Gut mucosal T cell lines from ankylosing spondylitis patients
are enriched with E 7 integrin
N. Van Damme1*, D. Elewaut1*, D. Baeten1, P. Demetter2, C. Cuvelier2,
G. Verbruggen1, H. Mielants1, E.M. Veys1, M. De Vos3, F. De Keyser1
Departments of Rheumatology1, Pathology2 and Gastroenterology3,
Ghent University Hospital, Ghent, Belgium.
Abstract
Objective
An intriguing link between gut and synovial inflammation exists in patients with spondyloarthropathy (SpA), illustrated by the high frequency of microscopically inflammatory gut lesions observed in these patients. We hypothesise
that aberrant homing of mucosal T cells might play a role in the induction/perpetuation of arthritis in SpA. Here, we
analyse the expression of the homing molecules 4 7 and E 7 on mucosal T cells from patients with ankylosing
spondylitis (AS) and controls, in view of the critical role of these receptors in the homing of mucosal lymphocytes.
Methods
Colonic biopsy specimens were obtained from patients with AS (n = 23) and controls (n = 30). Biopsy specimens
were immunostained, treated for extraction of intraepithelial lymphocytes (IEL) and lamina propria lymphocytes
(LPL) or cultured in the presence of IL-2. The expression of the 7 integrins was investigated.
Results
In situ no differences were observed in E 7 and 4 7 integrin expression in isolated IEL and LPL, whether
determined by flow cytometry or by immunohistochemical staining. In gut mucosal T cell lines, E 7 expression
was significantly higher in the mucosa of patients with AS compared with controls. 4 7 was highly expressed on T
cells in both groups studied. Mucosal T cells either expressed only the 4 7 integrin or co-expressed the 4 7 and
E 7 integrins. Almost none of them expressed only the E 7 integrin.
Conclusion
In gut mucosal T cell lines from patients with AS an increased expression of E 7 was observed.
Key words
E 7 integrin, ankylosing spondylitis, mucosal lymphocytes.
Clinical and Experimental Rheumatology 2001; 19: 681-687.
*These authors contributed equally to this
work.
N. Van Damme*, PhD; D. Elewaut*, MD,
PhD; D. Baeten, MD, PhD; P. Demetter,
MD; C. Cuvelier, MD, PhD; G. Verbruggen, MD, PhD; H. Mielants, MD,
PhD; E.M. Veys, MD, PhD; M. De Vos,
MD, PhD; F. De Keyser, MD, PhD.
This work was supported by FWO-Vlaanderen grants 3.0028.95 and 3.0022.96 and
by a concerted action grant GOA96001 of
the Ghent University.
Please address correspondence and reprint
requests to: Nancy Van Damme, PhD,
Ghent University Hospital, Department of
Rheumatology 0K12IB, De Pintelaan 185,
9000 Ghent, Belgium.
E-mail: [email protected]
© Copyright CLINICAL AND
EXPERIMENTAL RHEUMATOLOGY 2001.
Abbreviations: Mab= monoclonal
antibodies; SpA=spondyloarthropathy; AS= ankylosing spondylitis.
Introduction
The mucosal immune system plays an
important role in the pathogenesis of
the spondyloarthropathies (SpA), as
indicated by the high frequency of microscopically inflammatory gut lesions
unrelated to clinical gastrointestinal
symptoms observed in these patients
(1,2). Furthermore, an evolution to clinically overt inflammatory bowel disease (IBD), particularly Crohn’s disease (CD), has been observed in 7% of
SpA patients (3, 4).
Interestingly, a strong relationship between gut and joint inflammation was
observed in SpA: remission of joint
inflammation was always linked to a
disappearance of the gut inflammation,
whereas persistence of locomotor inflammation was usually associated
with the persistence of gut inflammation (2).
Although the precise origin of the link
between the gut and joints is unknown,
a recirculation of effector T cells has
been suggested (5,6). This process of
lymphocyte trafficking is regulated by
a set of adhesion molecules mediating
the interaction between lymphocyte receptors and their ligands on specialized
postcapillary venules termed “high
endothelial venules” (HEV). Lymphocyte homing to the intestine is mainly
regulated by 7 integrins. The 7 integrins comprise two members, 4 7 and
E 7. E 7 is strongly expressed on
intraepithelial lymphocytes (IEL) and
lymphocytes migrating to the mucosa;
4 7 is crucial for lymphocyte homing
to Peyer’s patches and lamina propria
through interaction with the mucosal
vascular addressin MAdCAM-1 (7).
