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CCL21 overexpressed on lymphatic vessels drives thymic hyperplasia in myasthenia.

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CCL21 Overexpressed on Lymphatic Vessels
Drives Thymic Hyperplasia in Myasthenia
Sonia Berrih-Aknin, PhD,1,2 Nathalie Ruhlmann, MSc,1,2 Jacky Bismuth, MSc,1,2
Géraldine Cizeron-Clairac, PhD,1,2 Einat Zelman,3 Idit Shachar, PhD,3 Philippe Dartevelle, MD,4
Nicole Kerlero de Rosbo, PhD,1,2 and Rozen Le Panse, PhD1,2
Objective: Myasthenia gravis (MG), a neuromuscular disease mediated by anti-acetylcholine receptor (AChR) autoantibodies,
is associated with thymic hyperplasia characterized by ectopic germinal centers that contain pathogenic antibody-producing B
cells. Our thymic transcriptome study demonstrated increased expression of CCL21, a recruiter of immune cells. Accordingly,
we are investigating its implication in MG pathogenesis.
Methods: The expression of CCL21 and its CCR7 receptor was analyzed by enzyme-linked immunosorbent assay and
fluorescence-activated cell sorting, respectively. Chemotaxis of T and B cells to CCL21 was measured by transwell assay. The
nature of the thymic cells overexpressing CCL21 was investigated by immunochemistry and laser-capture microdissection combined with real-time PCR.
Results: We demonstrate that CCL21 is overexpressed specifically in hyperplastic MG thymuses, whereas there is no variation
in CCR7 levels on blood cells. We show that although CCL21 attracts both human T and B cells, it acts more strongly on naive
B cells. CCL21 overexpression is normalized in corticoid-treated MG patients, suggesting that targeting this chemokine could
represent a new selective treatment, decreasing the abnormal peripheral lymphocyte recruitment. Moreover, we locate protein
and messenger RNA overexpression of CCL21 to specific endothelial vessels. Investigation of the nature of these vessels demonstrated different angiogenic processes in MG thymuses: high endothelial venule angiogenesis and lymphangiogenesis. Unexpectedly, CCL21 overexpression originates from afferent lymphatic endothelial vessels.
Interpretation: We postulate that thymic overexpression of CCL21 on specialized lymphatic vessels results in abnormal peripheral lymphocyte recruitment, bringing naive B cells in contact with the inflammatory environment characteristic of MG
thymuses, where they can be sensitized against AChR.
Ann Neurol 2009;66:521–531
Acquired myasthenia gravis (MG) is a neurological autoimmune disease caused by autoantibodies against
components of the neuromuscular junction and leading
to disabling fatigability. Seropositive MG is caused by
anti–acetylcholine receptor (AChR) autoantibodies and
represents 85% of patients.1 Functional and morphological abnormalities of the thymus occur frequently,
and 50 to 60% of the seropositive patients exhibit thymic hyperplasia of lymphoproliferative origin with ectopic germinal center (GC) development.2 These thymic abnormalities are correlated with the anti-AChR
antibody titer,2 which decreases after thymectomy.3
The hyperplastic thymus includes all the components
of the anti-AChR response: the AChR,4 B cells producing anti-AChR antibodies,5 and anti-AChR autore-
active T cells.6 The thymus thus plays a pivotal role in
MG. An understanding of the mechanisms leading to
ectopic GC formation should shed light on the pathogenesis of this disease and help us to identify new therapeutic targets to avoid thymectomy or the nonselective use of glucocorticoids.
Our analyses of the thymic transcriptome demonstrated an important chemotactic activity with an increased expression of CXCL13 probably involved in
the generalized B-cell infiltration observed for all MG
patients. Moreover, we also observed a specific overexpression of CCL21 in thymuses of MG patients with
thymic hyperplasia.7
CCL21 and CCL19 are two CCR7-binding chemokines that play an important role in thymopoiesis.
From the 1Centre National de la Recherche Scientifique-Unité
Mixte de Recherche 8162, Institut Paris-Sud Cytokines, Hôpital
Marie Lannelongue, Le Plessis-Robinson; 2Université Paris-Sud,
91405 Orsay, France; 3Department of Immunology, the Weizmann
Institute of Science, Rehovot, Israel; and 4Service de Chirurgie Thoracique et Vasculaire et de Transplantation Cardiopulmonaire, Hôpital Marie Lannelongue, Le Plessis Robinson, France.
Potential conflict of interest: Nothing to report.
