Emma Fiorini,
Ornella Poffe,
Monica Brentegani,
Antonello Villa,
Germana Cecchini,
Giuseppe Tridente, and
Pier Carlo Marchisio
From the Institute of Immunology and Infectious Diseases, University
of Verona, Verona, Italy; DIBIT, Department of Biological and
Technological Research, San Raffaele Scientific Institute, Milano,
Italy; Clinical Immunology, Azienda Ospedaliera Verona, Verona, Italy;
and the Department of Biomedical Sciences and Human Oncology,
University of Torino School of Medicine, Torino, Italy.
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ABSTRACT |
T-cell precursors develop within the thymus in contact with multiple
supportive elements, among which thymic epithelial cells (TEC) are
known to exert a dominant role in their homing, survival, and
functional differentiation. All these functions are supported by
cell-cell contacts and cytokine release. Signaling events triggered in
lymphoid cells by adhesion to TEC are well characterized, but little is
known about the opposite phenomenon. To address this issue, we derived
cultures of TEC from human normal thymus. TEC monolayers were
cocultured with thymocytes and immunostained with monoclonal antibodies
(MoAbs) to integrin  (2, 3, 4, and 6) and  (1 and
4) chains. Optical and confocal analysis showed that integrins were
polarized on TEC at discrete surface locations: 64 lined the
basal surface of TEC monolayers, whereas 31 was found mostly at
TEC-TEC contacts; it is noteworthy that both 31 and 64
became highly enriched also at the boundaries with adherent thymocytes.
Functional studies performed with MoAbs anti-1 and -4 integrins
showed that 1, and, to a much lower extent, 4 heterodimers are
involved in the TEC-thymocyte adhesion. Thymocyte contact or
MoAb-mediated ligation of 3, 6, 1, and 4 integrins was
investigated as a potential inducer of intracellular signaling in TEC.
Thymocyte adhesion or cross-linking of MoAbs bound to integrins
clustered at the TEC/thymocyte contact sites led to activation of
interleukin-6 (IL-6) gene transcription factors, namely NF-IL6 serine
phosphorylation and NF- B nuclear targeting, as well as to increased
IL-6 secretion. We propose that integrin clustering occurring during
TEC-thymocyte contacts modulates in TEC the gene expression of a
cytokine involved in thymocyte growth and functional differentiation.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE THYMIC EPITHELIUM is composed of
multiple cellular subsets of ectodermal or endodermal embryonic origin,
characterized by tonofilaments, surrounded by extracellular matrix, and
interconnected by desmosomes to form an intralobular meshwork filled
with developing thymocytes. Although the discrete functions of the
various subsets have yet to be elucidated in humans, it is known that
thymic epithelial cells (TEC) exert pivotal roles in the homing,
intrathymic migration, and differentiation of lymphoid precursors
through the release of cytokines (ie, interleukin-1 [IL-1] and
IL-1, IL-3, IL-6, IL-8, and transforming growth factor 3
[TGF3]),1-3 the secretion of extracellular matrix
proteins, and the establishment of adhesive interactions. Thymocytes
adhesion seems also effective at modulating functions and
differentiation process of TEC. Evidence in mouse models demonstrated
that thymocyte contact is required by thymic epithelium to properly
develop during embryogenesis.4 In addition, observations
obtained with human TEC derived in vitro showed that thymocyte adhesion
results in tyrosine phosphorylation of cytoplasmic proteins, increased
IL-6 mRNA transcription, and/or cytokine
production.4-6 The regulation of IL-6 gene expression in
TEC is of particular relevance within the thymic microenvironment as
regards its activities on lymphoid activation and cytotoxic
differentiation of thymic precursors as well as its antiapoptotic
functions in epithelial and lymphoid cells.7-10 The IL-6
gene expression is mainly regulated at a transcriptional level,
although mechanisms of posttranscriptional control have been also
described.3,11,12 To date, two major regulatory factors,
namely NF-B and NF-IL6, have been described to be involved in IL-6
transcription in different cell lineages.11 It is
noteworthy that transfection studies in murine carcinoma cell lines
have shown that overexpression of NF-IL6 and the p65 subunit of NF-B
synergistically activates an IL-6 promoter-reporter construct,
indicating that these two factors are sufficient to sustain the
activation of IL-6 gene.13 NF-IL6, a member of the CCAAT/enhancer binding (C/EBP) protein-related family, is a DNA binding
protein recognizing similar motifs in liver-specific, acute-phase
reaction, and cytokine genes.11,14 Its transactivation potential is regulated at translational and posttranslational levels.
Different usage of initiation codons accounts for expression of
isoforms, which differ in the N-terminal half of the protein containing
the activation domain and hence in transcriptional activity.15,16 Posttranslational modifications, consisting in serine/threonine phosphorylation of specific amino acids within the
functional domains, further regulate both the transcriptional and the
DNA binding activity, as demonstrated in epithelial and fibrosarcoma
cell lines.17,18 NF-B is an ubiquitous dimeric factor
composed of p50 and p65 (RelA) subunits sequestered inactive in the
cytosol bound to a family of inhibitors, IBs, that mask its nuclear
localization sequence.19 Activation relies on serine hyperphosphorylation and proteosomal degradation20 or on
tyrosine posphorylation of inhibitors,21 either one of
which is sufficient to disrupt the IB-NF-B complexes, thus
allowing NF-B to move into the nucleus.
Our previous data demonstrated that binding of lymphoid cells to TEC
increases the DNA binding activity of their endogenous NF-B as well
as their IL-6 production,22 prompting us to investigate whether IL-6 gene expression in TEC could be regulated at a
transcriptional level by signals delivered from adhesive interactions
with thymocytes. Several molecules have been identified that act as a
receptor or counter-receptor in TEC adhesion to thymocytes such as
CD6-100kD ligand, galectin, LFA3, intercellular adhesion molecule-1
(ICAM-1), vascular cell adhesion molecule (VCAM),
cadherin, and integrins.23-25 The latter are of particular
interest because of their signal transduction properties. Integrin
ligation with natural ligands or monoclonal antibodies (MoAbs)
initiates a variety of events including induction of Calcium
transients, changes in cAMP levels, activation of
Na+/H+ antiporter, or protein tyrosine
phosphorylation, which can ultimately lead to the expression of several
genes, including those of cytokines.26
In this report, we show that binding of normal thymocytes to TEC is
followed by the activation of IL-6 gene transcription factors, namely
NF-B nuclear translocation and serine phosphorylation of the 36- and
43-kD NF-IL6 isoforms, and is associated with increased IL-6
production. Moreover, we show that both activation of IL-6 transcription factors and increased IL-6 secretion are induced by
cross-linking of 31 and 64 integrin heterodimers, which cluster at the TEC/thymocyte contact sites and are functionally involved in their binding.