Whereas the lymphocyte/ HEV interactions in peripheral lymph nodes (PLNs)
are predominantly regulated by Lselectin, lymphocyte migration to
Peyer’s patch HEV and mesenteric
lymph nodes (MLNs) is mediated by
both L-selectin and 7 integrins (8).
We previously reported that synovial tissue of patients with early SpA (disease
duration of <1 year) is enriched with
7 integrins and especially E 7 (6).
The ligand of E 7 is E-cadherin, a
transmembrane glycoprotein localised
to the zonula adherens junctions of all
normal epithelia (9). In epithelial cells,
682
homophilic intercellular adhesion is
mediated by E-cadherin. Previously, an
upregulation of the E-cadherin/catenin
complex in acute and chronic active subclinical gut inflammation was found in
SpA patients (10), as well as in Crohn’s
disease (11).
The aim of the present study was to
investigate the levels of expression of
these crucial homing receptors 4 7
and E 7 on mucosal T cells from ankylosing spondylitis (AS) patients
compared with controls.
Materials and methods
Patients and samples
Fifty-three patients were included in
this study after informed consent was
obtained: 23 patients with AS and 30
controls. All of the patients with AS
fulfilled the New York criteria (12). The
clinical data on the patients are summarised in Table I. The controls conTable I. Clinical characteristics of patients
with ankylosing spondylitis included in this
study.
Pt.
Sex
Age
(years)
Treatment
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
F
F
M
M
M
F
F
M
M
F
M
M
F
F
M
M
M
M
M
M
M
M
M
37
74
32
40
37
38
34
57
27
17
56
58
40
45
38
38
26
31
44
20
40
36
47
Sulphasalazine
None
NSAID
None
None
None
None
NSAID
NSAID
None
NSAID
NSAID
NSAID
Sulphasalazine, NSAID
NSAID
None
Sulphasalazine, NSAID
NSAID
NSAID
None
Sulphasalazine, NSAID
Sulphasalazine, NSAID
Sulphasalazine, NSAID
Biopsy specimens from patients 1-5 were used
for immunohistochemistry; from patients 6-17
for isolation of intraepithelial and lamina propria
lymphocytes and from patients 18-23 for the
generation of T cell lines.
sisted of a group of patients that had
undergone colonoscopy as a screening
examination for colorectal cancer. No
bowel complaints or abnormalities in
the stool pattern were present in the
controls and all colonoscopies were
normal. This study was approved by
the Ethical Committee of the local Faculty of Medicine.
Immunohistochemistry
Colonic biopsy specimens from 5 AS
patients (patients 1-5 in Table I) and 9
controls were used for immunohistochemistry. Parallel 5 µm thick sections
of freshly frozen bowel mucosa were
immunohistochemically stained with
anti- E 7 (CD103, clone 2G5.1, ProBio, Margate Kent, UK) and with anti4 7 (Act-1, kindly provided by Dr.
Ringer, Leukosite, Cambridge, UK).
Cryostat sections were fixed in acetone
for 10 min. Each section was incubated
for 30 min with anti- E 7 (1/250) and
anti- 4 7 (1/100). Parallel sections
were incubated with irrelevant isotopematched monoclonal antibodies (Mab)
as a negative control. After rinsing, the
endogenous peroxidase was blocked
with 1% hydrogen peroxide. The sections were subsequently incubated for
15 min with a biotinylated anti-mouse
secondary antibody, followed by 15
min with a streptavidin-peroxidase
complex (LSAB + Kit, Dako, Glostrup,
Denmark). The colour reaction was
developed using 3-amino-9-ethylcarbazole (AEC) substrate (Dako) as chromogen. Finally, the sections were
counterstained with haematoxylin. All
incubations were carried out at room
temperature and the sections were
washed with phosphate-buffered saline
(PBS, Gibco BRL, Grand Ysland, NY,
USA) between all steps.
Stained sections were coded by two
independent observers using a semiquantitative method, where zero represented the lowest and three the highest
level of expression. Analysing E 7 in
the epithelial layer was done by counting the E 7+ cells per 100 epithelial
cells.
