Characterization of a novel set of resident intrathyroidal bone marrow-derived hematopoietic cells: potential for immune-endocrine interactions in thyroid homeostasis
Department of Basic Sciences, Dental Branch, University of Texas Health Science Center at Houston, Houston, TX 77030, USA
* Author for correspondence (e-mail: john.r.klein{at}uth.tmc.edu)
Accepted 9 September 2003
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Summary |
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Key words: immuneendocrine, bone marrow, homeostasis, thyroid
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Introduction |
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Other studies of hypothalamuspituitarythyroid axis mediated
immuneendocrine interactions have examined the involvement of dendritic
cells (DCs) in modulating thyroid cell function in normal non-autoimmune
animals. In vitro co-culture of thyrocytes with DCs restricted
thyrocyte growth and thyroid hormone synthesis
(Simons et al., 1998). This
may be mediated by interleukin-1ß or interleukin-6
(Simons et al., 1998
) produced
from DCs, or possibly by secretion of those monokines directly from the
thyrocytes themselves (Simons et al.,
1998
). Simons et al.
(1998
) showed that DCs were
obtained from splenic tissues, however. Given the phenotypic and functional
heterogeneity of splenic DCs, the relevance of those cells to intrathyroidal
DCs is unclear. In other studies, thyroid-derived low density mononuclear
cells isolated from rat thyroids expressed the rat monocyte ED1 marker, but
lacked the macrophage markers ED2 and ED3 and had little or no expression of
CD80 and CD86 (Simons et al.,
2000
). However, the inability to obtain sufficient numbers of
cells for flow cytometric analyses required that analyses be done in cytospin
preparation, and
50% of the cells were contaminated with thyrocytes
(Simons et al., 2000
). In
studies by Croizet et al.
(2000
,
2001
) porcine thyroid was used
as a source of both thyrocytes and DCs for in vitro analyses of
DCthyrocyte interactions. Thyroid-derived DCs were shown to proliferate
in the presence of TSH and to retain the ability to endocytose labeled ligands
(Croizet et al., 2001
).
Moreover, intrathyroidal DCs retained an immature phenotype until exposed to
tumor necrosis factor-
(Croizet et
al., 2000
), a cytokine commonly produced by DCs. An important
feature of that study pertains to its ability to compare DC-thyroid responses
using thyroid-derived rather than peripheral DCs; however, studies using
porcine tissues are limited by a paucity of immunological reagents for
extensive phenotypic analyses.
In the present study, we have used multiple approaches to characterize
intrathyroidal DCs in situ in mice. First, thyroid tissue sections from normal
mice were examined using immunofluorescent staining and a panel of monoclonal
antibodies (mAbs) to DC, macrophage (m), lymphocyte and granulocyte
markers (CD11b, CD11c, CD40, CD3, CD19, CD8
, F4/80, and Gr-1). This
permitted us to evaluate the presence and distribution of DCs as they
naturally occur in the thyroid, and to avoid problems associated with
isolating DCs from thyroid tissue digests that would be compromised by
difficulties in obtaining adequate numbers of DCs for in vitro
phenotypic analyses. Second, the trafficking of DCs to the thyroid was
examined to define the use in radiation chimeras of bone marrow cells from
transgenic donor mice expressing enhanced green fluorescent protein
(EGFP)+ were injected into irradiated EGFP host
animals. Direct fluorescence analyses of thyroid, kidney and liver tissues
were done to localize EGFP+ cells in non-lymphoid tissues. Third,
EGFP+ bone marrow-derived DCs generated in vitro and
transferred to EGFP host mice were used to confirm that
intrathyroidal EGFP+ cells were in fact DCs. Fourth, thyroid tissue
sections were stained with a mouse TSHß-specific mAb to determine the
spatial relationship between TSH-producing cells and CD11b+ cells
in the thyroid. Findings from these studies provide important new information
by demonstrating: (i) that intrathyroidal DCs bear an uncommon
`myeloid-related' CD11b+, CD11c,
F4/80, CD8
non-macrophage cell
phenotype; (ii) that they are seeded rapidly into the thyroid from the bone
marrow within 1 week of cell transfer and are present in the thyroid up to 20
weeks post-bone marrow transfer; (iii) that within the thyroid they tend to
cluster between and around thyroid follicles, and (iv) that they appear to
express high levels of TSH. A hypothesis is proposed whereby intrathyroidal
DCs in normal mice may be involved in the microregulation of thyroid hormone
activity, thus establishing a novel immune-endocrine network used to maintain
broad-spectrum physiological homeostasis.