Previous reports have shown that TGF- and epidermal growth factor
(EGF) induce IL-6 production in TEC through posttranscriptional control
mechanisms.3 We herewith propose that TEC IL-6 production can be regulated also at the transcriptional level through signals delivered by integrin recruitment during their adhesion to thymocytes.
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MATERIALS AND METHODS |
Cell cultures.
TEC primary cultures were derived from normal thymus of patients (<5
years of age) undergoing corrective cardiac surgery as previously
described.27 Briefly, thymus specimens were minced and
trypsinized (0.05% trypsin/0.01% EDTA) at 37°C for 3 hours. Cells
were collected every 30 minutes, pooled, plated onto lethally irradiated 3T3-J2 feeder-layer cells (a gift of Prof H. Green, Harvard
Medical School, Boston, MA) at 2.5 × 104/cm2
and cultured at 5% CO2 in growth medium composed as
follows: Dulbecco's modified Eagle's medium (DMEM) and
Ham's F12 media (3:1 mixture), 10% fetal calf serum (FCS), insulin (5 µg/mL), transferrin (5 µg/mL), adenine (0.18 µmol/L),
hydrocortisone (0.4 µg/mL), cholera toxin (0.1 nmol/L),
triiodothyronine (2 nmol/L), EGF (10 ng/mL), glutamine (4 mmol/L), and
penicillin-streptomycin (50 IU/mL). Confluent TEC primary cultures were
detached by trypsin-EDTA, plated on NIH-J2 feeder layer, and expanded
to confluent secondary cultures in growth medium. TEC obtained from
secondary cultures already devoid of NIH-J2 cells were replated without
the feeder layer at 1.2 × 104/cm2 cell
density, cultured in growth medium for 24 hours and maintained before
use for 48 hours in modified growth medium containing one third of the
concentration of insulin, transferrin, adenine, hydrocortisone, cholera
toxin, triiodothyronine, and EGF. Media were purchased from Seromed
(Berlin, Germany) and supplements were purchased from Sigma-Aldrich
(Milano, Italy). EGF was from Austral Biological (San Ramon, CA).
Human thymocytes were prepared by mechanical disruption of fresh thymus
specimens. At least 95% viable cells were isolated from the cell
suspension by Ficoll-Hypaque gradient centrifugation, washed, and used
immediately after preparation.
Antibodies.
The following antibodies were used for immunostaining, cell treatment,
Western blotting, electromobility shift assay (EMSA), and
Ca2+ mobilization assays: MoAbs anti-CD2, -CD3, -CD4, -CD8,
and -CD16 and fluorescein isothiocyanate (FITC)-labeled
F(ab)2 goat antimouse Ig from Becton Dickinson
(Mountain View, CA); anti-CD1a, anti-CD18, anti-ICAM-1, anti-3
(CD49c), and anti-a2 (Gi9) from Immunotech International (Marseille,
France); MoAbs B9-12 (anti-MCH class I) and D1-12 (anti-MHC class II)
provided by Dr R.S. Accolla (University of Pavia, Varese, Italy); MoAb
10.1.2 (gift of Dr G. Corte, CBA Genova, Genova, Italy); MoAb 3E1
(anti-4; gift of Dr E. Engvall, The Burnham Institute, La Jolla,
CA); MoAb MAR 4 (anti-1) and MAR 6 (anti-6; kindly provided by Dr
S. Ménard, Istituto Nazionale Tumori, Milano, Italy); MoAb Lam89
(anti-laminin 1) from Sigma; MoAb GB3 (anti-laminin 5; gift of Dr
Patrick Verrando, University of Nice, Nice, France); MoAb anti-VCAM
(British Biotechnology Laboratories Ltd, Oxford, UK) through the
courtesy of Dr R. Giavazzi (Istitut M. Negri, Bergamo, Italy);
F(ab)2 goat antimouse Ig from Pierce (Pierce, Oud
Beijerland, The Netherlands); swine antirabbit or rabbit antimouse IgG
for immunoistochemistry from Dako (Glostrup, Denmark); antiserum
anti-p105 recognizing both the p50 precursor (p105) and the p50 mature
form (kindly provided by Dr A. Israel, Institut Pasteur, Paris,
France); rabbit antiserum anticingulin (gift of Dr S. Citi, University
of Padova, Padova, Italy); antisera anti-NF-IL6 (C19) and
anti-NF-B p65 (A) from Santa Cruz Biotechnology (Santa Cruz, CA);
MoAb antiphosphoserine in agarose-coupled or uncoupled form,
horseradish peroxidase-labeled rabbit antimouse, goat antirabbit
antisera, and nonspecific mouse Igs from Sigma. Flow cytometry was
performed in a FacScan flow cytometer (Becton Dickinson).
Adhesion assays and MoAb treatment.
As detailed in the previous paragraph, morphological and functional
studies were performed with TEC grown to monolayers without the NIH-J2
cell support and adapted to low concentration of supplements in the
culture medium. Thymo-epithelial cocultures destined to immunohistochemical studies were performed with TEC plated at 1.2 × 104/cm2 on round glass coverlips fitting the
24-well Costar (Acton, MA) plates and grown to confluence.