Isolation of intraepithelial and lamina
propria lymphocytes
Colonic biopsy specimens obtained
from 12 AS patients (patients 6-17 in
Table I) and 12 controls were used for
the isolation of IEL and lamina propria
lymphocytes (LPL), as previously described (13). From each patient, 10 colonic biopsy specimens were collected
in 10 ml PBS containing CaCl2 (100
mg/l) (Sigma, Saint Louis, Missouri,
USA). The biopsies were placed between two cover glasses and squeezed
out to enrich the LPL. The cover glasses were rinsed with PBS containing
CaCl2 (100 mg/l). All the material was
collected and placed over a cellstrainer
(70 µm, Becton Dickinson (BD), San
Jose, CA, USA) to obtain a single cell
suspension. After the isolation of LPL,
the biopsies were placed in 5 ml without CaCl2 and stirred for 20 min at
37°C to obtain a mixture of LPL and
IEL. The remaining tissue fragments
were collected in 5 ml PBS without
CaCl2 and stirred for another 60 min a t
37°C. The material obtained was
placed over a cell strainer and the suspension constituted an enrichment of
IEL. The obtained suspensions were
centrifuged at 1800 rpm for 10 min and
the pellet was resuspended in PBS.
T cell lines from gut mucosal biopsies
Colonic biopsy specimens obtained
from 6 AS patients (patients 18-23 in
Table I) and 9 controls were transferred
into separate wells on a 24-well culture
plate (Gibco) and incubated in RPMI
1640 medium (Gibco) containing recombinant IL-2 (50 U/ml; Eurogenetics, Belgium), 10% autologous serum,
antibiotics (10 U/ml penicillin-G, 10
U/ml streptomycin sulphate, 0.025 µg/
ml amphotericin-B and 50 µg/ml gentamycin) and 0.5% L-glutamine. After
culturing, the cells were scraped with a
policeman, washed with PBS, and resuspended in PBS at 1-5 x 106 cells/ml
according to yield.
Flow cytometry
Phenotypic analysis of the expanded
and isolated lymphocytes was performed using Mab conjugated with either
fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll
protein (PerCP) or allophycocyanin
(APC). The following Mab were applied: anti-CD3 (Leu4, clone SK7;
683
BD), anti-CD4 (Leu3a, clone SK3;
BD), anti-CD8 (Leu24, clone SK1;
BD) and anti- E 7 integrin (CD103,
HML-1, clone 2G5, Immunotech, Marseille, France). Studies of 4 7 expression were performed by indirect staining with Act-1 Mab. Isotype-matched
immunoglobulins that did not react
with human leukocytes were used as
controls (BD).
For fluorochrome conjugated Mabstainings, aliquots (100 µl) were incubated with the appropriate amount of
Mab in the dark for 30 min at 4°C.
Cells were washed with 2 ml PBS and
centrifuged at 1800 rpm for 10 min.
Indirect labeling for 4 7 was performed as follows: (1) cells were incubated with unconjugated anti- 4 7
Mab in the dark for 30 min at 4°C; (2)
after washing with 2 ml PBS, cells
were incubated with goat anti-mouse
IgG FITC for 30 min at 4°C; and (3) after another washing step, PE-, PerCPand APC-conjugated Mab were added
and incubated for 30 min at 4°C, washed with 2 ml PBS and centrifuged at
1800 rpm for 10 min. After staining,
the cells were fixed with 500 µl PBS
containing 1% paraformaldehyde and
stored in the dark at 4°C until analysis.
The surface expression of antigens was
determined by quantitative three-colour
and four-colour analysis with a FACSort (BD). Isotype matched controls
were used to establish the non-specific
staining to the samples and to set the
fluorescent markers. Routinely, 10,000
events from each sample were collected. Data were analysed using Cellquest® and Attractors® software (BD).
Statistical analysis
Values are expressed as the percentage
of positive cells and the median and
range are displayed. The Mann-Whitney U test was used to compare groups.
A p < 0.05 was considered statistically
significant.
