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Departments of Endocrinology (M.Á.G.-L., M.M.) and Immunology (D.S., F.S.-M.), Hospital de la Princesa, Universidad Autónoma, Madrid 28006, Spain
Address all correspondence and requests for reprints to: Dr. Monica Marazuela, Servicio de Endocrinología, Hospital de la Princesa, Diego de León 62, Madrid 28006, Spain. E-mail: mmarazuela{at}hlpr.insalud.es
Abstract
To better understand the selective migration of lymphocytes in
autoimmune thyroid disorders (AITDs), we analyzed thyroid samples and
demonstrated an enhanced expression of the chemokines interferon
(IFN)-inducible protein (Ip)-10 and regulated on activation normal T
lymphocyte expressed and secreted (RANTES) in thyroids from AITD
patients. Ip-10 and monokine induced by IFN-
(Mig) were expressed
in vivo in thyroid follicular cells (TFCs) from AITD
thyroids. Interestingly, Ip-10 mRNA, although not basally detected in
cultured TFCs, was strongly induced by IFN- and synergistically
increased by TNF-
addition. Furthermore, high levels of Ip-10
protein were detected in the supernatants of IFN--stimulated TFCs.
Likewise, Mig protein was strongly induced in TFCs by the same stimuli
as Ip-10. Unlike Ip-10 and Mig, the expression of RANTES was induced
mainly by TNF-. In addition, intrathyroidal lymphocytes from
AITD patients showed higher expression of CXCR3, CCR2, and CCR5
chemokine receptors than autologous peripheral blood lymphocytes. T
lymphoblasts expressing CXCR3 showed an increased migration to
supernatants from stimulated TFCs, which was abolished by specific
antibodies to the chemokines Ip-10 and Mig, as well as to their
receptor CXCR3. Taken together, these data suggest a potential role of
TFCs, through the production of the chemokines Ip-10, Mig and RANTES,
in regulating the recruitment of specific subsets of activated
lymphocytes in AITDs.
HUMAN AUTOIMMUNE THYROID disorders (AITDs)—Graves’ disease (GD) and Hashimoto’s thyroiditis (HT)—are characterized by reactivity to self thyroid antigens, which may be expressed as destructive inflammatory or antireceptor autoimmune diseases (1). The term "autoimmune thyroiditis" has been redefined in recent years and can be viewed as a spectrum covering primary myxedema, HT, and GD. In this regard, some patients with GD will later develop thyroid failure and some patients with HT will develop hyperthyroidism or orbitopathy (2).
Intrathyroidal lymphocytes seem to play a central role in the pathogenesis of AITDs through thyroid antigen recognition in thyroid follicular cells (TFCs), as an essential step to T- and/or B-cell stimulation (1). In addition, they mediate important inflammatory effects, such as the release of cytokines (1). The recruitment of lymphocytes in AITDs is a multistep process involving adherence and migration across the endothelium, trafficking through the interstitium, and finally moving to the TFC (3, 4). Leukocyte extravasation involves the combined action of adhesion molecules such as selectin and integrins, and chemotactic factors, mainly chemokines (5). Infiltrating lymphocytes and endothelial cells (ECs) in AITD bear an enhanced expression of various adhesion molecules, pointing to lymphocyte function-associated antigen-1/intercellular adhesion molecule-1, very late antigen-4/vascular cell adhesion molecule-1, and selectin/selectin ligands adhesion pathways as predominant in leukocyte migration to the thyroid (6).
Chemokines are a family of cytokines initially characterized by their capacity to induce chemotaxis, or directed leukocyte migration (3, 7). The chemokines are divided into four subfamilies (C, CC, CXC, and CX3C) on the basis of the relative position of the cysteine residues in the protein (8). In addition, chemokines can be divided broadly into two categories: inducible, which recruit leukocytes in response to physiological stress, and homeostatic chemokines, which are responsible for basal leukocyte trafficking and forming the architecture of secondary lymphoid organs (9). Chemokine receptors are heptahelical receptors coupled to G proteins (8). The specificity of chemoattractants is regulated by the combinatorial distribution of their receptors on the cell surface (7).