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Materials and methods |
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Antibodies and immunofluorescence
Antibodies used in this study were: phycoerythrin (PE)-anti-CD3 (2C11);
PE-anti-CD8 (53-6.7); PE- and APC-anti-CD11b (M1/70); PE-anti-CD11c
(HL3); PE-anti-CD19 (1D3); PE-anti-CD40 (3/23); PE-anti-CD80 (B7-1, 16-10A1);
PE-anti-CD45 LCA (30-F11); biotinylated-anti-Ly-6G (RB6-8C5); streptavidin-PE;
purified anti-CD16/32 (Fc
III/II receptor) (2.4G2); PE-hamster and -rat
Ig control mAbs (BDPharMingen; San Diego, CA, USA, all reagents);
PE-anti-F4/80 (CalTag; South San Francisco, CA, USA); biotin-anti-mouse
TSHß (1B11) (Zhou et al.,
2002
).
Mice were killed by CO2 inhalation. Thyroid, mesenteric lymph nodes, kidney and liver tissues were recovered using a dissecting microscope. Tissues were frozen in liquid N2, embedded in Tissue Freezing Medium (Triangle Biomedical Sciences; Durham, NC, USA), and 5 µm sections were prepared on a Model 2800 Frigocut N cryostat (Cambridge Scientific Products; Cambridge, MA, USA). Tissues were fixed in acetone, air dried, and washed three times with phosphate-buffered saline (PBS). Tissues were blocked for 15 min at room temperature with Avidin Block (DAKO Corp.; Carpinteria, CA, USA), washed, and blocked for 15 min at room temperature with Biotin Block (DAKO). Slides were washed and reacted with anti-Fc receptor mAb for 30 min at room temperature. Tissues were washed with PBS and reacted with a PE- or biotin-labeled mAb for 3 h at 4°C and then washed again. For direct PE-labeled staining, tissues were washed and observed for immunofluorescence. For staining using biotin-labeled mAb, tissues were reacted for an additional 30 min with 1 µg ml1 of streptavidinPE, washed and examined. For analyses of EGFP+ cells in tissues, fresh-frozen tissue specimens were sectioned in the cryostat, fixed and examined directly without staining, or were reacted with PE-anti-CD45 LCA or PE-anti-CD11b for identification of hematopoietic cells and CD11b+ cells, respectively, in thyroid tissue sections from chimeric mice. For experiments with fluorescently labeled beads, mice were injected intraperitoneally (i.p.) with a solution of 6 drops ml1 of Fluoresbrite plain YG 1.0 µm microspheres (2.59% Solids-Latex) in PBS (Polysciences, Inc; Warrington, PA, USA). 18 h post-injection, mice were skilled and the spleen, mesenteric lymph nodes and thyroid were removed. Tissues were prepared by cryostat sectioning and observed directly for the presence of fluorescent beads. Fluorescence was analyzed using an Olympus BH-2 immunofluorescence microscope.
Bone marrow chimeras
12-week-old female EGFP BALB/c mice were given 900 rad
total body irradiation from a 60Co source at the University of
Texas M. D. Anderson Cancer Center, Houston, TX, USA. Whole bone marrow was
recovered from EGFP+ donor mice, washed with PBS and
17x106 cells were injected intravenously (i.v.) into the tail
vein of irradiated female EGFP recipient mice. At intervals
after bone marrow reconstitution (120 weeks), mice were sacrificed,
tissues were recovered and examined for immunofluorescence.