Results
Immunohistochemistry
Immunohistochemistry was performed
on colon biopsy specimens from 5 patients with AS and 9 controls. The
staining for E 7 integrin expression
in the epithelial layer was scored as the
number of E 7+ cells per 100 epithelial cells and was not different in AS
patients compared with controls (median: 8, range: 6-14 and median: 4, range:
0-14, respectively). In the lamina propria E 7 expression was not different
in patients with AS compared with controls (median: 1, range: 0-3 and median: 1, range: 0-2, respectively) (Fig.1).
4 7 integrin expression was only analysed in the lamina propria, as 4 7
integrin expression is absent on IEL.
No differences were observed in AS
compared with controls (median: 2,
range: 1-3 and median: 2.5, range: 1-3,
respectively) (Fig. 2).
Flow cytometric analysis of isolated
IEL and LPL
Intraepithelial and lamina propria lymphocytes were isolated from colonic
biopsies from 12 controls and from 12
patients with AS. The phenotype of the
lymphocytes was determined by flow
cytometry and the results are presented
in Table II. No differences were observed in E 7 and 4 7 integrin expression (Fig. 3).
Fig. 1. Immunohistochemical staining of E 7 in AS colon.
Adhesion molecule expression among
T cell lines from gut mucosal biopsies
The expanded mucosal T cells predominantly consisted of T cells with mixed
CD4+ and CD8+ cells (Table III). Characterisation of the adhesion molecule
profiles on expanded colonic lymphoid
cells was assessed, with special reference to the 7 integrins 4 7 and E7. In both AS and controls, 4 7 integrin expression was extremely high on
CD3+ T cells and on the CD4+ and
CD8+ T cell subsets. No differences were
Fig. 2. Immunohistochemical staining of 4 7 in AS colon.
Table II. Adhesion molecule expression on isolated LPL and IEL from colon in patients with ankylosing spondylitis (AS) and controls.
LPL
Cellular subsets
Controls (n = 12)
IEL
AS (n = 12)
Controls (n= 11)
AS (n = 12)
% CD3+CD4+/CD3+
71
(48-84)
66
(54-70)
15
(10-33)
12
(3-24)
% CD3+CD8+/CD3+
22
(11-42)
26
(20-38)
60
(49-78)
67
(53-85)
ratio CD4/CD8
3.2
(1.8-7.7)
2.5 (1.4-3.6)
0.3
(0.1-0.6)
0.2
(0.05-0.5)
% 4 7/CD3+
65
(55-82)
70
(52-73)
8
(1-40)
12
(4-78)
% E 7/CD3+
22
(15-48)
21
(14-32)
76
(63-83)
82
(71-93)
% E 7/CD4+
7
(0-14)
6
(3-12)
19
(12-35)
17
(9-40)
% E 7/CD8+
65
(34-93)
62
(35-76)
92
(80-98)
94
(88-99)
Results are expressed as the median and range. IEL: intraepithelial lymphocytes; LPL: lamina propria lymphocytes.
684
expressed only the 4 7 integrin or coexpressed the 4 7 and E 7 integrins,
indicating that these lymphocytes originated from the lamina propria. Almost
none of the expanded T cells expressed
only the E 7 integrin (Fig. 5).
Fig. 3. Histograms of E 7 and 4 7 positive IEL and LPL cells from normal controls (upper panel)
and from patients with AS (lower panel). Stained cells were gated for CD3+ cells. Representative histograms are shown.
Table III. Adhesion molecule expression on gut mucosal T cell lines in patients with ankylosing spondylitis (AS) and controls.
Cellular subsets
Controls (n = 9)
AS (n = 6)
% CD3+CD4+/CD3+
48
(2-64)
38
% CD3+CD8+/CD3+
36
(25-98)
47
(5-69)
ratio CD4/CD8
1.4
(0-6)
0.7
(0.4-20)
(51-99)
(24-92)
% 4 7/CD3+
93
(85-98)
93
% 4 7/CD4+
93
(65-98)
95
(61-99)
% 4 7/CD8+
94
(75-97)
97
(89-99)
(2-19)*
% E 7/CD3+
1.8
(1-9)*
11
% E 7/CD4+
0.8
(0.5-13)
5
(1-20)
% E 7/CD8+
5
(2-24)°
15
(12-40)°
Results are expressed as the median and range and were obtained by making the mean of the different
cell lines from the same patient. Statistical significant differences (p < 0.05) are noted:*p = 0.025,°p =
0.022.
observed between AS patients and controls (Table III). The other 7 integrin,
E 7 was upregulated in patients with
AS compared with controls on CD3+ T
cells (p=0.025) and on CD8+ T cells (p
= 0.022) (Table III, Fig. 4).