Ip-10 and Mig are CXC chemokines that are inducible by interferon
(IFN)- during inflammation and display potent lymphocyte chemotactic
activity (10, 11). In addition, the CC family members
macrophage chemoattractant (MCP)-1 and regulated upon activation normal
T lymphocyte expressed and secreted (RANTES) are potent monocyte and
lymphocyte chemoattractants (12). Inducible chemokines
recruit leukocytes that express the appropriate chemokine receptors to
an inflammatory focus (5, 13). The CXCR3 chemokine
receptor is specific for the CXC chemokines Ip-10, Mig, and T-cell
-chemoattractant (11, 13). Ip-10 and Mig binding to the
CXCR3 receptor is considered important in delivering specific signals
for selective homing of activated/effector cells to some inflammatory
sites (14). Recent studies have implicated CXCR3+
lymphocytes in the pathogenesis of inflammatory and autoimmune diseases
(9) such as rheumatoid arthritis (15),
ulcerative colitis (15), hepatitis C-infected liver
(16), and multiple sclerosis (17). However,
little is known about chemokines involved in AITDs. Expression of MCP-1
and IL-8 by TFCs has been reported in vitro (18, 19). In addition, macrophage inflammatory protein (MIP)-1 and
ß chemokines have been found to be increased in GD thyroid glands
in vivo (20). In this study, we have examined
the role of TFCs in the production of functionally active Ip-10, Mig,
and RANTES that attract activated T lymphocytes in vitro.
These findings were confirmed by in situ
immunohistochemistry performed on tissues from AITDs. In addition, an
up-regulated expression of the Ip-10/Mig receptor CXCR3, as well as the
RANTES receptor CCR5, was found in infiltrating lymphocytes in
vivo. These data suggest the direct involvement of TFCs in the
production of Ip-10, Mig, and RANTES that mediate the local
accumulation of T cells in AITDs.
Materials and Methods
Patients
Surgical thyroid tissue and peripheral blood were obtained from 16 patients with GD and 8 patients with HT. In addition, surgical tissue was obtained from six patients with multinodular goiter and three normal controls. The diagnosis was established on commonly accepted clinical, laboratory, and histological criteria (1). All GD patients had relapsed after antithyroid drug treatment and were euthyroid under carbimazole therapy at the time of surgery. All had received iodine preoperatively. Normal thyroid tissues were obtained from unaffected glands of patients undergoing parathyroidectomy who require biopsy of the thyroid to locate the parathyroid glands.
Cell preparation
TFCs were prepared as reported previously (6). The
specimens were minced with scissors and digested with collagenase (1
mg/ml; Roche, Mannheim, Germany) in RPMI 1640 (Whittaker
Bioproducts, Inc., Walkersville, MD) for 1 h at 37 C. Semidigested
follicles were removed, sedimented for 2 min, washed, and cultured in
RPMI 1640 medium supplemented with 10% FBS (Seromed, Biochrom, Berlin,
Germany), 2 mM glutamine, and 50 µg/ml
penicillin/streptomycin at 37 C and 5% CO2 in
plastic flasks (Flow Laboratories, McLean, VA). TFCs were incubated
from 24 h to 4 d with proinflammatory cytokines including
1000 U/ml IFN- (R&D systems, Minneapolis, MN), 800 mU/ml TNF-
(R&D), and 3700 U/ml IL-1ß (Calbiochem-Nobaviochem,
Darmstadt, Germany), alone or in combination. These concentrations were
selected after preliminary studies to yield the highest
responses.
Peripheral blood was collected into heparinized tubes, and mononuclear cells (PBMCs) were isolated by centrifugation on a Ficoll-Hypaque (Rafer S/L, Madrid, Spain) density gradient. To isolate thyroid mononuclear cells (TMCs) thyroid specimens were minced with scissors. Cells were sedimented twice for 1 h in RPMI supplemented with 10% FBS, and nonadherent cells were removed by density gradient centrifugation. To generate T lymphoblasts, PBMCs were incubated in RPMI with phytohemaglutinin (Sigma, St. Louis, MO) for 48 h and thereafter with IL-2 for 7 d (Eurocetus, Madrid, Spain).
Monoclonal antibodies (mAbs)
We have used the following mAbs anti-CXCR3 (R&D; clone 49801.11), anti-CCR2 (21), anti-CCR5 (PharMingen, San Diego, CA), anti-CXCR4 (R&D), anti-Ip-10 (PharMingen), and anti-Mig (PharMingen). In addition, polyclonal antibodies (pAbs) anti-Ip-10 (Preprotech, London, UK), anti-Mig (R&D), and anti-eotaxin (R&D) were used. Anti-CD45 for the leukocyte common antigen was used as control for leukocytes. The mouse myeloma cell line P3-X63 protein (IgG1, k) was used as negative control. The chemokines Ip-10 and Mig were purchased from PreproTech.