Generation of in vitro bone marrow-derived DCs and flow
cytometry
In vitro generation of DCs from bone marrow was done according to
published protocols (Inaba et al.,
1992). Briefly, whole bone marrow cells were flushed from the
femur canula of EGFP+ mice. Erythrocytes were lysed by hypotonic
shock with ammonium chloride. Cells were washed in PBS and seeded at a density
of 106 cells ml1 in 6-well tissue culture plates
(Fisher Scientific; Houston, TX, USA) in RPMI-1640 supplemented with FBS (10%
v/v), 100 U ml1 penicillinstreptomycin, 2 mmol
l1 L-glutamine, and 5x105
mol l1 2-mercaptoethanol (Sigma-Aldrich, St Louis, MO, USA),
containing mouse recombinant GMCSF (10 ng ml1)
(BDPharMingen, San Diego, CA, USA). On days 2 and 4 of culture, the medium and
floating cells containing many granulocytes were removed. DCs attached in
clusters to the bottom of the plate were re-fed with fresh supplemented medium
containing 10 ng ml1 GMCSF. By day 6 of culture, the
majority of floating cells had the morphology of DCs. These cells were
collected and some were stained for two-color flow cytometric analyses for
expression of CD11b-PE or CD11c-PE and endogenous green fluorescence using a
FACScalibur flow cytometer with CellQuest software (BD Biosciences). The
remaining cells were washed extensively with PBS and 3.3x106
cells were injected i.v. into each of two non-irradiated
EGFP recipient mice. Host mice were sacrificed 25 days
post-cell transfer, the thyroids were recovered, frozen sections were
prepared, and tissues were examined for the presence of cells having
endogenous green fluorescence.
Reverse transcription-polymerase chain reaction
Isolation of RNAs and reverse transcription-polymerase chain reactions
(RT-PCRs) were done using protocols we described previously
(Wang et al., 2002). Pituitary
tissues were purchased from Harlan Bioproducts for Science, Inc.
(Indianapolis, IN, USA); thyroid tissues were dissected from healthy BALB/c
mice. Primer sequences were: TSHß forward
5'-GTGGGTGGAGAAGAGTGAGC-3'; TSHß reverse
5'-TAGAAAGACTGCGGCTTGGT-3'; ß-actin forward
5'-ATGGATGACGATATCGCTG-3'; ß-actin reverse
5'-ATGAGGTAGTCTGTCAGGT-3'. Taq polymerase (Promega;
Madison WI) amplification consisted of 35 cycles at 95°C for 1 min,
55°C for 1 min and 72°C for 1 min using a Biometra T-Gradient
thermocycler (Whatman Biometra; Gottingen, Germany). Based on the published
sequence of the murine TSHß gene
(Gordon et al., 1988
), the
amplified TSHß product was expected to be 473 bp; PCR products were run
on a 1.5% agarose gel.
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Results |
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The CD11b+ cells may be either a myeloid-related DC population
with high CD11b and low CD11c expression
(Maraskovsky et al., 1999), or
they may be m
s, given that CD11b can be expressed on both DCs and
m
s (Maraskovsky et al.,
1999
; Ho and Springer,
1982
). To distinguish between those possibilities, thyroid tissue
sections from normal mice were stained with mAb F4/80, a reagent that
recognizes a determinant on nearly all mouse m
s
(Hirsch et al., 1981
;
Austyn and Gordon, 1981
). As
seen in Fig. 2, thyroid tissue
sections displayed a pattern of CD11b staining similar to that described in
Fig. 1, yet they were devoid of
F4/80+ cells. Additionally, immunofluorescent analyses indicated a
lack of staining with anti-CD40, anti-CD80, anti-CD19 and anti-CD3 mAbs.
Because CD40 and CD80 are acquired on DCs with activation
(Quadbeck et al., 2002
), the
lack of expression of those markers is reflective of the non-activated status
of the intrathyroidal DCs. Similarly, the lack of anti-CD19 and anti-CD3
staining further indicates the non-inflamed status of the thyroid given that
antigen-reactive lymphocytes, in particular T cells, would be expected to
accompany activated DCs, if present. The absence of CD8
staining
suggests that the intrathyroidal DCs are myeloid-related
(Maraskovsky et al., 1996
;
Pulendran et al., 1997
; Vrmec
and Shortman, 1997) and not lymphoid-related DCs
(Ardavin et al., 1993
;
Kronin et al., 1997
;
Saunders et al., 1996
;
Wu et al., 1995
); the lack of
expression of the Ly-6G antigen (Gr-1) suggests that they are not related to
plasmacytoid DCs (Nakano et al.,
2001
).