Address correspondence to Dr Le Panse, CNRS UMR 8162, Hôpital Marie Lannelongue, 133, Avenue de la Résistance, 92350 Le
Plessis Robinson, France. E-mail: rozen.lepanse@u-psud.fr
Received Jun 5, 2008, and in revised form Nov 27. Accepted for
publication Dec 5, 2008. Published online in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21628
© 2009 American Neurological Association
521
They are mainly secreted by medullary thymic epithelial cells and promote the migration of CD4⫹ or
CD8⫹ thymocytes toward the medullary zone.8 They
also play distinct roles because of their different presentation patterns on distinct stromal components:
CCL21 participates in prothymocyte recruitment in
the thymus,9 whereas CCL19 is involved in the export
toward the periphery of mature thymocytes.10
CCL21 is also known to play a central role in immune surveillance and defense by controlling the circulation of T cells and dendritic cells within lymphoid
and peripheral organs. In secondary lymphoid organs
(SLOs), CCL21 enables naive T cells to encounter sensitized dendritic cells by mediating their recruitment
through its expression on high endothelial venules
(HEVs) and afferent lymphatic vessels, respectively.11
Indeed, CCR7-deficient mice and paucity of lymph
node T-cell (plt) mice that do not express CCL19 and
CCL21 show defective migration of lymphocytes and
dendritic cells into T-cell zones.12
In view of the physiological role of CCL21 within
the thymus, we decided to investigate the pathological
relevance of CCL21 overexpression in hyperplastic MG
thymuses. We demonstrate that CCL21 is a potent recruiter of human naive B cells. We also show that different angiogenic processes, HEV angiogenesis and
lymphangiogenesis, occur in hyperplastic thymuses,
and that CCL21 overexpression is concentrated on
lymphatic endothelial vessels, suggesting a unique role
for these vessels in an abnormal recruitment of peripheral cells leading to thymic MG pathogenesis.
Subjects and Methods
Samples
Thymic fragments (50 –100mg) and sera were obtained from
MG female patients (16 – 42 years old) after thymectomy or
from babies (1 week to 1 year old) and women (16 –34 years
old) undergoing cardiovascular surgery at the Marie Lannelongue Chirurgical Center (Le Plessis Robinson, France).
Thymuses were thus classified as follows: babies (BB; n ⫽
13), non-MG adults (Ad; n ⫽ 12), MG patients with low
thymic hyperplasia (ML; with 2 or fewer GCs per section;
n ⫽ 10) or high thymic hyperplasia (MH; with 3 or more
GCs per section; n ⫽ 21), or corticosteroid-treated MG patients (Cortico, n ⫽ 9). Only seropositive MG caucasian female individuals, without thymoma or other known diseases,
were included. All MG patients included in this study had
received only anticholinesterase drugs.
Chemotactic assays and flow cytometry analyses were conducted with peripheral blood lymphocytes (PBL) from
healthy adult donors. All studies on thymuses and blood
samples were approved by the local ethics committee (CCPPRB, Kremlin-Bicêtre, France; agreement 06-018).
Immunohistochemistry on Thymic Sections
Frozen thymic sections (7␮m) were fixed in ice-cold acetone
for 20 minutes. At least three different non-MG or MG do-
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nors were used for each immunostaining. In immunohistological analyses performed with a Leica DMRB microscope
(Rueil-Malmaison, France), the relevant molecules were detected using the following combinations of antibodies: (1)
goat anti–human CCL21 (AF366; R&D Systems, Lille,
France) and Alexa-Fluor 488 rabbit anti–goat IgG (A21222;
Invitrogen, Cergy-Pontoise, France) or biotinylated horse anti–goat IgG followed by Alexa-Fluor 594 streptavidin
(S11227; Invitrogen, Eugene, OR), (2) mouse anti–human
keratin (A575; Dako, Trappes, France) and Alexa-Fluor 488
goat anti–mouse (GAM) IgG (A11029; Invitrogen), (3) rat
IgM anti–mouse (cross-reacting with human) peripheral
node addressin (PNAd) carbohydrate epitopes (MECA 79,
553863; BD Biosciences, Le Pont-De-Claix, France) and biotinylated mouse anti–rat IgM (550330; BD Biosciences)
followed by Alexa-Fluor 594 streptavidin, and (4) mouse anti–vascular endothelial growth factor receptor 3 (VEGFR3;
MAB3757, Abcys, Paris) and Alexa-Fluor 488-GAM IgG.
In immunohistological analyses using the microarray scanner, serial thymic sections were stained with mouse anti–
human CD21 antibody (555421; BD Biosciences) and
R-phycoerythrin (RPE)-GAM-IgG (R0480; Dako), or antiPNAd and biotinylated mouse anti-rat IgM followed by PE
streptavidin (349023; BD Biosciences). Sections were analyzed on the 428 Affymetrix scanner (Aviso, CroissyBeaubourg, France).
Enzyme-Linked Immunosorbent Assay
All antibodies and recombinant chemokines were from R&D
Systems. CCL21 (AF366) and CCL19 (MAB361) antibodies
were diluted at 1␮g/ml in coating buffer (50mM NaHCO3
and 50mM Na2CO3, pH 9.4) and incubated overnight at
4°C. Thymic extracts (1.5mg/ml proteins) or standards
(CCL21 or CCL19) were incubated for 3 hours, and subsequently, 0.25␮g/ml biotinylated anti-CCL21 (BAF366) or
anti-CCL19 (BAF361) and streptavidin-horseradish peroxidase (Beckman Coulter, Villepinte, France) were added. Tetramethylbenzidine was used for color development, and
plates were read at 450nm on an MRX-microplate reader
(DYNEX Technologies, ThermoLabsystems, Cergy-Pontoise,
France).