Flow cytometry
Cells (1–5 x 105) were suspended in 100-µl aliquots of PBS (pH 7.4). A specific mouse mAb was added, and cells were incubated for 30 min at 4 C, then washed twice and incubated with saturating amounts of fluorescein-conjugated F (ab')2 goat antimouse immunoglobulin (DAKO Corp., Glostrup, Denmark). After three washes, fluorescence was measured using a fluorescence-activated cell-sorting scan (Becton Dickinson and Co., Mountain View, CA). For double staining of TMCs, PerCP-conjugated CD45 (Becton Dickinson and Co.) and APC-conjugated CD3 (Becton Dickinson and Co.) were added to cells previously labeled with fluorescein isothiocyanate using an indirect immunofluorescent staining.
Fluorescence intensity for different mAbs was determined on linear and logarithmic scales. Data for linear and logarithmic mean fluorescence intensity and specific percentages of positive cells for different mAb were obtained.
Immunohistochemistry
Cryostat sections were cut from snap-frozen thyroid tissue embedded in Tissue-Tek OCT medium (Ames, Miles Laboratories, Elkhart, IN) stored at -80 C. The tissue sections were stained by an indirect immunoperoxidase method as described previously (6). Briefly, 5-µm acetone-fixed sections were sequentially incubated with mAb culture supernatants and peroxidase-conjugated rabbit antimouse Ig (DAKO Corp.). For pAbs, peroxidase-conjugated donkey antirabbit (Amersham Pharmacia Biotech, Buckinghamshire, UK) and rabbit antigoat (DAKO Corp.) were used. Each incubation was followed by three washes with Tris-buffered saline isotonic buffer (pH 7.6). Then, sections were developed with the Graham-Karnovsky medium containing 0.5 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and hydrogen peroxide. Sections were counterstained with Carazzi’s hematoxylin, dehydrated, and mounted by routine methods.
Each section was examined under code by two independent observers (M.A.G.-L., M.M.). The intensity of lymphocytic infiltration was determined in control sections in each case using the mAb against CD45. Sequential sections were used to study cell distribution.
Antibody capture ELISA
Ip-10 levels were measured from culture supernatants via a sandwich-type ELISA. Briefly, antihuman Ip-10 capture mAb (4 µg/ml in PBS, 100 µl/well) was adsorbed to microtiter plates (Maxi-Sorb, Nunc, Denmark). After saturation with 1% BSA, supernatants were added and incubated with affinity-purified rabbit anti-Ip-10 pAb (24 µg/ml in PBS, 100 µl/well). The reaction was developed using a peroxidase-labeled goat antirabbit immunoglobulin antibody and O-Phenylenediamine Dihydrochloride Tablet Sets (Sigma). The color reaction was stopped with 2.5 M H2 SO4, and absorbance was determined at 450 nm.
Ribonuclease (RNase) protection assay
Total RNA was extracted from thyroid tissue and thyrocytes using
ULTRASPEC (Biotex Laboratories, Inc. Houston, TX).
Multiprobe template set hCK-5 (containing DNA templates for Ltn,
RANTES, Ip-10, MIP-1ß, MIP-1, MCP-1, IL-8, I-309, L32, and
glyceraldehyde-3-phosphate dehydrogenase) was purchased from
PharMingen. The DNA templates were used to synthesize the
-[32P]UTP (3000 Ci/mmol, 10 mCi/ml;
Amersham Pharmacia Biotech)-labeled probes in the presence
of a GACU pool using a T7 RNA polymerase (PharMingen).
Hybridization with 5–15 µg of each target RNA was performed
overnight, followed by digestion with RNase A, according to the
PharMingen standard protocol. The samples were treated by
proteinase K-SDS mixture and then extracted and precipitated in the
presence of ammonium acetate. The samples were loaded on an
acrylamide-urea sequencing gel next to labeled DNA molecular weight
markers and to the labeled probes, and run at 50 W with 0.5x
Tris-borate/EDTA electrophoresis buffer. The gel was adsorbed to filter
paper, dried under vacuum, and exposed on film (X-AR;
Kodak, Rochester, NY) with intensifying screens at -70 C.
Densitometric analysis was performed using the Multianalyst software
from Bio-Rad Laboratories, Inc. (Hercules, CA). Results
are shown as optical density.