|
CD80 is expressed in the lymph nodes but not in the thyroid
To better address the issue of the activation characteristics of
intrathyroidal DCs, studies were done in which mice with a transgenic T cell
receptor for a HEL peptide (3A9 mice) were injected i.p. with 100 µl of 10
mg ml1 HEL. 48 h later mice were killed and the spleen,
lymph nodes and the thyroid were removed and stained for expression of CD11b
and CD80. In the spleen there was an abundance of both CD11b+
(Fig. 3A) and CD80+
(Fig. 3B) cells. Note the
presence of these in the marginal zones around the germinal centers
(Fig. 3A,B), suggesting that DC
in 3A9 mice had been activated as a consequence of exposure to HEL. Similar
findings also were observed for lymph node tissues (data not shown). Unlike
the spleen and lymph nodes, however, no CD80+ cells were present in
the thyroid of 3A9 mice injected with HEL
(Fig. 3C), despite an abundance
of CD11b+ cells in that tissue
(Fig. 3D). Of interest was the
finding that in HEL-primed mice, both CD11b+
(Fig. 3E) and CD80+
(Fig. 3F) cells were present in
the pericapsular lymph nodes associated with the thyroid, even though no
CD80+ cells were not present in the thyroid itself
(Fig. 3C). This suggests either
that CD80+ cells do not enter the thyroid, or that DC cells of the
thyroid are limited in their capacity to become activated in a conventional
manner.
|
Bone marrow origin of intrathyroidal cells
The findings described above indicate that intrathyroidal CD11b+
cells are present in the thyroid of normal mice. In an effort to understand
the origins of those cells, hematopoietic radiation chimeras were constructed
by injecting bone marrow cells from EGFP+ transgenic mice into
lethally irradiated EGFP recipient mice. At intervals
post-cell transfer, mice were killed, cryostat sections were made of the
thyroid, and sections were examined for green fluorescence without
staining to identify donor cells based on endogenous green fluorescence. As
seen in Fig. 4, donor
EGFP+ cells were readily detected in the thyroid as early as 1 week
post-bone marrow transfer, and were present at all points examined up to 20
weeks post-transfer. Typically, EGFP+ cells clustered near or
around thyroid follicles (Fig.
4) similar to that observed for CD11b+ cells (Figs
1 and
2). Note that the differences
in staining pattern seen in Figs
1 and
2 are because CD11b and CD11c
are membrane bound but EGFP fluorescence is cytoplasmic in EGFP+
cells. These findings are of particular interest for several reasons. First,
the rapid appearance of EGFP+ cells in the thyroid after bone
marrow injection suggests that some bone marrow cells traffic quickly to the
thyroid. Second, the fact that EGFP+ cells were present as late as
20 weeks post-bone marrow injection suggests either that bone marrow-derived
EGFP+ cells in the thyroid are long-lived cells, or that they are
continually replenished from the bone marrow.
|
To determine whether bone marrow-derived EGFP+ cells randomly
trafficked into the thyroid and other non-lymphoid tissues, or whether their
presence in the thyroid reflects a selective process of cell trafficking,
tissue sections were made from the thyroid, mesenteric lymph nodes, the kidney
and the liver of EGFP+ EGFP mice 2 weeks
after bone marrow transfer. As seen in Fig.
5, EGFP+ cells similar to those in
Fig. 4 were present in the
thyroid. Additionally, there was a high density of EGFP+ cells in
the lymph nodes, particularly in or near lymphocyte-enriched follicular
regions. In contrast, few if any EGFP+ cells were found in the
kidney or liver of EGFP recipient mice. These finding
strongly imply that at least some bone marrow CD11b+ cells traffic
directly to the thyroid, and that the distribution of those cells reflects a
specific rather than a stochastic process.
|
Intrathyroidal CD11b+ cells are not bone marrow stromal
cells
To better define the origins of EGFP+ cells in the thyroid of
EGFP+ EGFP chimeras, thyroid tissue
sections were made from mice 3 weeks post-bone marrow transfer. Unstained
tissues contained cells with endogenous green fluorescence
(Fig. 6) similar to that of
other chimeric mice (Fig. 5).