FACScan Flow Cytometry
The expression of CCR7 was analyzed with anti-CCR7
(MAB197; R&D Systems) and RPE-GAM (R0480; Dako)
or anti-CCR7-fluorescein isothiocyanate (FITC) (FAB197F;
R&D Systems) on T and B cells labeled with RPE-Cy5 antiCD4 (C7069; Dako), RPE anti-CD8 (R0806; Dako), and
RPE anti-CD19 (R0808; Dako).
Chemotaxis Assay
Human peripheral blood mononuclear cells from healthy
adults were recovered on Ficoll gradient and incubated overnight in culture flasks in a quiescent medium: RPMI-1640Glutamax-I supplemented with 0.5% fetal calf serum (Invitrogen). Chemotaxis assays were performed using transwell
chambers (5␮m pore size; Corning/Fisher ScientificBioblock, Illkirch, France). Transwell upper and lower chambers were loaded with 1 ⫻ 106 cells in 100␮l, and CCL21
or CXCL13 (R&D Systems) in 600␮l of quiescent medium,
respectively. After a 3-hour migration step at 37°C, cells in
the lower chambers were labeled with antibodies for T- or
B-lymphocyte subpopulations from BD Bioscience for
CD69-FITC (555530) and CD25-PE (555431), from
Amersham Bioscience (Orsay, France) for CD27-FITC
(M1764), or from Dako for CD3-FITC (F0818), CD19RPE, CD45RO-RPE (R0843), CD25-RPE (R0811), and
IgD (M0703). Cells were analyzed by fluorescence-activated
cell sorting (FACS) after 1-minute acquisition.
Chemotaxis assays on mouse lymphocytes were conducted
as described previously.13 In brief, spleen cells were separated
as CD45R (B220)-positive (B cells) or CD45-negative (T
cells) with antimagnetic beads using the magnetic-activated
cell sorting system (Miltenyi Biotec, Auburn, CA). Upper
and lower chambers of transwells were loaded with 5 ⫻ 106
cells in 100␮l, and ccl21 or cxcl13 (400ng/ml; PeproTech,
Rocky Hill, NJ) in 600␮l of quiescent medium, respectively.
After a 3-hour migration step at 37°C, cells in the lower
chambers were analyzed by FACS as described earlier.
Actin Polymerization Assay
Polymerization of actin filaments was analyzed as described
previously.13 In brief, mouse spleen cells collected as for chemotactic assays were incubated for 15 seconds at 37°C with
100ng/ml ccl21 or cxcl13, and fixed for 10 minutes in 3.7%
formaldehyde. Cells were then permeabilized, stained with
FITC-phalloidin, and cytoskeleton rearrangements were analyzed by FACS.
Laser-Capture Microdissection
Microdissection was conducted as described previously.14
Here sections were stained with anti-CCL21 (AF366) and
biotinylated rabbit anti–goat IgG (E0466; Dako), followed
by streptavidin-horseradish peroxidase and counterstained
with hematoxylin. CCL21-positive blood vessels and GCs
were microdissected separately before RNA extraction.
Reverse Transcription and Real-Time Polymerase
Chain Reaction
Total RNA was extracted as described previously,7 and 1␮ g
was reverse transcribed for 1 hour at 42°C using AMV (Eurobio, Courtaboeuf, France) with oligo-dT (Invitrogen).
RNA extracted after laser-capture microdissection was reverse
transcribed using SuperScript II with oligo-dT and random
primers (all from Invitrogen).
Real-time PCR reactions were performed on the LightCycler
apparatus as described previously.15 The primers used were as
follows: (1) for CCL21 (F: 5⬘-CCTTGCCACACTCTTTCTCCC-3⬘, R: 5⬘-CAAGGAAGAGGTGGGGTGTA-3⬘),
(2) for PROX1 (F: 5⬘-GGCTGAGACCTTGAAACAGG-3⬘,
R: 5⬘-ATTGTGGTTTTCCCCATTGA-3⬘), (3) for VEGFR3
(F: 5⬘-CCCACGCAGACATCAAGACG-3⬘, R: 5⬘-TGCAGAACTCCACGATCACC-3⬘), (4) for 28S (F: 5⬘-CGGGTAAACGGCGGGAGTAA-3⬘, R: 5⬘-GGTAGGGACAGTGGGAATCT-3⬘), and (5) for D6 (PPH01335A) from SuperArray Bioscience Corporation (TEBU, Le Perray en Yvelines,
France). All samples were normalized according to GAPDH or
28S amplification.
Statistical Analyses
In bar graphs, results are expressed as means of different experiments or duplicate values. Error bars represent standard
error of the mean. For two-by-two comparisons, nonparametric Mann–Whitney U or Wilcoxon tests were applied.
For multiparameter analyses (dose effect or subpopulation
analyses), analyses of variance followed by post hoc tests were
used.
Results
Thymic Overexpression of CCL21 in Hyperplastic
Myasthenia Gravis Thymuses
We previously demonstrated a specific increased expression of CCL21 messenger RNA (mRNA), by microarray and real-time PCR, in hyperplastic thymuses
of MG patients.7 To investigate in more detail the regulation of CCL21 expression in the thymus of MG
patients, we have analyzed by enzyme-linked immunosorbent assay its expression and that of CCL19, the
second ligand to CCR7 receptor. The data obtained
confirmed the high overexpression of CCL21 in all
MH thymuses (Fig 1A). An increased expression of
CCL19 was also observed in MH as compared with
thymuses from non-MG adults and ML patients but
not to those from babies (Fig 1B). Glucocorticoids are
widely used to treat MG patients, decreasing the number of ectopic GCs in the thymus,14 and for these patients, we did not observe an increased expression of
CCL21 and CCL19 compared with non-MG adults
(Figs 1A, B).
