Download Reduced Immunoregulatory CD31 T Cells in Patients With

Reduced Immunoregulatory CD31ⴙ T Cells in Patients With
Atherosclerotic Abdominal Aortic Aneurysm
Giuseppina Caligiuri, Patrick Rossignol, Pierre Julia, Emilie Groyer, Dikran Mouradian,
Dominique Urbain, Namita Misra, Véronique Ollivier, Marc Sapoval, Pierre Boutouyrie,
Srini V. Kaveri, Antonino Nicoletti, Antoine Lafont
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Background—Cell-mediated immunity is considered to contribute to the pathogenesis of abdominal aortic aneurysms
(AAA). In particular, infiltrating macrophages and CD8⫹ T lymphocytes participate in the destruction of the aortic wall
extracellular matrix and smooth muscle cells. We surmise that these pathological events are controlled by circulating
regulatory lymphocytes.
Methods and Results—Circulating CD4⫹/CD31⫹ cells were reduced in AAA patients (n⫽80, 8.9⫾0.6%) as compared with
controls (n⫽69, 13.7⫾0.8%; P⬍0.001) and inversely proportional to AAA size. Exclusion of the aneurysm by an
endoprothesis did not affect CD31⫹ T cell values. Reduction of blood CD4⫹/CD31⫹ cells was not attributable to their
enrichment in AAA tissue. In contrast, CD8⫹/CD31⫹ cells were slightly reduced in the blood while increased in the
aneurysmal tissue (29.2⫾0.5 versus 20.2⫾4.7% in blood, n⫽6; P⬍0.05). Remarkably, high percentages of CD4⫹/
CD31⫹ cells were able to regulate proliferation and cytokine production of CD8⫹ lymphocytes, as well as CD8⫹
cell-mediated cytotoxicity of aortic smooth muscle cells (P⬍0.01). Finally, CD4⫹/CD31⫹ cells reduced the production
and activity of metalloproteinase-9 by lipopolysaccharide-stimulated macrophages.
Conclusions—Circulating CD4⫹/CD31⫹ T cells regulate macrophage and CD8⫹ T cell activation and effector function in
the arterial wall. Their reduction might promote the development of AAA. (Arterioscler Thromb Vasc Biol. 2006;26:
618-623.)
Key Words: aortic diseases 䡲 immune system 䡲 blood cells
teases.7 Furthermore, the AAA infiltrating CD8⫹ T lymphocytes are able to produce cytotoxic mediators such as
cytokines, perforin, and Fas/FasL and thus contribute to
elimination of SMC, a source of elastin and collagen.5 This
hypothesis is further supported by a recent study showing that
IFN-␥– and perforin-producing CD28⫺/CD8⫹ T cells are
present in the vessel wall of a majority of AAAs.8
Although a number of autoreactive T cells are physiologically maintained in the peripheral lymphocyte pool, the
occurrence of such a T-cell mediated attack to autologous
vascular SMC with extensive tissue damage implies a loss of
the physiological self-tolerance allowing the release of pathologic autoreactive T cell responses.
Several studies suggest that defective immune control by
regulatory (suppressor) T cells is at the origin of pathologic
autoreactive immune responses.9 In addition to the well
known CD4⫹/CD25⫹ T cells,9 diverse and organ-selective
regulatory T cells appear to control distinct immune-mediated
diseases.10 CD4⫹/CD31⫹ T cells, which display immunoregu-
A
bdominal aortic aneurysms (AAA) are a common clinical manifestation of longstanding atherosclerosis.1 Although the similarity of risk factors for occlusive and aneurysmal aortic diseases2 suggests that atheroma is the initial
pathway, the opposite issue may depend on different local
responses to atherosclerosis in the abdominal aorta.3 Intimal
accumulation of material including lipids, matrix proteins,
and cells without thinning of the medial layer may predispose
to occlusive atherosclerotic disease. In contrast, extracellular
matrix degradation and smooth muscle cell (SMC) death
constitute the key features of the aneurysmal forms of
atherosclerotic aortic disease.4,5
Recent studies have shown that macrophages and lymphocytes are present in AAA tissue6 suggesting that cellmediated immune responses might be involved in the pathogenesis and progression of AAA. Lymphocyte–macrophage
cell– cell contact in the arterial wall can potentiate the
inflammatory response that leads to the destruction of extracellular matrix through enhanced local release of metallopro-
Original received May 5, 2005; final version accepted December 5, 2005.