Freshly isolated thymocytes were added at a 5:1 thymocyte:TEC ratio and
removed 12 hours later by gentle washing. Cocultures destined to
transcription factor studies were performed in 6-well Costar plates at
same cell ratio. Thymocytes were removed after 1, 3, or 5 hours of
contact by extensive vigorous washings. TEC were detached by
trysin-EDTA, scored for contamination with residual thymocytes by
immunostaining with MoAbs anti-CD1a and -CD2, and used as a source of
cytoplasmic and nuclear extracts.
TEC destined to binding assays with thymoctes were plated 24 hours
before use in 24-well Costar plates at high density
(5 × 105/well), so that their extremely tight
confluence could prevent the direct attachment to the plastic of
thymocytes.
Cocultures used for IL-6 production assays were performed for 12 hours.
After thymocyte removal, TEC were recovered by trypsin-EDTA, analyzed
for the absence of contaminants as described above, and plated onto new
plates. Cell supernatants were collected 24 and 72 hours from plating.
TEC destined to Ca2+ mobilization analysis were grown to
confluency on square glass coverslips fitting the spectrofluorimeter
cuvette. MoAbs anti-3, -6, -1, and -4 chains, anti-CD71,
and MHC class I were used purified at 5 µg/mL, previously identified
as the saturating concentration for all antibodies by flow cytometry.
Primary antibodies were cross-linked, if required, after 1 hour of
incubation, by 2 additional hours of incubation with F(ab)2
goat antimouse Ig at 10 µg/mL.
Thymo-epithelial coculture immunostaining.
Cocultures fixed in 3% paraformaldehyde (electron microscope grade),
2% sucrose in phosphate-buffered saline (PBS), pH 7.6, for 5 minutes
at room temperature were further treated as previously reported.28 Briefly, fixed monolayers were permeabilized (3 minutes at 4°C) in HEPES-Triton X-100 buffer (20 mmol/L HEPES, 300 mmol/L sucrose, 50 mmol/L NaCl, 3 mmol/L MgCl2, 0.5%
Triton X-100, pH 7.4). Staining for F-actin was performed with
fluorescein-labeled phalloidin (F-PHD; Sigma; 200 nmol/L for 20 minutes
at 37°C in the dark). Adhesion molecules were detected with the
relevant MoAbs (see above), followed by rhodamine-tagged swine
antirabbit or rabbit antimouse IgG. Primary antibodies were replaced by
mouse IgG or preimmune rabbit sera in control samples. Coverslips were incubated with the appropriate rhodamine-tagged secondary antibodies routinely supplemented with 200 nmol/L F-PHD. After a final thorough washing, coverslips were mounted in Mowiol 4-88 (Hoechst, Frankfurt am
Main, Germany) and observed in a Zeiss (Oberkochen,
Germany) Axiophot microscope equipped for epifluorescence
and a 63× planapochromatic lens. Stained coverslips were photographed
with Kodak T-MAX 400 films (Eastman Kodak, Rochester, NY)
exposed at 1000 ISO and developed at 1600 ISO in T-MAX developer for 10 minutes at 20°C. The same coverslips were analyzed in parallel with a
confocal laser scanning microscope (CLSM Bio-Rad 1024; Bio-Rad,
Hercules, CA). Image files were recorded on different
channels and digitally reconstructed to provide z-axis views. Files
obtained from confocal microscopy were assembled and printed with Adobe
Photoshop 3.5 (Adobe Systems Inc, Mountain View, CA).
TEC-thymocyte binding assay.
Binding assays were performed with freshly isolated, unfractionated
normal thymocyte prestained with the PK H26-GL red fluorescent dye
(Sigma). Dye loading was achieved according to the manufacturer's recommendations. Thymocytes were added to TEC monolayer grown to
confluence at 5:1 thymocyte/TEC ratio and allowed to adhere for 1 hour
at 37°C in humidified atmosphere of 5% CO2 in binding medium composed of incomplete medium and 3% bovine serum albumin (BSA;
Sigma). At the end of the coculture, the nonadherent thymocyte were
gently removed by three subsequent washings performed with binding
medium. TEC monolayer and bound thymocytes where then detached with
trypsin-EDTA treatment, washed, vigorously resuspended to disrupt
aggregates, and analyzed by flow cytometry. TEC monolayers and PKH 26 GL-labeled thymocytes were separately incubated with the various MoAbs
or with the aspecific mouse Ig (all used at 5 µg/mL) for 30 minutes
at 37°C before binding. Assays in the absence of
divalent cations were performed in PBS
Ca2+Mg2+-free buffer at 3% BSA. TEC and
thymocytes were washed twice with the buffer before binding. The
results of each experiment were expressed as the ratio of TEC and
thymocytes recovered and as the percentage of the ratio of the
untreated control.
EMSA.
Nuclear and cytoplasmic extracts were prepared from TEC cells as
previously described,30 with minor modifications. Protein concentration was assessed by Coomassie protein assay reagent (Pierce,
Rockford, IL). DNA binding activity was determined by using a 32P  -ATP end-labeled double-strand
oligonucleotide encompassing the NF-B binding site located in the
IL-6 promoter region as probe. Cell extracts (6 µg) were incubated
for 30 minutes at room temperature with the probe
(1 × 105 cpms/sample) in 20 µL of binding buffer (20 mmol/L Tris-HCl, pH 7.5, 0.1 mol/L NaCl, 1 mmol/L
dithiothreitol [DTT], 1 mmol/L EDTA, 1 mg/mL BSA, 0.1% NP-40, 4%
glycerol) containing 1 µg of Poly(dI-dC) (Pharmacia, Uppsala,
Sweden). Cytoplasmic extracts were pretreated with 0.2% deoxycholic
acid in binding buffer for 10 minutes at room temperature before
probing. Antisera anti-NF-B subunits were preincubated with nuclear
extracts for 30 minutes on ice before probing. Nucleoprotein complexes
were electrophoresed on a 5% polyacrylamide (30:1.2) gel in 0.05 mol/L
Tris-borate/1 mmol/L EDTA at 150 V. Gels were dried and exposed to
Amersham Hyperfilm films (Amersham, Little Chalfont, UK). Film
densitometry was performed with an UltroScan Densitometer and the
built-in software (LKB, Bromma, Sweden).
Cell labeling, immunoprecipitation, and Western blotting.