Western blot
TFCs previously activated with proinflammatory cytokines were lysed in a detergent buffer (0.5% Triton X-100 in PBS, with protease inhibitors) for 30 min at 4 C with continuous stirring, then centrifuged (15,000 x g, 15 min). Protein extracts were separated in 15% SDS-PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed as described (22), using 10% nonfat dry milk in Tris-buffered saline as a blocking agent. After washing with PBS, the membrane was incubated with pAb anti-Mig with agitation overnight at 4 C, followed by rabbit-antigoat peroxidase (1:2000 dilution). The reaction was developed using enhanced chemiluminescence assay (Amersham Pharmacia Biotech). Protein loading was controlled in all cases by reblotting with anti-ß1 integrin TS2/16 mAb, since ß1 integrin expression is not altered by these stimuli in TFCs (23).
In vitro chemotactic assay
Assays for T lymphoblasts chemotaxis were performed in polycarbonate membrane, 6.5-mm diameter, 5-µm diameter pore size transwell cell culture chambers (Costar Corp., Cambridge, MA). Cells (100 µl at 5 x 106/ml) suspended in RPMI 1640 and 1% BSA were added to the upper chamber, and chemokines at the appropriate concentration in the same medium (50 ng/ml for Ip-10 and 100 ng/ml for Mig) were added to the lower well. In the other wells, supernatants from TFC cultures were incubated for 30 min at room temperature with pAb against Ip-10 (3 µg/ml) and Mig (4 µg/ml) and mAb against eotaxin (5 µg/ml). In addition, cells were incubated for 30 min at 37 C with anti-CXCR3 mAb (5 µg/ml). Cells were allowed to migrate for 2 h at 37 C in 5% CO2 atmosphere, and then migrated cells were recovered and stained with propidium iodide (Ip) 10 µg/ml just before counting with the FACScan. Experiments were performed by duplicate.
Statistical analysis
Flow cytometry and in vitro chemotactic assay data were compared using the t test for paired samples.
Results
Chemokine expression in thyroid tissues from AITD patients
To explore the mechanisms by which activated lymphocytes are
recruited to the thyroid in AITDs, we studied the chemokine expression
pattern in pathologic thyroid samples. A RNase protection assay was
used to detect chemokine mRNA in thyroid tissues from 8 HT, 12 GD, 6
multinodular goiter, and 3 healthy donors. Ip-10, RANTES, and MCP-1
were detected only in tissue samples derived from AITDs, but not in
normal controls or multinodular goiter (Fig. 1
, A and B). The
chemokine expression was significantly higher in HT (Fig. 1B) and
correlated with the presence of tissue inflammatory infiltrate as
assessed in parallel by immunohistochemical staining (data not shown).
In GD, chemokine expression was highly variable and did not reach
statistical significance. In this regard, while some GD patients with
intense inflammatory infiltrate showed high chemokine expression,
others, with no inflammatory infiltrate, had absent Ip-10 and RANTES
expression (Fig. 1A, lanes 8 and 6, respectively).
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, a and b). Moreover, Ip-10 and
Mig expression in TFCs was related to the degree of inflammation and
was mainly found in thyrocytes in close proximity to lymphocytic
infiltrates. In contrast, Ip-10 and Mig were not detected in thyrocytes
of normal thyroid glands (data not shown). In addition, a strong Ip-10
and Mig staining was also found in ECs from some GD and all HT glands
(Fig. 1C, c and d), but not in control thyroids (data not shown). TFCs express proinflammatory chemokines
We next explored the regulation of chemokine expression in TFCs
obtained from normal controls and patients with multinodular goiter.
TFCs basally expressed MCP-1 and IL-8 mRNA. This expression was
strongly up-regulated by TNF-, but not by IFN- (Fig. 2A). IL-1ß induced an up-regulation of
MCP-1 and IL-8 mRNA levels. The chemokine RANTES, although not basally
present, was induced in the same way after the treatment of TFCs with
TNF- and to a lesser degree with IFN-. IFN- and TNF- had a
synergistic effect in RANTES production (Fig. 2B).