Institut National de la Santé et de la Recherche Medicale (INSERM), EMI0016 (G.C., D.U., A.L.), U681 (G.C., E.G., N.M., S.V.K., A.N.), U765 (P.R.),
U698 (V.O.), and U652 (P.B.); Faculté de Médecine René Descartes Paris 5 (G.C., P.R., P.J., D.U., A.L.); Universite Pierre et Marie Curie (UPMC) Paris
6 (E.G., N.M., S.V.K., A.N.); Service de Médicine Vasculaire et Hypertension artérielle (P.R., D.M.), Service de Chirurgie Cardio-vasculaire (P.J.), and
Service de Cardiologie (M.S., A.L.), Hôpital Européan Georges-Pompidou, AP-MP, Paris, France.
Correspondence to Giuseppina Caligiuri, MD, PhD, INSERM U681-Institut des Cordeliers, 15, rue de l’Ecole de Médecine, 75006 Paris, France.
E-mail [email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
618
DOI: 10.1161/01.ATV.0000200380.73876.d9
Caligiuri et al
Reduced Immunoregulatory CD31ⴙ T Cells in AAA
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latory properties in vitro,11,12 may be critical in regulation of
vascular immune-mediated pathologies such as atherosclerotic arterial aneurysms.
CD31 is a transmembrane glycoprotein present on the
surface of endothelial cells, platelets, monocytes, granulocytes, and lymphocytes. Initially CD31 was classified as an
adhesion molecule,13 however, it is now recognized that
CD31 signaling results in cell inhibition.14 Among blood
cells, only T cells lose CD31 in human adults.15 Lack of
CD31 signaling allows enhanced T cell activation16 and a
greater ability to infiltrate atherosclerotic arteries17 where
they are likely to exert a pathogenic role. Our data show an
enrichment of CD4⫹/CD31⫺ T cells in the blood and the
arterial tissues of AAA patients. These cells enhance the
effector capacity of macrophages and of CD8⫹ T cells and
can thus contribute to the pathogenesis of the disease. On the
other hand, the presence of CD31 on the surface of circulating
T cells likely allows not only their own constraint but also
regulation of the interacting blood or vascular cells through
homophilic CD31-CD31 inhibitory signaling. Here we report
that AAA patients display a significant reduction in the
percentage of circulating CD4⫹/CD31⫹ T cells that may be
responsible for an altered regulation of local macrophage and
CD8⫹ T cell responses in the aneurysmal arterial wall.
Methods
Patients and Samples
Patients with a diameter of the abdominal aorta (measured by
echography) larger than 3 cm (AAA, n⫽80 men, 50 to 80 years old)
were prospectively recruited into the study. Age- and sex-matched
Controls (n⫽69) were either patients with essential peripheral
arterial blood hypertension (n⫽32) or apparently healthy subjects
(n⫽37). Subgroup analysis was performed according to the presence
of hypertension and/or atherosclerosis (ie, the presence of carotid
atherosclerotic plaques detected by high definition echotracking).
Exclusion criteria were: cancer, infection, and any other immunemediated disease. The protocol was approved by the local ethical
committee (CCPPRB Paris-Cochin n° 2095, n° 1930 and n° 1931)
and informed consent was obtained from all subjects before sampling. Of the 80 AAA patients included in the study, 30 (37.5%)
were on medical treatment only, 44 (55%) were treated by endoprothesis placement, and 6 (7.5%) by aneurysmectomy. In the latter
(n⫽6), specimens of the anterior AAA wall were collected at the
time of surgical intervention and were freshly treated with collagenase under optimal conditions to obtain cell suspensions for flow
cytometry analysis. Cell viability of these cell suspensions was
⬎90% as detected by propidium iodide positive cell exclusion by
flow cytometry. Heparinized blood samples were obtained in
fasting conditions from all subjects and were immediately processed for flow cytometry analysis.
Flow Cytometry
Blood leukocytes and tissue cell suspensions were incubated with a
cocktail of 4 fluorescent monoclonal antibodies (BD Biosciences)
directed to human CD25 (fluorescein isothiocyanate [FITC]), CD31
(PE), CD4 (PE-Cy5), and CD8 (allophycocyanin [APC]). A minimum of 2000 (AAA tissue) or 10 000 (blood) events in the
lymphocyte gate (FSC-SSC) were acquired and analyzed on a
FacsCalibur (BD Biosciences).