TEC monolayers were labeled overnight with 25 µC/mL of
14C-leucine (324.9 mC/mmol; NEN Research Dupont, Boston,
MA) in incomplete growth medium. Cell lysates were prepared as
previously described for EMSA. Twenty micrograms of cytoplasmic or
nuclear extracts was precleared with 20 µL of swollen protein
A-Sepharose beads (Pharmacia) in lysis buffer (20 mmol/L Tris-HCl, pH
7.2, 0.5 mmol/L DTT, 1 mmol/L EDTA, 0.5 mmol/L phenylmethylsulfonyl
fluoride [PMSF] adjusted to 0.15 mol/L NaCl) for 1 hour
at room temperature and incubated with the anti-NF-IL6 antiserum at
1:103 dilution for an additional 2 hours on ice.
Immunocomplexes were precipitated with 20 µL of protein A-Sepharose
beads for 1 hour at room temperature and extensively washed in 0.05 mol/L Tris-HCl, pH 7.2, 0.15 mol/L NaCl, 0.1% NP40. Cell lysates (20 µg/sample) or immunoprecipitates (loaded at equal amount of counts)
were fractionated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane (Bio-Rad, Milano, Italy), and probed with antiphosphoserine MoAbs in
TBST 1.5% wt/vol powdered low fat dry milk. Cross-immunoblotting was
performed by immunoprecipitation of 20 µg of precleared cell extracts
with 30 µL of antiphosphoserine MoAb agarose-coupled (Sigma) for 1 hour on ice probed with the anti-NF-IL6 antiserum at
1:5,000 dilution. Membranes were washed with TBST buffer and specific
bands were shown by enhanced chemiluminescence system (ECL; Amersham).
Intracellular calcium assays.
Free cytosolic Ca2+ concentration was determined by using
Indo-1 acetoxymethyl ester (Indo-1-AM; Molecular Probes Europe BV, Leiden, The Netherlands).31 Optimal cell loading was
achieved with 3 µmol/L Indo-1-AM in DMEM at 1% FCS without phenol
red for 40 minutes at 37°C. Coverslips washed in fresh buffer were
immobilized in the test cuvette in working buffer consisting of 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L
NaH2PO4, 20 mmol/L HEPES, 5 mmol/L glucose, 0.5 mmol/L EGTA, or 1 mmol/L CaCl2, when indicated, at pH 7.4. MoAbs were used at 5 µg/mL. Relative fluorescence intensity was
measured on a Hitachi F-2000 spectrofluorimeter (Hitachi, Ltd, Tokyo,
Japan) at excitation and emission wavelengths of 340 and 400 nm,
respectively, with slits of 20 nm. The
[Ca2+]in was calculated according to the
following formula (Grynkiewicz et al31):
[Ca2+]in = kd
(F  Fmin)/(Fmax Fmin), where kd, the Ca2+-Indo-1
dissociation constant, was assumed to be 250 nmol/L; Fmax is
fluorescence intensity obtained upon addition of 0.05% Triton X-100 in
the presence of 1 mmol/L CaCl2; and Fmin is the level of
fluorescence obtained after the addition of 1 mmol/L MnCl2. Because we observed that TEC treated with 100 µmol/L ATP increased intracellular Ca2+ independently from extracellular
Ca2+, this responsiveness was assumed to be a criterion of
cell suitability for analysis and, hence, was evaluated in all
MoAb-unresponsive samples.
IL-6 production.
Cell supernatants were assayed for IL-6 production using an
enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (CLB, Amsterdam, The Netherlands). Optical density (OD) values were plotted on the standard
curve and expressed as nanograms per 106 cells recovered.
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RESULTS |
Thymo-epithelial cocultures.
Activation of IL-6 transcription factors in TEC after contact with
thymocyte was investigated by adhesion assays with time-course. Cocultures were performed by using TEC monolayers at the third passage
of culture grown to confluence and unfractionated thymocytes. Flow
cytometry of seven cultures derived from five independent donors (Table
1) consistently showed that greater than
90% of TEC expressed a high amount of MHC-class I, 1, and 3
molecules and moderate amounts of 5; 75% to 80% expressed 6 and
4 chains; ICAM-1 ranged between 9.5% and 20%; all cultures lacked
CD18, CD16, and MHC-class II molecules (not shown); VCAM, recently
described as a marker of cortical epithelium in vivo,32 was
also undetectable in our TEC since the primary culture, whereas greater
than 90% of TEC of all cultures were stained by the 10.1.2 MoAb,
previously shown to recognize an 3 variant expressed by medullary
epithelium in vivo.29
TEC monolayers were maintained in modified growth medium (see Materials
and Methods) and in the absence of feeder layer for at least 48 hours
before the adhesion assays to minimize the strong stimulating activity
of the variety of growth factors required to raise and expand the
primary cultures. Culturing TEC in modified growth medium did not
affect the quantitative or qualitative features of their phenotype, the
polarization of integrins, or the extent of thymocyte binding (see
below). Freshly isolated, unfractionated thymocytes were used at 5:1
thymocyte/TEC ratio, allowed to adhere to TEC for 1, 3, and 5 hours at
37°C in humidified atmosphere of CO2, and ultimately
removed by extensive and vigorous washings with medium. Control TEC
were washed in the same way.
Thymocyte adhesion induces NF-B nuclear translocation
and NF-IL6 phosphorylation in TEC.