In the normal thymus, CCL21 and CCL19 play an
important role in prothymocyte recruitment and thymocyte export, respectively.9,10 Comparison of the thymic CCL21/CCL19 ratios indicated a major representation of CCL19 in babies, which might be related to
the high thymic cell mass and the need to export mature thymocytes to the periphery (Fig 1C). Accordingly, in involuted adult thymuses, the level of CCL19
decreased (Fig 1C). In MH thymuses, the high level of
CCL21 compared with CCL19 could lead to changes
in the patterns of thymocyte recruitment and export.
In this context, these changes could explain the increased number of CD4⫹ observed in MG thymuses.16
CCL21 overexpression in hyperplastic thymuses could
also trigger an abnormal recruitment of peripheral lymphocytes.
CCR7-deficient mice are prone to develop generalized multiorgan autoimmunity with lymphocytic infiltration in peripheral organs and has an altered thymic
architecture.17 Moreover, variations in CXCR3 and
CXCR5 expression have been detected on T lymphocytes in MG patients.18,19 Accordingly, we measured
CCR7 expression on peripheral B, CD4⫹, or CD8⫹ T
lymphocytes by FACS. Both fluorescence means (data
not shown) and percentages of CCR7-positive cells
were similar between non-MG adults and MH patients
Berrih-Aknin et al: CCL21 in MG Thymic Hyperplasia
523
Fig 1. Specific thymic overexpression of CCL21 in hyperplastic myasthenia gravis (MG) thymuses. Determination by enzyme-linked
immunosorbent assay (ELISA) of (A) CCL21 and (B) CCL19 concentrations in thymic extracts from babies (BB), non-MG adults
(Ad), MG patients with low (ML) or high (MH) thymic hyperplasia, or corticosteroid-treated MG patients (Cortico). (C) Distribution of log(10) ratios of the concentrations of CCL21 versus CCL19 determined by ELISA. (D) Fluorescence-activated cell sorting
(FACS) analysis of the percentage of CCR7-positive cells in peripheral CD4⫹, CD8⫹, or CD19⫹ cells from non-MG adults (Ad;
n ⫽ 6) and MH patients (n ⫽ 6). p values are assessed by the Mann–Whitney U test and indicated when less than 0.05.
in all populations of peripheral lymphocytes tested (Fig
1D). We also did not observe variations in thymic expression of CCR11, a decoy receptor for CCL21 that
could have scavenged and controlled CCL21 overexpression in MG thymuses (data not shown).
Altogether, these observations show that thymic hyperplasia in MG patients is specifically associated with
the thymic overexpression of CCR7 ligands (CCL19
and CCL21) rather than with variations of CCR7 or
CCR11 expression. Because CCL21 is largely overrepresented in MH patients compared with CCL19, we
have focused our investigations on the role of CCL21
in MG patients.
Potent Chemotactic Effect of CCL21 on B Cells
To address the consequences of CCL21 overexpression
in the thymus of MG patients, we investigated the nature of peripheral lymphocytes preferentially chemoattracted by CCL21. Surprisingly, we observed that human CCL21 was significantly more potent in
recruiting B cells than T cells (Fig 2A). For a representative experiment, in terms of absolute number, the recruited cells correspond to 7,704 ⫾ 119 and 195,000
⫾ 420 T cells or 1,820 ⫾ 3 and 16,400 ⫾ 69 B cells
in controls and with 500ng/ml of CCL21, respectively.
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To determine whether these observations were specific
to human CCL21, we compared its effects with those
of mouse ccl21. In a representative experiment, the recruited mouse T cells corresponded to 2,512 ⫾ 376 in
control and 10,644 ⫾ 816 with 400ng/ml of ccl21.
The recruited mouse B cells corresponded to 1,785 ⫾
223 in control, 3,422 ⫾ 186 with 400ng/ml of ccl21
or 3,645 ⫾ 186 with 400ng/ml of cxcl13. Consequently, mouse B cells are also attracted by ccl21, although this chemokine is more efficient in recruiting
mouse T cells as shown by chemotactic and actin polymerization assays (Figs 2C, D). We next compared
the chemotactic effect of human CCL21 to that of
CXCL13, the well-known B-cell chemoattractant that
is also overexpressed in MG thymuses.7,14 CXCL13
triggered B-cell migration from concentrations greater
than 0.05ng/ml with an optimum effect around 0.5 to
5ng/ml (average migration indexes around 200%; Fig
2B). The effect of CCL21 on B-cell migration was observed from concentrations greater than 5ng/ml with
average migration indexes of 323 and 875% at 50 and
500ng/ml, respectively (Fig 2B). Thus, high concentrations of CCL21 can trigger B-cell migration more efficiently than any concentrations of CXCL13 tested. In
contrast, the mouse chemokines, ccl21 and cxcl13,
Fig 2. Preferential migration of human B cells toward CCL21. (A, B) Migration of T (CD3⫹) or B (CD19⫹) cells from human
PBL toward human CCL21 or CXCL13. The percentage of migrating cells analyzed by fluorescence-activated cell sorting (FACS) is
calculated from the number of cells in lower wells as the ratio of (chemokine/control) ⫻ 100. Control subjects corresponding to
basal migration are thus equal to 100. (A) One representative experiment out of three with bars corresponding to means of duplicate values for T-cell (black bars) and B-cell (gray bars) migration. A two-way analyses of variance followed by a Bonferroni post
hoc test shows that B- and T-cell migration is significantly different from concentrations more than 5ng/ml with p values ⬍ 0.05.