Regulation of CD8ⴙ Lymphocyte Activation by
CD4ⴙ/CD31ⴙ T Cells
CD8⫹ lymphocyte proliferation was elicited by mixed lymphocyte
reaction using allogeneic dendritic cells (DC) as challenger. DC were
619
generated from peripheral blood monocytes of a single healthy
donor, as previously described.18 CD8⫹ and CD4⫹ lymphocytes were
prepared from 6 healthy individuals on the same day and were
isolated by negative selection (DynalBiotech) rather than by positive
sorting to avoid the potentially biasing effect of the cell-bound
antibody on the subsequent functional assays.19 We had initially
performed functional experiments using either total CD4⫹ T cells or
CD4⫹/CD31⫹ and CD4⫹/CD31⫺ cell fractions obtained by positive
fluorescent cell sorting. Significant differences were found between
cultures containing total CD4⫹ as compared with those containing
either CD4⫹/CD31⫹ or CD4⫹/CD31⫺ T cells, whereas no difference
was detected between CD4⫹/CD31⫹ and CD4⫹/CD31⫺ positively
selected cells. Indeed, the binding of the antibody to the CD31 at the
surface of the CD4⫹ cells tended to inhibit the CD31-mediated
interaction with the cognate CD8⫹ cells. Preincubation of total CD4⫹
T cells with the concentrations of anti-CD31 antibody equivalent to
those used for positive cell sorting abolished the observed (regulatory) effect of total CD4⫹ T cells. Therefore, negative selection was
used to deplete CD31⫹ cells from CD4⫹ lymphocyte preparations to
be compared with total CD4⫹ T cells. A monoclonal mouse antihuman CD31 antibody (BD Biosciences) was added at 0.01, 0.1, and
1 ␮g/106 target (CD4⫹) cells to the antibody mix to obtain different
percentages of CD31⫹ cells within CD4⫹ cell preparations. Purity of
enriched CD8⫹ and CD4⫹ T cell populations was always ⬎90% as
assessed by flow cytometry. DC (104 cells per well), CD8⫹ (105 cells
per well), and CD4⫹ (0.5⫻105 cells per well) cells were cocultured
in complete RPMI 1640 containing 10% human male AB serum in
96-well plates. After 5 days, one fourth of cell culture supernatant
was collected and frozen for subsequent ELISA measurement of
IFN-␥ and interleukin (IL)-2 (DuoSet, R&D system); and replaced
with fresh complete medium containing 10␮Ci/mL 3H thymidine.
Proliferation was evaluated after 16 hours in a ␤-counter. Either
CD8⫹ or CD4⫹ cells were omitted in control cultures. CD4⫹ cell
proliferation data were subtracted from CD8⫹ cell proliferation data
and results are expressed as cpm (mean⫾SEM of triplicate values).
Regulation of CD8ⴙ Cell-Mediated Cytotoxicity by
CD4ⴙ/CD31ⴙ T Cells
CD8⫹, CD4⫹, and CD31-depleted CD4⫹ cells were prepared as
described above. Human SMC (ATCC-LGC) were labeled overnight
with a lipophilic green fluorescent dye (DiOC 18 from the Live/Dead
kit, #L7010; Molecular Probes) to be used as allogeneic target (T)
cells (105 cells per well in 8-well glass Labtek culture plates). After
two washes of the adherent SMC, CD8⫹ effector (E) cells were
plated at 4⫻105 cells per well (E:T ratio⫽4:1) either alone or in the
presence of total CD4⫹ or CD31-depleted CD4⫹ T cells (2⫻105 cells
per well) and in the presence or absence of neutralizing antibodies
for Fas-L (0.1 ␮g/mL, MAB126; R&D Systems) and IL-2 (0.1
␮g/mL, MAB202; R&D Systems). SMC alone were used as control
for spontaneous cytotoxicity. After 4 hours, the culture medium was
collected for ELISA measurement (Fas-L, DuoSet; R&D system)
and replaced by a buffer containing a low dose of propidium iodide
(PI). Five minutes later, cells were detached and analyzed by flow
cytometry according to the manufacturer’s instructions (Live/Dead
kit; Molecular Probes). The percentage of cytolysis (%CTL) was
calculated as the number of PI⫹ target cells among total DiOc 18⫹
target cells. Duplicate cultures were cover-mounted (VectashieldDAPI; Vector laboratories) and observed under fluorescent
microscopy.