NF-B binding activity was assayed by EMSA in cytoplasmic and nuclear
extracts prepared from TEC previously scored negative for residual
CD1+ or CD2+ cells by flow cytometry. As shown
in Fig 1A, the constitutive nuclear NF-B
binding activity found in untreated TEC was increased more than
3.5-fold after 3 hours of thymocyte contact. This increase was
consistent with the two thirds decrease of NF-B activity observed in
the cytoplasm at the same time point, indirectly indicating that
hyperphosphorylation and subsequent proteolysis of NF-B inhibitors
had occurred.20 The subunit composition of translocated complexes was investigated by binding assays performed in the presence
of antisera that recognize the p65 or the p50 proteins. As evidenced by
the changes of NF-B gel retardation pattern specifically induced by
each antiserum, the translocated complexes found to be increased after
3 hours of thymocyte contact contained both p65 and p50 subunits, which
have been previously shown to constitute transcriptionally active
heterodimers.19 TEC extracts were next examined for the
presence of serine-phosphorylated NF-IL6 isoforms, because previous
findings have demonstrated that NF-IL6 protein and DNA interactions are
positively regulated by serine/treonine phosphorylation of its
functional domains.11,17,18 We therefore performed Western
blot analysis of (1) cytoplasmic and nuclear lysates immunoblotted with
an anti-C-terminal NF-IL6 antiserum recognizing all protein isoforms
(Fig 1B) and (2) NF-IL6 immunoprecipitates probed with
antiphosphoserine MoAbs (Fig 1C). Cell extracts were prepared from
14C-metabolically labeled cells so that immunoprecipitates
could be loaded at equal amounts of counts. As shown in Fig 1B, the cytoplasmic NF-IL6 pool was composed of 24-, 36-, and 43-kD isoforms, whereas the nuclear pool was mainly constituted of the 43-kD form. The
cytoplasmic pool was increased after 1 hour of thymocyte contact, whereas the nuclear 43-kD isoform remained quantitatively unchanged in
the same experimental conditions. By contrast, as shown in Fig 1C,
increasing amounts of 43- and 36-kD phosphorylated proteins were
immunoprecipitated from cytoplasmic (2-fold over control) and, more
importantly, from nuclear extracts after 3 and 5 hours of adhesion with
thymocytes (more than 10-fold over control). The latter results were
further confirmed by cross-immunoblotting of antiphosphoserine MoAb
immunoprecipitates probed with anti-NF-IL6 antiserum (not shown).
Supernatants of thymocytes cocultured with TEC for 12 hours failed to
induce NF-B and NF-IL6 activation (Fig 1A, B, and C). These results
demonstrated that posttranslational regulation of NF-B and NF-IL6
transcription factors had occurred in TEC after signals delivered by
thymocyte adhesion rather than by the release of soluble factors during
the contact and led us to investigate whether they were associated with
topographical rearrangements of adhesion molecules at cell contact
sites.
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| Fig 1.
NF-B binding activity and NF-IL6 phosphorylation in
TEC after adhesion to normal thymocytes. (A) NF-B binding activity
of cytoplasmic (Cyt) and nuclear (Nuc) extracts of TEC untreated,
cocultured with freshly isolated thymocyte for 1 and 3 hours or
incubated with thymocyte supernatant (Thy.sup) for 3 hours. Thymocyte
supernatant was obtained from thymocytes cocultured with TEC for 12 hours. EMSA were performed with 6 µg of cell extracts probed with the
32P-labeled IL6-B oligonucleotide. Cytoplasmic proteins
were treated with 0.2% deoxycholic acid before binding to dissociate
NF-B/IB inhibitors. Nuclear complexes contained p50 and p65
subunits, as assessed by the band supershifting obtained with anti-p105
or anti-p65 antisera. DNA binding activity was quantitated by gel
densitometry and expressed as a percentage of untreated controls (top
of the gels). (B and C) PAGE analysis of NF-IL6 phosphorylation and
isoform composition of cytoplasmic and nuclear extracts prepared from
14C-leucine-labeled TEC untreated and cocultured with
thymocytes for 1, 3, and 5 hours or incubated with thymocyte
supernatants for 5 hours. (B) Immunoblotting of cytoplasmic and nuclear
lysate (20 µg) probed with the anti-NF-IL6 antiserum. (C)
Immunoblotting of cytoplasmic and nuclear NF-IL6-immunoprecipitates
(103 cpm/lane) probed with antiphosphoserine MoAbs.
Arrowheads indicate the positions of proteins with apparent molecular
weights of 24, 36, and 43 kD. Sections of gels are shown. The results
are representative of at least three experiments performed
independently by using TEC and thymocytes derived from different
donors.
|
|
Thymocyte adhesion to TEC monolayers induces repolarization of
31 and
64 heterodimers.
The immunofluorescence analysis of TEC monolayers or TEC-thymocyte
cocultures performed for 12 hours at 37°C is shown in Fig 2A. Analysis of TEC monolayers (Fig 2A, a
through d) demonstrated that both 31 and 21 (the latter is
not shown) lined the TEC intercellular boundaries (c shows F-actin and
d shows the corresponding pattern of 3), whereas 64 was
localized at the adhesion surface of TEC to the plastic (a and b show
F-actin and 4, respectively). It is noteworthy that, as previously
described for keratinocytes,28 spread and attached TEC
displayed a submembraneous F-actin feltwork and few organized
microfilament bundles (a) that exclude 64 (compare a and b),
whereas 31 codistributed with the microfilamentous feltwork
(compare c and d) at intercellular boundaries indicated by arrowheads.
As a positive control of TEC cell-to-cell junctions, we used an
antibody to cingulin as a marker of tight junctions (not shown).
Immunofluorescence analysis of TEC-thymocyte cocultures showed that
thymocyte adhesion partially redistributed 31 (h obtained with
MoAb to 3 and g showing the corresponding F-actin pattern) and
64 (f obtained by an MoAb to 4 and e showing the corresponding
F-actin pattern) at the TEC-to-thymocyte contact sites (e through h).
In fact, 64 (f) and 31 (h), but not 21 (not shown),
became enriched at the interface between TEC and engulfed thymocytes.
Note that the focus of frames (e) through (h) is removed from the
substratum adhesion to highlight TEC-to-thymocyte interface.
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| Fig 2.