(B) Each point corresponds to the mean of three different experiments analyzing B-cell migration with CCL21 (solid line) and
CXCL13 (dashed line). p values are assessed by the Mann–Whitney U test for each concentration, and p values are ⬍ 0.05 from
50ng/ml. (C, E) Migration of purified mouse T or B cells toward mouse ccl21 or cxcl13 at 400ng/ml (means of duplicate values ⫾ standard error of the mean). Cell migration was analyzed as described earlier. (D, F) Measure of actin-filament polymerization in purified mouse T or B cells treated 15 seconds with 100ng/ml of ccl21 or cxcl13. Actin polymerization was analyzed by
FACS on cells stained with FITC-phalloidin. The percentage of increase in actin polymerization is calculated from the FITC-phalloidin–positive cells as follows: (treated cells ⫺ control cells/control cells) ⫻ 100. (C–F) Bar graphs correspond to the mean of two
different experiments. p values are assessed by the Mann–Whitney U test, and only p values ⬍ 0.05 are indicated.
triggered mouse B-cell migration similarly (Figs 2E, F).
These results demonstrate that, in the human system,
CCL21 recruits B cells preferentially over T cells and is
even more efficient than CXCL13.
Potent Chemotactic Effect of CCL21 on
Naive B Cells
We also investigated the chemotactic properties of
CCL21 on different human B- and T-cell subpopulations. Using CD69 and CD25 as makers of T-cell activation and CD45RO to distinguish between naive
(CD45RO⫺) and memory (CD45RO⫹) T cells, we
analyzed the percentages of T cells recruited with
CCL21 (500ng/ml). CCL21 recruited all T-cell subpopulations studied, albeit preferentially naive T cells
(Fig 3A). In addition, we observed that CCR7 was
preferentially expressed on naive T cells as compared
with memory T cells (Fig 3B), as Sallusto and colleagues20 described previously.
Using CD27 to distinguish between naive or GC B
cells (CD27⫺, about 37–59% of B cells) and memory/
plasma B cells (CD27⫹), we observed that CCL21 recruited preferentially CD27⫺ B cells (Fig 3C). To determine whether within the CD27⫺ B-cell population
CCL21 had comparable chemotactic effects on naive
and GC B cells, we analyzed the migration of
CD27⫺IgD⫺ or CD27⫺IgD⫹ B cells corresponding,
respectively, to naive and GC B cells.21 As shown in
Figure 3D, CCL21 triggered especially the recruitment
of naive B cells. By analyzing expression levels of
CCR7 on CD19⫹IgD⫹ (naive B cells) and
CD19⫹IgD⫺ (differentiated B cells), we also observed
that CCR7 expression was higher on naive B cells (Fig
3E). Altogether, these data confirm that, although
CCL21 can recruit all T- and B-cell subtypes analyzed,
its chemotactic activity is more pronounced on naive B
cells.
Berrih-Aknin et al: CCL21 in MG Thymic Hyperplasia
525
CCL21 Overexpression by Endothelial Vessels
The next step was to determine the nature of the cells
overexpressing CCL21 in MG thymic hyperplasia. Onodera22 reported a strong CCL21 immunoreactivity in
thymic stromal cells in hyperplastic MG thymuses. Although thymic epithelial cells express CCL21, we did
not detect any increased expression of CCL21 by these
cells in MG patients (data not shown). Immunohistochemical analysis indicated that CCL21 is overexpressed on endothelial vessels in hyperplastic thymuses
(Fig 4). To determine whether the CCL21 increase in
hyperplastic MG thymuses was due only to CCL21positive vessels, we performed laser-capture microdissection to isolate CCL21-positive vessels from the thymic stroma and also from GCs. Analysis of CCL21
mRNA expression in the microdissected fragments and
in the remaining tissue section confirmed that CCL21
overexpression in hyperplastic thymuses originates from
endothelial vessels (Fig 4D), as well as partly from
GCs, rather than from the thymic stroma. Altogether,
these observations demonstrate that CCL21 is overexpressed at the mRNA and protein level by endothelial
vessels in hyperplastic MG thymuses.
Overrepresentation of High Endothelial Venules in
Hyperplastic Myasthenia Gravis Thymuses
In SLOs and in chronically inflamed tissues, lymphocyte homing is directed through HEVs, a specialized
endothelium bearing on its luminal surface diverse chemokines and expressing high levels of PNAd carbohydrate ligands.23 Using anti-PNAd antibody, we observed only a few HEVs in thymuses of babies and
non-MG adults (Figs 5A–C). In contrast, in the thymus of MG patients, we observed increased numbers
of HEVs, located around GCs, which correlated with
the degree of thymic hyperplasia (Figs 5D, F).