Additionally, however, thyroid tissue sections stained with PE-anti-CD11b mAb
or PE-anti-CD45 LCA, and examined for yellow fluorescence, had many
CD11b+ and CD45 LCA+ cells
(Fig. 6). Because LCA is
expressed on all hematopoietic cells except erythrocytes
(van Ewijk et al., 1981
), and
is not expressed on bone marrow stromal cells, this indicates that the
intrathyroidal EGFP+ cells in EGFP+
EGFP chimeras were hematopoietic cells and were not derived
from bone marrow stromal cells.
|
Adoptively transferred DCs localize in the thyroid
To further confirm that intrathyroidal DCs originated from bone
marrow-derived DCs, bone marrow cells from EGFP+ mice were cultured
for 6 days with GM-CSF according to published protocols
(Inaba et al., 1992) as
described in the Materials and methods. Floating cells with the morphology of
DCs were collected and stained for two-color PE-anti-CD11b expression plus
endogenous green fluorescence, and PE-anti-CD11c expression plus endogenous
green fluorescence. As seen in Fig.
7A, 96.5% of the cells showed endogenous green fluorescence and
also expressed high levels of CD11b. In contrast, only 38.9% of the cells
expressed CD11c and showed green fluorescence, whereas 58.7% of the cells did
not express CD11c but showed green fluorescence
(Fig. 7B). Hence, the day-6 DCs
consisted of a population of CD11b+ CD11c+ cells, and a
population of CD11b+ CD11c cells. Cells were
collected, washed extensively in PBS, and 3x106 cells were
injected into each of two non-irradiated EGFP recipient
mice. 25 days later, thyroid tissue sections were prepared and examined for
the presence of green fluorescent cells. As seen in
Fig. 7C-F, intrathyroidal
EGFP+ cells were present adjacent to and surrounding thyroid
follicles as seen in EGFP+
EGFP radiation
chimeras (Fig. 4). It is
important to note that because the host mice in this experiment were not
irradiated, the homing of bone marrow-generated DCs into the thyroid cannot be
attributed to tissue damage imparted by ionizing radiation. Additionally, and
perhaps most important, these findings indicate that DCs grown using
conventional in vitro methods migrate to the thyroid after cell
transfer, thus reinforcing the notion that the EGFP+ intrathyroidal
cells present in EGFP+
EGFP chimeras were
derived from a bone marrow DC precursor population.
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CD11b+ thyroid cells produce TSHß
The finding that there is an abundance of CD11b+ intrathyroidal
cells, and the homing of bone marrow cells to the thyroid of normal mice, were
unexpected and surprising. Although DCs can be found in nearly all tissues of
the body (in part because of their natural ability to sample and transport
antigens to secondary lymphoid tissues during the early phase of an adaptive
immune response) the presence of large numbers of DCs in internal organs of
normal animals with no evidence of pathology has, to our knowledge, not been
described. The rare but not totally atypical phenotype of the intrathyroidal
DCs described here therefore prompted us to explore the possibility that they
may function in some capacity other than in antigen handling and processing,
given that the thyroid is not a tissue known to have excessive amounts of
foreign antigens. Similarly, we have recently demonstrated that bone marrow
cells, in particular a CD11b+ population, produce high levels of
TSH (Wang et al., 2003). To
further explore the possibility that intrathyroidal CD11b+ cells
might be a source of TSH production within the thyroid, thyroid sections were
stained with APC-labeled anti-CD11b mAb to identify CD11b+ cells,
and with biotin-labeled anti-mouse TSHß-specific mAb
(Zhou et al., 2002
) plus
streptavidin-FITC to identify TSH-producing cells in the thyroid. Of many
sections examined, the general pattern was one in which CD11b+
cells (Fig. 8A) also expressed
TSHß (Fig. 8B), strongly
suggesting that CD11b+ cells routinely produce TSHß.
Occasionally, however, some CD11b+ cells did not express
intracellular TSHß (Fig.