Immunostaining of thymo-epithelial cocultures. All the
experiments were performed on cells cultured at 37°C under standard
incubation conditions as described in Materials and Methods. (A) a
through d: immunostaining of TEC monolayers for F-actin (a and c), 4
(b), and 3 (d). 4 and 3 stainings are shown coupled to their
F-actin stainings (a and c, respectively). Frames a and b were focused
at the basal surface of TEC monolayers to show the complementary
distribution of F-actin with 4, showing that 4 is excluded from
F-actin-rich areas. Frames c and d were focused slightly above the
plane of adhesion to show intercellular boundaries. e through l:
immunostaining of TEC thymocytes cocultures for F-actin (e and g), 4
(f), and 3 (h). Frames e through h were all focused above the basal
surface of TEC monolayers to show the aggregation of 4 (f) and 3
(h) at TEC-thymocyte interface. Arrowheads in g and h show a TEC
intercellular boundary that is still in focus. (B) Confocal analysis of
4 enrichment at TEC-thymocyte interface (a). Digital reconstruction
and rotation at the dotted line (a) to show the z axis (b) confirm 4
enrichment at the TEC-thymocyte interface. Arrowheads in a and b
indicate the same position before and after rotation.
|
|
This finding prompted the search for laminin 1 and 5, both substrates
for either integrin heterodimers, in between TEC and thymocytes. Both
laminin types were expressed at TEC attachment surface (not shown),
whereas neither was demonstrated at the TEC-thymocyte interface. The
same applied for fibronectin and collagen type IV.
This very unusual localization of 64 at TEC-thymocyte contact
sites was further investigated by confocal analysis (Fig 2B, a and b). Upon the removal of background fluorescence by optical sectioning, it was confirmed that 64 was specifically enriched at
the boundary between thymocytes and the engulfing TEC. Because the
latter are the only source of 64 in our cellular system, we
conclude that the contact of thymocytes induced clustering of 64
within the TEC membrane just where thymocytes came in touch. The
31 confocal pattern was identical (not shown).
These observations indicated that 31 and 64, among the
considered surface antigens, were selectively induced to cluster at the
TEC-thymocyte contact sites by an unknown mechanism that apparently
excluded any known bridging component of the extracellular matrix. The
next step was to investigate whether these integrins were also
functionally involved in the TEC adhesion to thymocytes, in the
assumption that they might specifically provide signals leading to
activation of IL-6 transcription factors.
TEC adhesion to thymocytes is inhibited by MoAbs
anti-1 and, to a lesser exent, anti-4
integrins.
The involvement of 1 and 4 heterodimers in the TEC-thymocytes
adhesion was evaluated by inhibition assays performed using MoAbs
recognizing their extracellular domain. Assays were performed for 1 hour at 37°C in humidified atmosphere of 5% CO2 between TEC monolayers grown to tight confluence to prevent the direct attachment of thymocyte to the plastic and freshly isolated thymocytes previously labeled with the PKH26-GL red fluorochrome dye. As shown in
Fig 3, the absence of divalent cations
(PBS ) in the binding medium yielded 70% inhibition
(70 ± 3.3 SE), indicating that integrin functions were largely
involved in TEC-thymocyte adhesion. Precoating both TEC and thymocytes
with MoAbs anti-1 or anti-4 reduced the thymocyte binding
(58% ± 3% SD and 41% ± 3.5% SD, respectively) when compared
with the untreated controls. A considerable inhibition
(39.5% ± 12% SD) was exerted by MoAbs anti-ICAM-1, whereas a
15% ± 2% SD inhibition was observed in the presence of nonspecific
mouse Ig.
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| Fig 3.
Inhibition of TEC-thymocyte binding by MoAbs anti-1
and anti-4 integrins. Binding assays were performed between TEC
monolayers plated and grown to tight confluence and PKH26-GL
dye-labeled thymocytes (1:5 TEC/thymocyte ratio) for 1 hour at 37°C
in humidified atmosphere of 5% CO2. Purified antibodies
were separately incubated with TEC and thymocytes for 30 minutes before
binding at 5 µg/mL and maintained during the test. PBS
indicates assays performed in PBS
Ca2+Mg2+-free. Nonadherent thymocytes were
removed by gentle washings. TEC and bound thymocytes were detached by
trypsin-EDTA, washed, vortexed to disrupt aggregates, and counted by
flow cytometry. Results were expressed as the ratio of TEC:thymocytes
recovered and as a percentage of the ratio of the untreated controls.
Shown are the mean values ± SD of four independent experiments
performed with TEC and thymocytes obtained from different donors.
|
|
Based on the finding that integrins clustered at the TEC-thymocyte
contact sites were also functionally involved in their adhesion, we
investigated whether MoAb-mediated ligation and/or aggregation
of 3, 6, 1, and 4 could signal to NF-B and NF-IL6 activation. To this purpose, all MoAbs were initially examined as
regards their ability to functionally interact with these integrins on
TEC monolayers by measuring [Ca2+]in fluctuation
elicited by their binding. As summarized in Table 2, an increase in intracellular calcium was
elicited by binding of MoAbs to both and subunits. This
increase required extracellular Ca2+, but not MoAbs
cross-linking (not shown), thereby indicating that signals accross the
membrane were delivered by the divalent binding of the MoAbs to the
3, 6, 1, and 4 extracellular domains.
Cross-linking of MoAbs bound to 3, 6,
1, and 4 integrins is required for
activation of both NF-B and NF-IL6 transcription
factors.
Integrin ligation and cross-linking were then investigated as inducers
of signals leading to NF-B nuclear translocation (Fig 4A) and NF-IL6 phosphorylation (Fig 4B and
C). Nuclear extracts were prepared from TEC incubated at 37°C with
anti-3, -6, -1, or -4 MoAbs for 3 hours with or without the
subsequent addition of F(ab)2 goat antimouse Ig (GAM) and
washed. Control TEC were washed in the same way. NF-B binding
activity and NF-IL6 phosphorylation were evaluated as described for Fig
1. As shown in Fig 4A, considerable increases
(>2.8-fold the GAM-treated controls) of NF-B binding activity were
induced by cross-linking of both anti- or anti- MoAbs, whereas
the activity observed after their divalent binding was comparable to
that observed in the presence of the GAM reagent alone. MoAbs
cross-linking was also required to induce NF-IL6 serine phosphorylation
(3- to 8-fold higher than the GAM-treated controls, depending on the
MoAbs used; Fig 4C), with the exception of the anti-4, whose
divalent binding was followed by a threefold increase of phosphorylated
36- and 43-kD isoforms. As previously observed in cell extracts
prepared from TEC stimulated by thymocyte adhesion, the amounts of
NF-IL6 proteins in the nucleus remained unmodified after binding
and/or cross-linking of the various MoAbs (Fig 4B).