We thus wondered whether HEVs were the vessels
overexpressing CCL21. Double labeling for CCL21
and PNAd antibodies (Figs 5G–I) did not show any
colabeling, indicating that the endothelial vessels overexpressing CCL21 in hyperplastic MG thymuses were
not HEVs.
Characterization of the Endothelial Vessels
Overexpressing CCL21
We further investigated the phenotype of CCL21positive endothelial vessels in the thymus of MH pa-
Fig 3. Preferential migration of human naive B cells toward CCL21. (A) Migration of different T-cell subpopulations toward
CCL21 (500ng/ml). (B) Analysis of the percentage of CCR7-positive cells in naive (CD3⫹CD45RO⫺) and memory
(CD3⫹CD45RO⫹) T cells. (C) Analysis of the migration of B-cell subpopulations toward CCL21 (500ng/ml). CD19⫹ B cells
were labeled with CD27 to distinguish naive- and germinal center (GC)–selected B cells (CD27⫺) from memory and plasma B
cells (CD27⫹). (D) To determine whether, within the CD27⫺ B-cell population, CCL21 had comparable chemotactic effects on
naive- (IgD⫹) and GC-selected B cells (IgD⫺), we labeled B cells with both CD27 and IgD. (E) Analysis of the percentage of
CCR7-positive cells in naive- (CD19⫹IgD⫹) and memory (CD19⫹IgD⫺) B cells. (A, C, D) Percentage of migrating cells is calculated from the number of cells in lower wells as the ratio of (chemokine/control) ⫻ 100. Each bar corresponds to the mean of different experiments with PBL from five to seven different donors. p values are assessed by Kruskal–Wallis test followed by Dunn’s
multiple-comparison test, showing a significant recruitment of naive T cells with CCL21 compared with control. (B, E) Each bar
corresponds to the mean of eight different donors ⫾ standard error of the mean. p values are assessed by the Wilcoxon test.
Fluorescence-activated cell sorting histograms correspond to representative experiments. FITC ⫽ fluorescein isothiocyanate.
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tients by double-labeling analysis of CCL21 and
VEGFR3, a lymphatic endothelial cell (LEC) marker.
We then observed that all CCL21-positive vessels were
also VEGFR3-positive (Figs 6A–G) and corresponded
to small vessels. We also detected large VEGFR3positive vessels in the thymus of babies, non-MG
adults, and MG patients, but these were not positive
for CCL21 (data not shown). Interestingly, these observations made in humans differ from what has been
observed in mice. Indeed, Odaka and coauthors24 have
demonstrated that both thymic lymphatic and blood
vessels, together with thymic epithelial cells, express
CCL21.
To confirm the LEC origin of CCL21 expression in
hyperplastic thymuses, we analyzed the expression of
PROX1, a specific LEC transcription factor involved in
endothelial cell differentiation into LECs,25 in the microdissection extracts described earlier. We show that
the microdissected CCL21-positive vessels expressed
PROX1, whereas we could not detect its expression on
the remaining part of the sections (Fig 6H). The level
of PROX1 expression in CCL21-positive vessels appeared to be similar to the one observed in primary
cultures of human dermal LECs used as a positive control. In hyperplastic MG thymuses, the CCL21positive lymphatic vessels could correspond to afferent
or efferent vessels, allowing the import or export of peripheral cells, respectively. We then analyzed the expression of D6, a decoy receptor for most inflammatory chemokines known to be expressed on afferent
lymphatic vessels of peripheral organs.26 We observed
that CCL21-positive vessels strongly expressed D6
compared with the thymic stroma (Fig 6I).
To determine whether this overexpression of CCL21
in hyperplastic thymuses is related to a greater number
of lymphatic vessels, we analyzed thymic expression of
Fig 4. CCL21 overexpression by endothelial vessels. Immunohistochemical analyses on thymic sections from (A) babies, (B)
non–myasthenia gravis (non-MG) adults (Ad), and (C) high
thymic hyperplasia (MH) patients. Thymic epithelial cells were
labeled in red with an anti-keratin antibody and CCL21 in
green. Sections were viewed with a Leica DMRB microscope,
and images were captured with a Sony DXC-930P color CCD
camera (Tokyo, Japan) connected to a Leica MSP 930 image
analysis system (Leica Microsystems, Rueil-Malmaison, France)
(bars ⫽ 20␮m). (D) Real-time polymerase chain reaction
(PCR) on laser-capture microdissection. Cryostat sections of human thymic tissues were peroxidase-labeled to detect CCL21
and counterstained with hematoxylin. CCL21-positive blood
vessels identified by a dark brown color and germinal centers
(GCs) with mantle zones were isolated separately by lasercapture microdissection. The microdissected regions (CCL21⫹
vessels and GCs), the rest of the sections (whole sections without
CCL21⫹ vessels and GCs), and also whole independent sections
(WS) were collected, and CCL21 RNA was quantified by realtime PCR. For all samples, the CCL21 RNA level was reported
to 28S. Each bar corresponds to the mean ⫾ standard error of
the mean from three different non-MG (white bars) or MH
(black bars) samples. One-tailed p values are assessed by
Mann–Whitney U test in each microdissected region to determine whether CCL21 is significantly overexpressed in MG patients; only p values ⬍ 0.05 are indicated.