Regulation of Macrophage Activation by
CD4ⴙ/CD31ⴙ T Cells
Monocytes were enriched by negative magnetic selection (Dynal
Biotech) from the peripheral blood of 3 healthy donors and cultured
at 5⫻104 cells per well for 6 days in RPMI 1640 (10% fetal calf
serum, 20 mmol/L HEPES, 2 mmol/L L-Glu, 50 ng/mL rHu
M-colony stimulating factor [CSF]) in 8-well glass Labtek culture
plates. On day 7, autologous total CD4⫹ or CD31-depleted CD4⫹ T
cells prepared as above were added (2⫻105 cells per well) in the
presence of fluorescence-quenched collagen type IV (100 ␮g/mL,
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Arterioscler Thromb Vasc Biol.
March 2006
Oregon Green 488 conjugate, Molecular Probes), and of the reactive
oxygen species sensor dihydroethidium (20 ␮mol/L; Molecular
Probes) and in the presence or absence of LPS (10 ng/mL). After 3
hours incubation, MMP1/TIMP1 and MMP9/TIMP2 complexes
(DuoSet; R&D Systems) were measured on the supernatant by
ELISA. Adherent cells were thoroughly washed in PBS, detached,
incubated with APC-conjugated anti-CD14 antibody, and analyzed
by flow cytometry. Duplicate cultures were cover-mounted
(Vectashield-DAPI) and observed under fluorescent microscopy.
Statistical Analysis
Results are expressed as Mean⫾SEM. Comparison of groups and
experimental conditions were performed by ANOVA and correlation
between variables by simple regression using Statview 5.0 software
(SAS Institute Inc). Differences between groups were considered
significant if P⬍0.05.
Results
ⴙ
CD31 T Cells Are Reduced in AAA Patients
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The number of total leukocytes, lymphocytes, granulocytes,
and monocytes was similar in AAA patients and controls
(Table I, available online at http://atvb.ahajournals.org). Similarly, no difference was found in the number and percentage
of CD4⫹ and CD8⫹ lymphocytes, as well as that of CD4⫹/
CD25⫹ cells (Table I). Four-color simultaneous staining
allowed determining that ⬎94% of CD4⫹/CD25⫹ cells were
negative for the expression of CD31 at their surface (data not
shown).
The number and percentage of circulating CD4⫹/CD31⫹
and CD8⫹/CD31⫹ lymphocytes was significantly decreased
in AAA patients as compared with controls (Table I), and
reciprocal changes were observed for the CD31⫺ fractions of
CD4⫹ and CD8⫹ blood cells which increased in AAA patients
as compared with controls (Table I). No difference was
detected in CD31⫹ T lymphocyte percentages between patients eligible for endoprothesis placement whether enrolled
before (n⫽18) or after (n⫽26) the treatment (8.9⫾2.0 versus
7.4⫾0.7% of CD4⫹/CD31⫹ cells, NS; and 16.0⫾2.0 versus
18.7⫾1.3% of CD8⫹/CD31⫹ cells, NS).
The reduction of circulating CD4⫹/CD31⫹ cells in AAA
patients was not dependent on the presence of either hypertension or atherosclerosis detected at the level of the carotid
arteries (Table II, available online at http://atvb.ahajournals.
org). Circulating CD8⫹/CD31⫹ lymphocyte reduction in the
blood of AAA patients as compared with controls (Table I)
was particularly evident when neither AAA patients nor
controls showed hypertension (Table II). Linear regression
analysis between the cross-section surface area of the aneurysm (in cm2, calculated from the antero-posterior and laterolateral diameter of the AAA, by echography) and the percentage of CD31⫹/⫺ T lymphocytes showed that blood CD31⫺ T
lymphocytes percentages were directly proportional to the
AAA size, whereas an inverse correlation existed between
blood CD31⫹ T cell percentages and AAA size. These
correlations reached statistical significance in the case of
CD4⫹/CD31⫺ and of CD8⫹/CD31⫹ cells (Figure 1) and not in
the case of CD4⫹/CD31⫹ and of CD8⫹/CD31⫺ cells (data not
shown).