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| Fig 4.
NF-B binding activity and NF-IL6 phosphorylation in
TEC untreated and treated with anti-3, -6, -1, and -4 MoAbs
in the presence or absence of cross-linking. (A) NF-B binding
activity of nuclear extracts (6 µg) prepared from TEC untreated or
treated for 3 hours with the various MoAbs at 5 µg/mL. When
indicated, bound MoAbs were cross-linked by the addition of
F(ab)2 GAM at 10 µg/mL for 2 hours. (B and C) PAGE
analysis of NF-IL6 phosphorylation and isoform composition in nuclear
extracts prepared from 14C-leucine-labeled TEC untreated
or treated as previously mentioned. (B) Immunoblotting of nuclear
lysate (20 µg) probed with anti-NF-IL6 antiserum. (C) Immunoblotting
of nuclear NF-IL6 immunoprecipitates (103 cpm/lane) probed
with antiphosphoserine MoAbs. Arrowheads indicate the positions of
proteins with apparent molecular weight of 36 and 43 kD. Sections of
gels are shown. Results shown are representative of three experiments
performed independently by using TEC and thymocytes derived from
different donors.
|
|
IL-6 production by TEC is enhanced after adhesion of thymocytes or
cross-linking of MoAbs bound to 3, 6,
1, and 4 integrins.
IL-6 production by TEC challenged with thymocyte adhesion was
consistent with the observed functional activation of IL-6 gene transcription factors (Fig 5). Thymocyte
contact increased TEC IL-6 secretion, as assessed in the culture
supernatants collected 24 hours after the removal of the cell stimulus
(1.96- ± 0.4-fold SE over controls), whereas supernatants of
thymocytes cocultured with TEC or cultured in TEC medium for 12 hours
exerted negligible activity. The increase of IL-6 production induced by
thymocyte contact was impaired by the incubation of TEC and thymocytes
before adhesion with MoAbs anti-1 or anti-ICAM-1 (30% ± 8% SE
and 25% ± 15%, respectively), whereas 4 activity seemed very
close to the effect of nonspecific mouse Ig (8% ± 6% and
9% ± 4%, respectively).
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| Fig 5.
IL-6 production of TEC after coculture with
unfractionated thymocytes or treatment with MoAbs anti-3, -6,
-1, and -4 integrins in the presence or absence of cross-linking.
Cocultures were performed for 12 hours at 1:5 cell TEC-thymocyte ratio.
Thymocyte supernatants were incubated for 12 hours. Treatment with the
various MoAbs during the thymocyte adhesion was performed as described
in Fig 3. Direct treatment with the same MoAbs of TEC monolayers was
performed as described in Fig 4. Cross-linking with GAM was performed
overnight. IL-6 production was measured by ELISA in culture
supernatants withdrawn 24 and 72 hours after the removal of stimuli and
the replating. Because of the variability of the basal level of IL-6
production by the different TEC cultures (range, 2 to 20 ng/106 cell/d; mean, 8.4 ± 2.6 SE), IL-6 production was
expressed as the ratio of sample/untreated control. Shown are the means ± SE of the ratios observed in two independent experiments performed
with TEC obtained from different donors.
|
|
IL-6 production by TEC was also increased by cross-linking of MoAbs
bound to and integrins ( 2-fold over the GAM-treated controls), whereas the divalent binding was ineffective with the exception of anti-4, followed by a 0.5-fold increase at 72 hours. The analysis of IL-6 accumulation at 72 hours
confirmed the enhancing activity of thymocyte contact, the impairment
exerted by the presence of MoAbs anti-1 (28% ± 12% SE) and
anti-ICAM-1 (23% ± 9% SE), the similarity between the effect of
the presence of MoAbs anti-4 (15% ± 7%) and nonspecific mIg
(14% ± 4% SE), and highlighted the stronger activity exerted by
rather than molecules cross-linking. These results indicate
that the activation of NF-B and NF-IL6 transcription factors induced
by thymocyte adhesion was associated with induced IL-6 gene expression.
Moreover, thymocyte induction could be impaired by MoAbs anti-1 or
-4 integrins and mimicked by clustering of or subunits of
31 and 64 heterodimers.
| |
DISCUSSION |
A great deal of interest has arisen on intracellular signaling and the
mechanisms of gene expression induced by cell-cell and
cell-extracellular matrix adhesion.33 Cell-cell adhesion was investigated here in a coculture system of TEC and thymocytes obtained from normal donors, aimed at mimicking in vitro some of the
thymo-epithelial interactions occurring physiologically within the
thymus. With our culture procedure, we reproducibly obtained the
development of organized monolayers of TEC that display a homogeneous
morphology, retain their surface phenotype throughout the various
culture passages, and produce their own matrix. Their lack of VCAM,
together with the uniform expression of the 3 integrin recognized by
the 10.1.2 MoAb, is suggestive of a medullary, rather than cortical,
origin.28,31 TEC cultures maintained in vitro the
constitutive production of variable amounts of IL-6. It has been
reported that cytokines and growth factors induce IL-6 production in
TEC through mechanisms most likely affecting the messenger stability.3,4 In the present report, we
suggest that IL-6 production can be upregulated at the transcriptional
level by signals delivered by integrin recruitment after thymocyte
adhesion. In fact, adhesion to thymocytes triggered in TEC the
intracellular cascade leading to the simultaneous presence in the
nucleus of activated NF-B and NF-IL-6 transcription factors, which
are known to synergize in the inducible IL-6 gene
expression.13,35 It is noteworthy that the nuclear pool of
phosphorylated NF-IL6 was mainly constituted by the 43- and 36-kD
isoforms previously shown to be endowed with transcriptional activity
in humans.15 Additionally, NF-B and NF-IL6 activation
was followed by increased cytokine secretion, thus indicating that the
two phenomena were associated.