Berrih-Aknin et al: CCL21 in MG Thymic Hyperplasia
527
VEGFR3 and PROX1 in non-MG and MG patients.
Using real-time PCR analyses, we observed a high interindividual variation for these markers. Nevertheless,
significant increased expressions of VEGFR3 (3.1
times) and PROX1 (3.6 times; Figs 6J, K) were demonstrated in the thymus of MH patients compared
with non-MG adults. Altogether, these data indicate
that CCL21-positive vessels correspond to afferent
lymphatic vessels and show for the first time lymphangiogenesis in hyperplastic thymuses.
Fig 5. Overrepresentation of high endothelial venules (HEVs)
in hyperplastic myasthenia gravis (MG) thymuses. Immunohistochemical analyses of germinal center (CG) and HEV localization in thymuses. Serial thymic sections from (A) babies,
(B, C) non-MG adults, (D) seropositive MG patients with
low thymic hyperplasia (ML), and (E, F) seropositive MG
patients with high thymic hyperplasia (MH) were stained with
antibodies against CD21 (red), anti-PNAd (green), and antikeratin (blue) to localize CG, HEVs, and the epithelial cells,
respectively. The whole sections were then scanned using the
428 Affymetrix scanner with a resolution of 10␮m, and images were superimposed. Immunohistochemical analyses of
HEV and CCL21 localization in thymuses. Thymic sections
from (G) babies, (H) non-MG adults, and (I) MH patients
were labeled in red with an anti-PNAd antibody for HEVs
and in green for CCL21. Scale bars ⫽ 200␮m (A, B, D, E);
10␮m (G–I).
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Annals of Neurology
Vol 66
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October 2009
Discussion
Role of CCL21 in Ectopic Germinal Center
Development in the Thymus of Myasthenia
Gravis Patients
In MG, thymic hyperplasia is characterized by the
presence of numerous GCs,2 and here we demonstrate
that it is specifically associated with overexpression of
CCL21. The overexpression of this chemokine has also
been observed in nonlymphoid organs, especially in autoimmune diseases characterized by ectopic GC development.27,28 Transgenic mouse models with a specific
expression of CCL21 in islet ␤ cells or in thyroid cells
showed that ectopic expression of CCL21 is sufficient
to trigger lymphoid neogenesis in pancreas or thyroid,
respectively.29,30
To sustain the role of CCL21 in GC development within the thymus of MG patients, we observed
that CCL21 expression was strikingly reduced in
glucocorticoid-treated compared with untreated patients, and glucocorticoids are known to decrease the
number of ectopic GCs in MG thymus.14 We suggest
that the efficiency of glucocorticoids in MG is at least
partly due to their effects on CCL21 expression.
By analyzing in detail the chemotactic properties of
CCL21 on different T- and B-cell subpopulations, we
demonstrate that although CCL21 recruits T cells efficiently, it is also a potent chemoattractant for B cells.
Moreover, in humans, CCL21 could even be more efficient than CXCL13 in recruiting B cells, especially
naive B cells. We hypothesize that CCL21 overexpression is probably decisive in allowing an additional recruitment of peripheral cells, in particular, naive B
cells. Once in contact with the inflammatory environment characteristic of MG thymuses, these naive B
cells lead to ectopic GC development characteristic of
thymic hyperplasia. CCL21 may also play a final role
in the segregation of lymphoid compartments necessary
for the complex cell organization of GCs, as Hopken
and coworkers31 suggested.
Altogether, these observations suggest that the specific thymic overexpression of CCL21 in hyperplastic
thymuses plays a central role in ectopic GC development. Consequently, we can envisage that therapies inhibiting CCL21 overexpression selectively could lead to
a decrease in thymic hyperplasia.
Neoangiogenic Processes with Specific Overexpression
of CCL21 by Lymphatic Endothelial Vessels in
Hyperplastic Thymuses
Our investigation of the nature of the cells involved in
thymic overexpression of CCL21 in MG demonstrated
a specific overexpression of CCL21 by endothelial vessels in hyperplastic thymuses. In parallel, we observed
the development of numerous HEVs around GCs in
hyperplastic thymuses. The protein CCL21 is known
Fig 6. CCL21-positive vessels correspond to lymphatic endothelial vessels. Immunohistochemical analyses on thymic sections from seropositive MG patients with high thymic hyperplasia (MH): staining with (A, D) green-labeled anti–vascular endothelial growth factor receptor 3 (VEGFR3) antibody, (B, E) red-labeled anti-CCL21 antibody, and (C, F) merge and (G) representative merge of isotype control (G). Real-time polymerase chain reaction (PCR) on laser-capture microdissection as detailed in Figure 2. (H) PROX1 and (I) D6
messenger RNA expression in CCL21⫹-microdissected vessels and in the rest of the sections (whole sections without CCL21⫹ vessels and
germinal centers [GCs]; WS⫺) were quantified by real-time PCR and reported to 28S expression. Each bar corresponds to the mean of
experiments with samples from different non-MG adults (n ⫽ 3) or MH patients (n ⫽ 3). Messenger RNA (mRNA) expression for
these genes was also analyzed using lymphatic endothelial cell and human umbilical vein endothelial cell (HUVEC) cultures as positive
and negative controls, respectively. p values are assessed by the Mann–Whitney U test for non-MG, MG, or cell culture samples; p
values ⬍ 0.1 are indicated. (J, K) Real-time PCR for mRNA expression level of (J) vascular endothelial growth factor receptor 3
(VEGFR3) and (K) PROX1 in the thymus of non-MG adults (n ⫽ 5) and MH patients (n ⫽ 7). mRNA expression level was reported to GAPDH. p values were obtained by the Mann–Whitney U test. Scale bars ⫽ 10␮m.