CD8ⴙ/CD31ⴙ But Not CD4ⴙ/CD31ⴙ T Cells Are
Sequestered in AAA Tissue
To evaluate whether the reduction of CD31⫹ T cells in the
peripheral blood of AAA patients is a consequence of their
Figure 1. Correlation between circulating CD31⫹ T cells and
AAA size. Left, The percentage of CD4⫹/CD31⫺ lymphocytes by
flow cytometry was directly proportional to the size (crosssection surface area) of the aortic aneurysm as calculated from
the antero-posterior and latero-lateral diameter obtained by
echography. Right, The percentage of CD8⫹/CD31⫹ lymphocytes in the blood was inversely proportional to the AAA size
calculated as above.
sequestration in the aneurysmal wall where they can infiltrate,
the percentages of CD8⫹/CD31⫹ and CD4⫹/CD31⫹ lymphocytes in blood were compared with those in autologous AAA
tissue in 6 patients that were treated by aneurysmectomy. The
percentage of CD8⫹/CD31⫹ lymphocytes was significantly
increased in the AAA tissue as compared with the peripheral
blood, whereas the percentage of CD4⫹/CD31⫹ lymphocytes
reflected the data from circulating cells (Figure 2).
CD4ⴙ/CD31ⴙ Cells Regulate CD8ⴙ Lymphocyte
and Macrophage Effector Function
DC-stimulated CD8⫹ T cell proliferation and cytokine (IFN-␥
and IL-2) production in CD4 –CD8 cocultures was increased,
in a dose-dependent manner, as CD31⫹ (%CD4⫹ cells)
Figure 2. CD8⫹/CD31⫹ and CD4⫹/CD31⫹ cells in the blood and
tissue of AAA patients. Representative dot-plots of CD8⫹/CD31⫹
(left) and CD4⫹/CD31⫹ (right) cells in the blood (top) and in the
autologous aneurysmectomy tissue (bottom). Data (mean⫾SEM)
for the 6 patients are indicated in the upper right quadrant. The
percentage of CD8⫹/CD31⫹ T cells was significantly enriched in
the aneurysmal tissue as compared with the blood of these AAA
patients (*P⬍0.05 vs blood) whereas the proportion of CD4/
CD31⫹ T cells was similar in the blood and AAA tissue.
Caligiuri et al
Reduced Immunoregulatory CD31ⴙ T Cells in AAA
621
tive oxygen species (Figure 4) were quantified by flow
cytometry within CD14⫹ cells. Values were not affected by
the presence of total CD4 lymphocytes whereas they were
significantly increased in the presence of CD4 lymphocytes
depleted of CD31⫹ cells (Figure 5).
Discussion
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Figure 3. CD4⫹/CD31⫹ T cells regulate CD8⫹ T cell activation
and cytotoxic function. Left, The presence of increasing numbers of CD31⫹ among CD4⫹ cells (0, 20, 40, or 60%) leads to
significantly decreased CD8⫹ cell proliferation (top) and cytokine
production (bottom) induced by allogeneic dendritic cell stimulation. Mean⫾SEM, n⫽6, *P⬍0.05 vs lower dose. Right, Spontaneous cytolysis (CTL, top) of SMC in the absence of T cells was
⬇10%. The addition of CD8⫹ cells alone or in the presence of
CD4⫹/CD31⫺ cells increased the percentage of SMC cytolysis
to a similar extent ⬇50%). Neutralizing antibodies for Fas-L
(MAB126) abrogated more that 90% of CD8 T cell–mediated
cytotoxicity and neutralizing antibodies for IL-2 (MAB202)
reduced SMC cytotoxicity in CD8⫹/CD4⫹CD31⫺ cocultures by
30%. Coculture of CD8 cells with CD4 total lymphocytes (of
which 48.5⫾12% were CD31⫹) significantly reduced the extent
of SMC cytolysis (*P⬍0.01 vs CD8⫹CD4/CD31⫺ cells). Similarly,
soluble Fas-L production (bottom) in the supernatant of these
experiments was reduced in the presence of cocultured CD4
total cells as compared with the presence of CD4⫹/CD31⫺ cells
or to SMC/CD8 cocultures devoid of CD4 cells. Mean⫾SEM,
n⫽6, *P⬍0.05 vs lower dose; n.d. indicates not determined.
decreased (Figure 3). Similarly, soluble Fas-L production and
CD8⫹ T cell-mediated SMC cytotoxicity were significantly
reduced when CD8⫹ T cells were cocultured with total CD4⫹
lymphocytes (48.5⫾12% CD31⫹) as compared with CD31depleted CD4⫹ cells (Figure 3). Neutralizing antibodies for
Fas-L abrogated more that 90% of CD8-mediated cytotoxicity and neutralizing antibodies for IL-2 reduced SMC cytotoxicity in CD8⫹/CD4⫹CD31⫺ cocultures by 30% (Figure 3).