The finding that thymocyte supernatants failed to activate NF-B or
NF-IL6 transcription factors ruled out the functional involvement of
cytokines released by thymocytes in the activation of IL-6 gene
expression and led us to investigate the inducing activity of adhesion
molecules. Among them, we focused on 31 and 64 integrins
because of (1) their repolarization at the TEC-thymocytes cell
boundaries in the cocultures and (2) the involvement of their subunits in the TEC-thymocyte binding.
TEC grown up to organized monolayers displayed, in fact, two sets of
integrins polarized at discrete locations, 21 and 31 heterodimers, mostly located at the TEC-TEC boundaries, and the laminin
receptor 64, sharply polarized at the basal domain of the
monolayer and strictly facing the basement membrane. This pattern was
remodeled by thymocyte contact so that 31 and 64, but not
21 heterodimers, clustered around individual thymocytes, therefore indicating that the latter trigger the local aggregation of
otherwise located molecules without any evidence of simultaneous local
enrichment of laminin 5, their natural common ligand.36 The
functions of 31 and 64 heterodimer in TEC-thymocyte binding could not be directly investigated (ie, by blocking experiments with
inhibiting peptides), because both heterodimers recognize conformational structures,37,38 and it was hence evaluated by MoAbs recognizing their extracellular domains. The strongest inhibition, reaching almost a 60% reduction of the untreated controls, was exerted by MoAbs anti-1 integrins. The inhibition observed in
the presence of anti-4 MoAbs was also considerable, particularly if
compared with the activity of MoAbs anti-ICAM-1 molecules, which are
well known components of the molecular pairs mediating the
lympho-epithelial adhesions.33,39 Consistent with these results, the selective ligation of 1 and ICAM-1 receptors during the
thymocyte adhesion impaired the overall adhesion potential of the
TEC-thymocyte interaction, hence decreasing the TEC IL-6 production
induced by thymocyte contact. Anti-4 MoAbs seemed uneffective in the
same experimental context. However, it has to be considered that any
inhibitory effect due to 4 ligation would be counteracted by the
peculiar signaling properties of this integrin (see below). The
thymocyte ligands that may recognize 31 and 64 heterodimers
are still unknown. It has been reported that 31 heterodimers can
interact homotypically in keratinocytes.40 If a similar
interaction occurs between TEC and thymocytes, the predominant
expression of this integrin on very immature (double negative) and
mature (single positive) thymocytes41 could affect the
intrathymic migration and hence the differentiation of discrete thymocyte subsets. On the other hand, the inhibition exerted by MoAbs
anti-4 chains, together with the lack of detectable laminins interposed at the cell boundaries, raise the hypothesis of a novel laminin-like thymocyte ligand, possibly expressed by a restricted thymocyte subset. Further studies aimed at investigating these points
are required.
Functional studies performed with the same MoAbs also demonstrated that
recruitment of 31 and 64 heterodimers at the TEC surface
can lead to activation of IL-6 transcription. Cross-linking of or
subunits in fact yielded both NF-B translocation and NF-IL6
phosphorylation. By contrast, with the exception of 4, which induced
NF-IL6 phosphorylation and a detectable increase of IL-6 production,
the divalent ligation of 3, 6, and 1 was ineffective. This
occurred despite the fact that all MoAbs used for the analysis were
functionally active, as demonstrated by the calcium transients elicited
by their binding. The peculiar behavior of 4 might be possibly
related to the properties of its large cytodomain, endowed with many
potential docking sites for kinase activity42 and
indirectly connecting the extracellular domain with the
cytoskeleton.43 Our observations disagree with a recent
report showing NF-B translocation in monocytic cells upon 1
ligation with F(ab)2 MoAbs.44 However, the
different cell lineages used in our and in the reported experiments may possibly explain this discrepancy.
Consistent with the requirement of cross-linking for inducing NF-B
and NF-IL6 activation, integrin cross-linking was also required for
induction of IL-6 production. The finding that single subunits of the
heterodimers may signal in a similar or even greater way than
thymocytes could seem paradoxical. Rather, it has to be considered that
MoAb cross-linking can theoretically recruit all molecules expressed at
the TEC surface, whereas TEC interaction with thymocyte ligands can be
affected by ligand(s) expression and concentration, the number of cells
expressing the molecule(s) at the surface, the affinity-avidity state
of integrin receptors, and the adhesive/deadhesive functions of
proximal receptors within the membrane environment.
Although we did not investigate the signaling pathway(s) linking
integrin recruitment at the TEC surface with posttranslational regulation of IL-6 gene transcription factors, current evidences indicate that thymocytes can trigger 31 and 64 recruitment and/or aggregation at the TEC surface. This phenomenon, among many others occurring at the same time, may, in turn, initiate the
intracellular cascade leading to IL-6 gene expression.
Because IL-6 receptors are shared by both thymocytes and
TEC45 and IL-6 is implicated in TEC growth4 and
EGF-mediated differentiation,46 our results imply that
thymocytes can regulate the expression of a cytokine exerting autocrine
and paracrine activities within their microenvironment. Moreover, our
findings may be also of help to investigate the molecular mechanisms
regulating IL-6 gene expression in autoimmune diseases such as
miasthenia gravis,6 where the IL-6 overproduction
has been related to the thymic morphological and functional
abnormalities.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge Dr M. Merola and Prof M. Palmieri
(Institute of Biological Chemistry, University of Verona, Verona,
Italy) for discussion and helpful suggestions concerning IL-6
transcription factor experiments.
| |
FOOTNOTES |
Submitted December 15, 1997;
accepted July 10, 1998.
Supported by Grants ISS-AIDS No. 9306.34 (1995) to G.T. and 9404-20 (1996) to P.C.M. P.C.M. was also supported by grants from CNR target
project "Applicazioni Cliniche della Ricerca Oncologica" (ACRO),
from AIRC (Associazione Italiana per la Ricerca sul Cancro, Milano),
and from Telethon (Grant No. 762).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Dunia Ramarli, MD, Istituto di Immunologia
e Malattie Infettive, Policlinico Borgo Roma, Via delle Menegone, 37134 Verona, Italy; e-mail: dunia{at}borgoroma.univr.it>.
| |
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Abstract
Introduction