to be present on HEVs, even if Carlsen and colleagues32 demonstrated species-specific differences in
its expression: mouse HEVs produce and express ccl21,
whereas human HEVs lack detectable CCL21 mRNA.
However, we did not detect any colabeling between
CCL21 and thymic HEVs, suggesting that, unlike in
SLOs, CCL21 in the thymus is not involved in the
recruitment of peripheral cells through HEVs; rather,
and similarly to what was recently described in other
human diseases with lymphoid neogenesis, such as
rheumatoid arthritis, Sjögren syndrome, and ulcerative
colitis,33 CCL21-positive thymic vessels corresponded
to lymphatic endothelial vessels. In addition, we observed that CCL21-positive vessels expressed D6, a de-
coy receptor for inflammatory chemokines considered
as a marker for afferent lymphatic vessels.26
The increased expression of lymphatic markers
(VEGFR3 and PROX1) in hyperplastic thymuses also
suggests an expansion of the lymphatic system. Lymphangiogenesis occurs throughout life in homeostasis
and diseases. It has been described in lymph nodes
after immunization, where it was shown to be dependent on the entry of B cells.34 In a model of transgenic mice overexpressing CCL21 within the thyroid,
lymphangiogenesis has also been observed and was related to the influx of CD4⫹ T cells.35 Altogether,
these results demonstrate active angiogenic processes
within the thymus of MG patients with HEV develop-
Berrih-Aknin et al: CCL21 in MG Thymic Hyperplasia
529
ment and lymphangiogenesis associated with the overexpression of CCL21.
Role of Lymphatic Overexpression of CCL21 in
Lymphocyte Trafficking
Hyperplastic MG thymuses present the major characteristics of SLOs and may behave similarly. First, the
large number of HEVs observed in hyperplastic thymuses suggests an entry of peripheral cells through
these specialized vessels, and we recently demonstrated
a significant increase in migration of PBL toward hyperplastic thymic extracts in chemotactic assays.7 Second, the overexpression of CCL21,7 and also
CXCL13,14 could account for an abnormal recruitment of peripheral B and T cells; however, the main
difference between hyperplastic thymus and SLOs is
that CCL21 and CXCL13 (unpublished data) were not
detected on thymic HEVs, suggesting that B- and
T-cell recruitment in the thymus is different from that
in SLOs. According to Pearse,36 there are no afferent
lymphatic vessels in the normal thymus; however, we
can hypothesize that, in hyperplastic thymuses,
CCL21-positive vessels represent a pathological development of afferent lymphatic vessels, which, in addition to thymic HEVs, could correspond to an entrance
of circulating peripheral cells, in particular, naive B
cells, indispensable for triggering ectopic GC development. These thymic afferent lymphatic vessels could
also mediate the recruitment of sensitized dendritic
cells as Nagane and colleagues37 suggested.
In the human system, CCL21 is also strongly expressed by afferent lymphatic vessels in peripheral tissues, where it mediates dendritic and T-cell exit, and
their entry into afferent lymph, allowing their migration to SLOs.38 Consequently, another hypothesis is
that hyperplastic MG thymuses behave similarly to an
inflamed peripheral organ where CCL21-positive afferent lymphatic vessels may behave as an exit signal for
CCR7-positive cells.
Conclusion
We show for the first time that the extent of thymic
hyperplasia in MG correlates with pathological angiogenic processes, ectopic HEV angiogenesis and lymphangiogenesis, which suggest abnormal peripheral lymphocyte recruitment. In vivo, overexpression of CCL21
on lymphatic endothelial vessels could trigger a greater
recruitment of naive B cells to the inflamed MG thymus, where they could be sensitized against AChR. Because CCL21 overexpression is normalized upon corticoid treatment, we postulate that molecules inhibiting
the effects of CCL21 could represent a new way to treat
MG patients by decreasing the abnormal recruitment of
peripheral lymphocytes, especially B cells, and limiting
the development of thymic hyperplasia.
530
Annals of Neurology
Vol 66
No 4
October 2009
Grants from the European Community LSHM-CT-2006-037833
and the “Association Francaise contre les Myopathies” (AFM, CGR
37500711) obtained by Dr. S. Berrih-Aknin, and from the “Agence
Nationale de la Recherche” ANR-06-MRAR-001-01 obtained by
Dr. R. LePanse.
We thank R. Alon for helpful discussions, F. Truffault
for her technical assistance, S. Mussot and A. Serraf for
the thymic samples, and E. Dulmet and V. de Montpreville for histological analyses.
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