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, but not TNF-, was able to induce the
expression of Ip-10 mRNA. This response was synergized with the
addition of TNF- (Fig. 2, A and B). Hence, the chemokines RANTES and
Ip-10 are selectively and strongly regulated by TNF- and IFN-,
respectively, with a synergistic action of both cytokines, thus
suggesting a role of activated TFCs in mediating the migration of
activated lymphocytes to inflammatory foci in the thyroid in AITDs. Induction of Ip-10 and Mig expression on TFCs stimulated with proinflammatory cytokines
To further confirm the role of TFCs in the secretion of
proinflammatory chemokines, we studied the kinetics of Ip-10
production. Culture supernatants from TFCs from controls and patients
with multinodular goiter, incubated for 24 h in the presence of
different stimuli, did not contain detectable levels of Ip-10.
Incubation of TFCs with IFN- significantly increased secretion of
Ip-10, reaching a peak at d 2 (Fig. 3A).
Combinations of IFN- with TNF-, and to a lesser degree with
IL-1ß, synergistically increased Ip-10 secretion when compared with
each cytokine alone (Fig. 3A). Moreover, IFN- was able to induce Mig
expression as determined by Western blot of cytokine-treated TFCs (Fig. 3B). TNF- had a synergic effect with IFN- in this induction.
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To determine whether the chemokines studied might have a
functional role in lymphocyte recruitment to the thyroid, we next
studied the expression of chemokine receptors on thyroid-infiltrating
lymphocytes using both immunohistochemistry and flow cytometry in eight
HT and eight GD patients, which had sufficient TMC to analyze. Since
CXCR3 and CCR5 can be up-regulated on lymphocytes by activation
in vitro, we used freshly isolated cells that had not been
activated or expanded ex vivo. Two-color flow cytometry was
used on CD45+ mononuclear cells and/or CD3+ lymphocytes. Interestingly,
the percentage of CXCR3+ cells among CD45+ TMCs and CD3+ lymphocytes
was significantly higher than in autologous PBMCs (Fig. 4). In addition, CCR5 and CCR2 were also
significantly increased on TMCs compared with PBMCs from the same
patients (Fig. 4). In contrast, CXCR4 expression was significantly
lower in TMCs when compared with PBMCs (Fig. 4). These results indicate
that thyroid gland lymphocytes probably represent in
vivo activated lymphocytes selectively located at sites of organ
injury.
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), being more marked for CXCR3.
Dendritic cells (DCs) present in follicular centers were also stained
with anti-CXCR3, CCR2, and CCR5 mAbs, respectively (insets
a, b, and c). In addition, CXCR3 staining was found in ECs (data not
shown), in accordance with previous studies (22). Staining
for CXCR4 was found in TFCs (inset d) and DCs
(inset e), with nearly absent staining in infiltrating
mononuclear cells (Fig. 4). Supernatants from cultures of TFCs induce migration of activated lymphocytes expressing CXCR3
The presence of both Ip-10 and Mig in the thyroid of AITDs
suggests that they might be involved in recruiting the infiltrating
lymphocytes. We, therefore, tested the ability of supernatants from
cultures of TFCs, to induce chemotaxis in cells that express the CXCR3
receptor. We found that T lymphoblasts, which expressed high quantities
of CXCR3 receptor (80 ± 10%), showed significantly higher
chemotactic responses when exposed to supernatants from TFCs
previously activated with proinflammatory cytokines, being maximal with
the combination of IFN- with TNF- (Fig. 5A). These chemotactic responses were
abolished when the assays were performed in the presence of pAb to
Ip-10 and Mig, as well as with the anti-CXCR3 mAb (Fig. 5B). Although
we cannot exclude a role in chemotaxis of IFN- and TNF-, which
could have been present in low quantities in the supernatants, the
abolishment of the chemotactic responses by anti-Ip-10- and anti-Mig
blocking antibodies suggests an important role of these chemokines in
TFC-mediated recruitment of activated T lymphocytes.
|
In this study, we provide evidence for the role of TFCs as an important source of the CXC chemokines Ip-10 and Mig, which can mediate the recruitment of activated CXCR3+ lymphocytes to the thyroid in AITDs. Expression of Ip-10 and Mig was found in thyroid glands from patients with AITDs, but not in normal controls. Because these chemokines play a role in selective recruitment of activated lymphocytes, chemokine expression was correlated with presence of tissue inflammatory infiltrate, independently of the clinical diagnosis of GD or HT.