We also determined the levels of 4 7
and E 7 expression on CD4 and CD8
T cells at the single cell level by fourcolour flow cytometry: T cells either
685
Discussion
The pathogenesis of IBD and SpA remains unresolved, but there is evidence
that mucosal inflammation and tissue
damage are mediated by substances
released from activated immune cells,
including T lymphocytes. The role of T
lymphocytes in IBD and SpA is suggested by the following observations:
a) the immune infiltrate in synovial
membrane and gut mucosa (mainly
lamina propria) in patients with SpA
and IBD predominantly consists of T
cells (14,15); b) activated mucosal T
cells are able to induce epithelial lesions resembling those observed in
IBD (16); c) MHC linkage of the SpA
disease complex suggests a role for
antigen-specific T cell activation (17,
18); and d) HLA-B27 transgenic rats
develop a systemic inflammatory disease resembling human SpA (19). In
this model, T lymphocytes play a critical role as athymic rats are resistent to
the disease.
A strong relationship exists between
gut and joint inflammation, although
the precise origin of this link is unknown. A process of lymphocyte trafficking has been suggested and lymphocyte homing to the intestine is mainly
regulated by the 7 integrins (20,21).
Previously, upregulated E 7 integrin
expression was found on activated synovial T cells from patients with SpA (6)
and on mucosal T cells from patients
with IBD (22). The ligand of the E 7
integrin is E-cadherin, expressed by
epithelial cells in the gut mucosa. An
upregulation of the E-cadherin/catenin
complex was found in patients with
SpA (10) and in Crohn’s disease (11).
In the present study the in situ expression of E 7 and 4 7 was analysed
by immunohistochemistry and by flow
cytometry on isolated IEL and LPL. No
differences in E 7 and 4 7 expres sion were observed.
The pathogenic relevance of IL-2 expanded T cell lines in autoimmune dis-
ating lymphocytes (27).
The present study documents a significant increase in E 7 integrin on gut
mucosal T cell lines from patients with
AS compared with controls. The increase in E 7 integrin expression was
present on CD8+ T cells, but not on the
CD4+ T cell subset. In previous studies, increased E 7 integrin expression
was also observed in patients with
Crohn’s disease (22). In the present
study, increased E 7 integrin expression was observed in patients with no
histological signs of gut inflammation,
pointing to the presence of “Crohnlike” immune alterations in the mucosa
of SpA patients preceding the occurrence of histological inflammation.
Fig. 4. Increased expression of E 7 integrin on mucosal T cells expanded from the mucosa of ankylosing spondylitis (AS) patients. Diagram shows flow cytometric expression of E 7 integrin on T
cells expanded from the colon of patients with AS and controls. Box plot: dots represent values of the
different T cell-lines obtained; solid line = median, box = interquartile rang e, error bar = rang e. Data
for CD3+ and CD8+ T cells are shown.
orders is supported by several observations. Firstly, an important argument
for the selective enrichment of diseaserelated T lymphocytes under IL-2 stimulation comes from T cell studies in
multiple sclerosis (MS) (23). In this
disease, myelin basic protein (MBP) is
considered to be an important autoantigen. Several reports have pointed to a
pathogenic role of MBP reactive T cells
in this disease (23-26). In humans MBP
reactive T cells can be found both in
MS patients and in controls (23). Interestingly, the relative frequency of MBP
reactive T cells in the cerebrospinal
fluid and peripheral blood of MS patients increases significantly after expansion with IL-2 compared to nonexpanded T cells (23-26). Secondly,
clonally restricted IL-2 expanded T
cells from rheumatoid nodules have
been shown to contain highly related
CDR3 sequences, strongly suggesting
an antigen-driven T cells activation in
situ (27). Those IL-2 expanded T cells
predominantly represent in situ activated IL-2R positive cells, and they are
enriched for tissue inflammation-medi-
Acknowledgements
We are indebted to J. Vermeersch for
excellent technical assistance.
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