An example of the fluorescence microscopic appearance of
the cytotoxicity is shown in Figure 4. Secretion of MMP9/
TIMP2 (ELISA) was significantly increased in supernatants
of LPS-stimulated macrophage cultures in the presence of
CD4⫹/CD31⫺ cells n⫽3, 4323⫾257 pg/mL, P⬍0.05) as
compared with total CD4⫹ cells (n⫽3, 3595⫾236 pg/mL) or
to macrophages alone (n⫽3, 3284⫾229 pg/mL). No changes
were detected for MMP1/TIMP1 complexes (data not
shown). MMP2/MMP9 activity and the production of reac-
A growing body of evidence suggests that lymphocytemediated responses are involved in the pathogenesis of
AAA.8,20 –22 Locally infiltrating T cells produce inflammatory
cytokines that in turn activate proteolysis of the extracellular
matrix. These T cells also secrete death-promoting molecules
such as perforin and Fas/FasL, that can mediate cytotoxicity
of the resident cells and consequently reduce the local
production of extracellular matrix components by these cells.5
Lack of appropriate immune regulation is responsible for
immune-mediated self-tissue damages.9,23 The type of immune regulating cells varies according to the diversity of the
diseased organ.10 In the present study, a significant reduction
in the percentage of the circulating immunoregulatory CD4⫹/
CD31⫹ T cells was a hallmark of patients with AAA. The
proportion of circulating CD4⫹/CD25⫹ T cells was not
significantly different between patients and control subjects
suggesting a crucial regulatory role in AAA for the CD31
itself, which is nearly absent at the surface of CD4⫹/CD25⫹ T
cells.
CD4⫹/CD31⫹ circulating T cell reduction was not attributable to cell sequestration in the aneurysmal wall, because the
percentage of CD4⫹/CD31⫹ T cells in the aneurysmal tissue
was similar to that observed in the autologous circulating
blood of the patients. Furthermore, CD4⫹/CD31⫹ T cell data
were similar in patients whether evaluated before or after
aneurysm exclusion by endoprothesis placement. Therefore,
the reduced number of CD4⫹/CD31⫹ cells is not an epiphenomenon but rather a primitive event in AAA disease and
might possibly play a pathogenetic role.
CD31 is a self-interacting molecule expressed both on cells
of the innermost layer of vascular wall and on leukocytes, and
CD31-mediated inhibitory signals might be critical in regulation of vascular immune-mediated pathologies. Among
human adult blood nucleated cells, T lymphocytes are the
only circulating leukocytes to lose partially (CD8) or consistently (CD4) the expression of CD31 at their surface,15 and it
has been previously demonstrated that CD4⫹/CD31⫹ T cells
possess immunoregulatory properties.12 The regulatory role
of both CD4⫹/CD25⫹ and CD4⫹/CD31⫹ T cells has been
documented in CD4-mediated immune responses,9,12 but the
role of CD4⫹/CD31⫹ T cells in the regulation of macrophages
and CD8⫹ T cell-mediated immune responses remains unknown. We found that CD8⫹/CD31⫹ T cells are reduced in
the blood and increased in AAA wall of patients suggesting
that these cells are sequestered into the aneurysmal tissue
where they could actively participate in the tissue damage by
mediating cytolysis of the smooth muscle cells, a key feature
of the evolution of atherosclerosis toward aneurysm. Interestingly, the decrease in circulating CD8⫹/CD31⫹ T cells (and
a fortiori their increase in AAA tissue) is directly proportional
to the size of the aneurysm and therefore to the severity of the
disease. Interestingly, the increased percentage of blood
622
Arterioscler Thromb Vasc Biol.
March 2006
Figure 4. Fluorescent microscopy of
SMC subjected to the cytotoxic assay
and LPS-stimulated macrophages. Representative images of fluorescent
microscopy of SMC subjected to the
cytotoxic assay and of LPS-stimulated
macrophages. Cells were counterstaining with DAPI before microscopy. Left,
Target (SMC) cell membranes were incubated with DiOC18 (green), with propidium iodide added after the cytotoxic
assay. Dead target cells appear yellow
and nuclei result violet. Right, Activated
macrophages produce reactive oxygen
species detected by the fluorescence of
dihydroethidium (red) and show MMP2/
MMP9 activity detected by fluorescencequenched collagen type IV (green). Bottom, Details of the overlay for the cells
indicated by the arrowheads.