Possible sources of Ip-10 and Mig in the thyroid gland could be
intrathyroidal inflammatory cells, such as lymphocytes, macrophages
and/or DCs, as well as ECs and TFCs. Our results show that activated
epithelial cells represent an important source of Ip-10 and Mig in
AITDs. Ip-10 and Mig were expressed by TFCs both in vivo in
AITDs and in vitro when stimulated with proinflammatory
cytokines, mainly IFN-. In this regard, the proinflammatory
cytokines IFN-, TNF-, and IL-1ß have been repeatedly detected
in thyroid tissues from AITD patients (24, 25, 26). Other
activated epithelial cells, such as bronchial epithelial cells
(27), and intestinal epithelia (28) have been
reported to secrete Ip-10 and Mig in vitro when treated with
IFN-. Our findings support an important role of epithelial cells in
lymphocyte recruitment not only in inflammation, but also in autoimmune
diseases.
Another question is whether Ip-10 and Mig act in concert with other
chemokines in regulating the inflammatory infiltrate in AITDs. The
highly organized infiltrate present in AITD glands suggests that
several chemoattractant proteins are probably involved in a multistep
navigation mode (30). Although we found basal expression
of IL-8 by TFCs in vitro as previously reported
(29), no IL-8 expression was demonstrated in thyroid
glands in vivo. We have not confirmed the presence of
MIP-1 and ß chemokines as previously reported in GD
(20). These conflicting data could be related to different
chemokines analyzed (Ashab et al. studied only CC
chemokines), the techniques used (PCR vs. RNA protection
assay) and to the patients studied (GD vs. patients with
AITDs, including HT). Interestingly, we have demonstrated expression of
the chemokines RANTES and MCP-1 in biological samples from HT thyroids
in vivo when compared with normal controls. In addition,
RANTES and MCP-1 production were also found when TFCs were stimulated
in vitro, not with IFN- but with TNF-, pointing also
to resident TFCs as an important source of these chemokines. RANTES has
been reported to be secreted by other epithelial cells, such as renal
tubular epithelium (31) or bronchial epithelium
(32). In addition, MCP-1 production by pulmonary
epithelial cells (33), and by tubular epithelium has also
been reported (34). Like Ip-10, RANTES is known to recruit
and activate specific T-cell subsets (35).
In AITDs, the subset of intrathyroidal infiltrating lymphocytes displays a CD45RO+, CD69+, L-selectin+/-, lymphocyte function-associated antigen-1high, vascular cell adhesion molecule-4high, effector/activated phenotype (6). Accordingly, we found herewith increased expression of the Ip-10/Mig receptor CXCR3+, the RANTES receptor CCR5, and the MCP-1 receptor CCR2 in intrathyroidal lymphocytes when compared with autologous peripheral blood lymphocytes. In addition, our immunohistochemical studies revealed that a majority of intrathyroidal lymphocytes were CXCR3+, with the highest Ip-10 and Mig expression by thyrocytes adjacent to CXCR3+ lymphocytic infiltrates. Most CXCR3+ and CCR5+ T cells are type 1 helper cells, that have been reported to predominate in AITDs (36). On the other hand, a decreased expression of the CXCR4 receptor was found in infiltrating lymphocytes in AITD. CXCR4 is highly expressed by peripheral blood naive T cells, but at lower levels on CD45RO+ memory T cells, suggesting CXCR4 is progressively down-regulated after repeated stimulations (37). In addition, CXCR3, CCR5, CCR2, and CXCR4 chemokine receptors were found in DCs from germinal centers, but not in follicular DCs in AITD thyroids. DCs, which are capable of presenting antigen more efficiently than any of the other antigenpresenting cells, are key cells in the immune response, with a central role in antigen-specific immunity (38). DCs have been shown to both produce and respond to chemokines (38). These cells are increased at the site of an organ-specific autoimmune disease (39), and an active involvement in AITDs has been suggested (40).
Recent studies have implicated different chemokines and their receptors
in the pathogenesis of different inflammatory diseases. In multiple
sclerosis, where an increase in IFN- production precedes the
relapses, expression of the chemokines Ip-10, Mig, and MIP-1 and
their ligands has been reported, pointing to these chemokines as
responsible for the recruitment of specific T cells (17).
In rheumatoid arthritis, a critical role has been suggested for
TNF-, as indicated by the success of anti-TNF--based therapy
(9). In addition, antagonism of MCP-1 and RANTES
chemokines has reduced the prevalence and severity of
adjuvant-collagen-induced arthritis in rats (41). In
AITDs, release of Ip-10, Mig, RANTES, and MCP-1 by TFCs could be
induced through chronic IFN--dependent stimulation by infiltrating T
cells. On the other hand, as herein demonstrated, because lymphocytes
express CXCR3, CCR5 and CCR2 receptors could be considered both
inducers of production and targets of these chemokines. The
inducibility and high expression of the IFN-/chemokine axis in AITDs
could create an anomalous high inflammatory scenarium.