8A, circled area), though overall these were rare. It is possible
either that those cells constitute a population of CD11b+ cells
that do not normally secrete TSH, or that they were not producing TSH at the
time of analyses.
|
Owing to the high phagocytic potential of most DCs, the possibility was
considered that the presence of intracellular TSH could reflect a process of
TSH engulfment rather than one in which TSH is manufactured by intrathyroidal
DCs. Two experiments were done to test this. First, normal BALB/c mice were
injected with green fluorescent microspheres as described in the Materials and
methods. 18 h later, the mice were killed and the thyroid, mesenteric lymph
nodes and spleen were examined directly in cryostat sections for the presence
of green fluorescent particles. As seen in
Fig. 8C, there was a distinct
rim of green fluorescence in the marginal zones around follicular regions in
the spleen, as would be indicative of a phagocytic process of microspheres by
DCs and ms that are present at high density in those areas. A similar
pattern was also noted for the lymph node tissues surrounding germinal centers
and follicles (data not shown). Notably, however, fluorescent spheres were not
present in the thyroid (Fig.
8D).
In the second experiment, RT-PCR analyses was done using thyroid tissues from normal mice to determine whether the TSHß gene was expressed locally. Control material consisted of tissue from the anterior pituitary, which actively expresses the TSHß gene. As seen in Fig. 9, a clearly detectable TSHß band of the correct size (473 bp) was amplified from thyroid tissue and the pituitary tissue. Collectively, these findings make a strong case for TSH in intrathyroidal DCs not being the consequence of phagocytosis but, rather, the result of local synthesis of TSH within the thyroid itself.
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Discussion |
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Studies from the adoptive transfer experiments using in
vitro-derived DCs indicate strongly that some cells with DC
characteristics traffic to the thyroid. It will be of interest to determine
whether this occurs from the CD11b+ CD11c+ or the
CD11b+ CD11c population of bone marrow cells.
Experiments are underway to differentiate between those possibilities using
cell-sorted populations of in vitro-generated DCs. Also of interest
is the identification of thyroid-specific homing markers that might be
involved in regulating the trafficking of DCs to the thyroid, possibly
involving integrins similar but different from those used to direct
hematopoietic cells to mucosal and other tissue sites
(Kilshaw and Murant, 1991;
Streeter et al., 1988
).
An interesting finding from this study was the extensive TSHß staining
of cells with features very similar to the CD11b+ intrathyroidal
cells, and the location of those cells near thyroid follicles as occurred with
CD11b+ cells (Figs 1
and 2) and bone marrow-derived
EGFP+ cells (Figs 3,
4,
5). The implication of this is
that TSHß+ intrathyroidal cells are a potential source of
nonendocrine TSH that is directly available to the thyroid itself. Consistent
with this, studies from our laboratory recently demonstrated that some bone
marrow cells are a potent source of TSH as seen from intracellular TSHß
staining and by the presence of secreted TSH in the supernatants of cells
cultured in vitro (Wang et al.,
2003). Moreover, the primary TSH-producing cells were
CD11b+ cells, although CD11b cells also produced
TSH albeit at significantly lower levels. Bone marrow cells expressing TCR,
B220 or Thy-1 did not synthesize TSH (Wang
et al., 2003
). In an opposite manner, the TSH receptor was heavily
expressed on a population of CD11b bone marrow cells
distributed among monocyte precursors, as well as lymphocyte and granulocyte
precursors (Wang et al.,
2003
). Stimulation of CD11b (TSH receptor
positive) bone marrow cells with TSH resulted in a dose-dependent release of
TNF
, whereas CD11b+ cells produced TNF
independent of
TSH stimulation (Wang et al.,
2003
). Those observations are particularly curious in light of the
findings by Croizet et al.
(2001
), demonstrating an effect
of TNF on the phenotype of intrathyroidal DCs. Finally, the possibility that
intrathyroidal DCs have phagocytosed or pinocytosed TSH within the thyroid
seems unlikely in light of the finding that those cells failed to ingest
fluorescent microspheres in a manner comparable to DCs in lymphoid tissues,
and because of evidence from RT-PCR analyses suggesting that TSHß may be
produced locally within the thyroid.
In summary, this study has provided detailed phenotypic analyses of intrathyroidal DCs in normal mice, and has traced the origins of those cells to populations of bone marrow hematopoietic cells. The unconventional phenotype of this DC population, plus the observation of TSH production by intrathyroidal DC-like cells, suggest a role for the immune system in the maintenance of thyroid homeostasis in ways not appreciated before.
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Acknowledgments |
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References |
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