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CD4⫹/CD31⫺ T cells in AAA patients is proportional to the
extent of disease and the analysis of tissue-infiltrating lymphocytes suggests that these cells could play a pathogenic
role in AAA pathology. Indeed, our in vitro data suggest that
CD4⫹/CD31⫺ T cells may indirectly damage the aortic wall
because they enhance CD8⫹ cell-mediated smooth muscle
cell cytolysis and macrophage-derived MMP2/MMP9
activity.
CD8⫹/CD31⫺ T cells are also slightly increased in the
blood of AAA patients, but circulating as well as tissueinfiltrating CD8⫹ cells are predominantly CD31⫹ and therefore a contribution by CD31⫺CD8⫹ T cells to the pathogenesis of AAA seems unlikely. In fact, sequestration of CD8⫹/
CD31⫹ T cells within the lesions might explain the relative
increase in CD8⫹/CD31⫺ T cells in the blood.
Figure 5. Quantification of reactive oxygen species and MMP2/
MMP9 activity. Adherent macrophages were detached by trypsin/EDTA and gentle scraping for cytometry analysis. A minimum of 2000 events were acquired in the CD14⫹ macrophage
gate. FL1 (MMP9 activity) and FL2 (reactive oxygen species)
median fluorescence were calculated within the CD14⫹ gate.
MØ indicates macrophages. Mean⫾SEM of 3 donors. *P⬍0.05
vs CD4 Tot and MØ alone.
Unfortunately, the number of T lymphocytes and smooth
muscle cells recoverable from AAA tissue is not sufficient to
perform functional ex vivo experiments in patients. However,
our data on commercially available aortic smooth muscle
cells and blood donor– derived leukocytes show that CD8⫹
cell-mediated SMC cytolysis as well as CD8⫹ cell proliferation can be efficiently regulated by CD4⫹/CD31⫹ T cells,
possibly via a modulation of the production of Fas-L by
CD8⫹ T cells and of IL-2 by CD4⫹/CD31⫺ T cells. The
presence of enriched CD4⫹/CD31⫺ T cells in CD8/CD4/SMC
cocultures was also associated with an increased IFN-␥
production. Human24 as well as experimental25 aneurysm
have been suggested to be associated with a Th2 environment. However, it has been shown that the Th1 cytokine
IFN-␥ alone is able to restore aneurysm formation in CD4
knockout mice, which are otherwise resistant to aneurysm
formation in the CaCl2 model.26
Recent experimental studies suggest that the death of
medial smooth muscle cells alone is not enough to cause
aneurysm formation in allo-mismatched aortic allograft
recipients.27 Local elastolytic and collagenolytic activity
consistently contribute to AAA formation. In this perspective, our study points out a regulatory role for CD4⫹/
CD31⫹ cells also on production and activity of macrophage
MMP9 which is considered to be critical in the formation
of AAA.28,29
Together, our findings suggest that the reduction in the
percentage of blood CD4⫹/CD31⫹ T cells observed in patients with atherosclerotic abdominal aneurysm might play an
important role in the pathology because their reduction leads
to an altered regulation of the cell-mediated immune responses involved in aneurysmal disease.
Acknowledgments
This study was supported by the “Institut National de la Santé Et de
la Recherche Médicale” (INSERM), the “Assistance PubliqueHopitaux de Paris” (AP-HP), the University of Paris V and VI, and
Caligiuri et al
Reduced Immunoregulatory CD31ⴙ T Cells in AAA
by grants from the “Fondation de France” and “Fondation pour la
Recherche Médicale”.
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Reduced Immunoregulatory CD31+ T Cells in Patients With Atherosclerotic Abdominal
Aortic Aneurysm
Giuseppina Caligiuri, Patrick Rossignol, Pierre Julia, Emilie Groyer, Dikran Mouradian,
Dominique Urbain, Namita Misra, Véronique Ollivier, Marc Sapoval, Pierre Boutouyrie, Srini
V. Kaveri, Antonino Nicoletti and Antoine Lafont
Arterioscler Thromb Vasc Biol. 2006;26:618-623; originally published online December 15,
2005;
doi: 10.1161/01.ATV.0000200380.73876.d9
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