Leukocyte-mediated injury may induce higher expression of these or even
new chemokines resulting in a more extensive tissue damage,
self-perpetuating AITDs (9). Although this hypothesis
seems attractive as a possible source of recruitment of activated
lymphocytes in AITDs, it may not reflect what actually happens in the
development of AITDs. The capacity to control precisely the movement of
inflammatory cells suggests that these chemokines and their receptors,
independently of whether they are primary or secondary in the
inflammatory response, might provide novel targets for therapeutic
intervention.
Acknowledgments
Footnotes
This work was supported by grants from INSALUD (FIS 98/1099). D.S. was supported by the "Severo Ochoa" fellowship from the Fundación Ferrer.
Abbreviations: AITD, autoimmune thyroid disorder; EC,
endothelial cell; DC, dendritic cell; GD, Graves’ disease; HT,
Hashimoto’s thyroiditis; IFN, interferon; Ip-10, IFN-inducible protein
10; mAb, monoclonal antibody; MCP, monocyte chemotactic protein; Mig,
monokine induced by IFN-; MIP, macrophage inflammatory protein; pAb,
polyclonal antibody; PBMC, peripheral blood mononuclear cell; RANTES,
regulated on activation normal T lymphocyte expressed and secreted;
TFC, thyroid follicular cell; TMC, thyroid gland mononuclear cell.
Received April 2, 2001.
Accepted June 28, 2001.
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A. Antonelli, P. Fallahi, M. Rotondi, S. M. Ferrari, P. Romagnani, M. Grosso, E. Ferrannini, and M. Serio Increased serum CXCL10 in Graves' disease or autoimmune thyroiditis is not associated with hyper- or hypothyroidism per se, but is specifically sustained by the autoimmune, inflammatory process. Eur. J. Endocrinol., May 1, 2006; 154(5): 651 - 658. [Abstract] [Full Text] [PDF]
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A. Antonelli, M. Rotondi, S. M. Ferrari, P. Fallahi, P. Romagnani, S. S. Franceschini, M. Serio, and E. Ferrannini Interferon-{gamma}-Inducible {alpha}-Chemokine CXCL10 Involvement in Graves' Ophthalmopathy: Modulation by Peroxisome Proliferator-Activated Receptor-{gamma} Agonists J. Clin. Endocrinol. Metab., February 1, 2006; 91(2): 614 - 620. [Abstract] [Full Text] [PDF]
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G Aust, M Kamprad, P Lamesch, and E Schmucking CXCR6 within T-helper (Th) and T-cytotoxic (Tc) type 1 lymphocytes in Graves' disease (GD) Eur. J. Endocrinol., April 1, 2005; 152(4): 635 - 643. [Abstract] [Full Text] [PDF]
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M. Rotondi, A. Falorni, A. De Bellis, S. Laureti, P. Ferruzzi, P. Romagnani, A. Buonamano, E. Lazzeri, C. Crescioli, M. Mannelli, et al. Elevated Serum Interferon-{gamma}-Inducible Chemokine-10/CXC Chemokine Ligand-10 in Autoimmune Primary Adrenal Insufficiency and in Vitro Expression in Human Adrenal Cells Primary Cultures after Stimulation with Proinflammatory Cytokines J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2357 - 2363. [Abstract] [Full Text] [PDF]
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A. Antonelli, M. Rotondi, P. Fallahi, P. Romagnani, S. M. Ferrari, A. Paolicchi, E. Ferrannini, and M. Serio Increase of interferon-{gamma} inducible {alpha} chemokine CXCL10 but not {beta} chemokine CCL2 serum levels in chronic autoimmune thyroiditis Eur. J. Endocrinol., February 1, 2005; 152(2): 171 - 177. [Abstract] [Full Text] [PDF]
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A. Antonelli, M. Rotondi, P. Fallahi, P. Romagnani, S. M. Ferrari, A. Buonamano, E. Ferrannini, and M. Serio High Levels of Circulating CXC Chemokine Ligand 10 Are Associated with Chronic Autoimmune Thyroiditis and Hypothyroidism J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5496 - 5499. [Abstract] [Full Text] [PDF]
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