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,
Departments of
*
Internal Medicine,
Molecular Microbiology and Immunology, and
Pathology, University of Missouri School of Medicine, Columbia, MO 65212;
Veterans Affairs Research Service, Columbia, MO 65212; and
¶ Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Fas/FasL system is one of an expanding family of receptor-ligand pairs involved in cell fate determination in a variety of cells (7). Fas can be expressed by many cell types, whereas FasL is tightly regulated, expressed mainly by activated T cells and NK cells as well as some nonlymphoid cells (8, 9, 10, 11, 12, 13, 14). When FasL binds to Fas on Fas-sensitive target cells, target cells die by apoptosis (7, 8). FasL expression in nonlymphoid tissue is important for protecting immune privileged sites from immune-mediated damage (10, 11). Peripheral deletion of T cells activated by superantigen was predominantly dependent upon inducible expression of FasL on nonlymphoid cells (14). Thus, the Fas signaling pathway is involved in immune regulation, and is one pathway by which cytotoxic CD8 T cells can induce apoptosis in target cells (15).
The Fas/FasL pathway has received considerable attention in autoimmune diseases such as autoimmune thyroiditis, experimental allergic encephalomyelitis, experimental autoimmune uveitis, diabetes, and arthritis (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). Accumulating data obtained using transgenic and genetically deficient mice suggest that Fas-FasL interactions can be responsible for inducing tissue damage in autoimmunity (18, 19, 20, 21, 22, 23, 24, 28), and may also be important for recovery or resolution of autoimmune disease (21, 22, 23). What cells express Fas or FasL and the level of FasL expression may also contribute to the outcome of autoimmune disease (20, 21, 28). There have been conflicting results regarding the expression and the role of Fas/FasL on normal and diseased thyroids in human and mouse autoimmune thyroiditis (17, 24, 25, 26, 28, 29), indicating that the precise role of this pathway in autoimmune thyroid disease needs further clarification.
Identification of the cellular expression of Fas and FasL is important to address the role of the Fas/FasL signal pathway in autoimmune thyroiditis. Our previous results showed that FasL mRNA was detected in G-EAT thyroids, but not in normal thyroids, and the level of FasL mRNA expression generally paralleled inflammation severity (5). To determine whether FasL-mediated apoptosis may play a role in G-EAT resolution, the present study was undertaken to analyze the in situ localization of Fas and FasL in G-EAT thyroids with or without depletion of CD8+ cells. The results suggest that CD8+ cells may regulate G-EAT by inducing up-regulation of FasL on thyrocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBA/J mice were obtained through C. Reeder at the National
Institutes of Health (Bethesda, MD). Female mice, 6–8 wk old, were
used for all experiments. AKR mice and
2-microglobulin (2m)
gene knockout mice on the AKR background (The Jackson Laboratory, Bar
Harbor, ME) were also used for some experiments.
EAT induction and depletion of CD8+ cells
EAT was induced as previously described (1).
Briefly, donor mice were injected i.v. twice at 10-day intervals with
150 µg MTg and 15 µg LPS (Escherichia coli 011:B4;
Sigma-Aldrich, St. Louis, MO). Seven days later, donor spleen cells
were restimulated in vitro with 25 µg/ml MTg in the presence of 5%
final concentration of culture supernatant containing mAb specific for
IL-2R (M7/20) and 5 ng/ml IL-12 (1, 4). Cells were
harvested after 72 h and washed twice with balanced salt solution,
and 3.5 x 107 cells were transferred i.v.
to 500-rad irradiated syngeneic recipients. Recipient thyroids were
collected at different times after cell transfer for histologic
evaluation of EAT. CD8+ cells were
depleted by i.p. injection of 300 µg anti-CD8 mAb
(anti-Lyt-2.1) (ATCC HB-129). In most experiments, mice were given
anti-CD8 mAb beginning 1 day after cell transfer, and injections
were repeated every 10 days until termination of the experiment. In
other experiments, anti-CD8 injections were begun 6, 12, or 20 days
after cell transfer; these injections were also repeated every 10 days
until termination of the experiment (3, 5). Since
anti-CD8 mAb (anti-Lyt-2.1) can deplete both
CD8+ T cells and CD8+
dendritic cells (DC) (30), anti-CD8 mAb (YTS 156)
(31) was used to deplete CD8 T cells, but not DC. Mice
received 300 µg YTS 156 beginning 1 day after cell transfer with
subsequent injections every 10 days until termination of the
experiment. For each experiment, depletion of splenic
CD8+ cells was found to be complete by flow
cytometry at the time thyroids were removed.
In vivo administration of anti-FasL mAb
The neutralizing hamster anti-mouse FasL mAb MFL1 (32, 33) was purified using protein G. Mice were given 0.5 mg MFL1
i.p. twice weekly beginning 2 or 4 days after cell transfer and
continued until thyroids were removed, as indicated in Table IV
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Control mice were given the same amount of hamster Ig (Jackson
ImmunoResearch, West Grove, PA).
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Thyroids were scored quantitatively for EAT severity (the extent
of thyroid follicle destruction) using a scale of 1+ to 5+, as
described previously (1, 4, 6). The 1+ thyroiditis is
defined as an infiltrate of at least 125 cells in one or several foci;
2+ is 10–20 foci of cellular infiltration involving up to 25% of the
gland; 3+ indicates that 25–50% of the gland is infiltrated; 4+
indicates that >50% of the gland is destroyed by infiltrating
inflammatory cells; and 5+ indicates virtually complete destruction of
the thyroid with few or no remaining follicles. Thyroid lesions were
also evaluated qualitatively. Granulomatous lesions are characterized
by proliferation and enlargement of thyrocytes and small to moderate
numbers of neutrophils in the perifollicular stroma, lymphocytes,
epithelioid histiocytes, and multinucleated giant cells (1, 4, 6). Thyroids of mice with very severe (5+ severity) G-EAT at day
45 or 55 (see Table IV
) also had extensive fibrosis, with increased
collagen deposition determined using Masson’s trichrome staining
(6, 34).
Immunohistochemistry
Paraffin sections of thyroids were deparaffinized in xylene and dehydrated in graded alcohol, or cryostat sections were fixed in acetone for 10 min at 4°C. The slides were washed in PBS (0.1 M, pH 7.6), blocked for 1 h with 1.5% normal goat serum, and washed and incubated with affinity-purified rabbit polyclonal anti-Fas (M20, 1/200) or anti-FasL (N20, 1/400; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-cytokeratin (PCK-26) (rat IgG; Sigma-Aldrich) for 30 min. Following incubation with a secondary biotinylated goat anti-rabbit Ab (1/500; Jackson ImmunoResearch) or goat anti-rat Ab (1/500; Caltag Laboratories, Burlingame, CA), endogenous peroxidase was quenched with 0.3% hydrogen peroxide in 0.1 M PBS (pH 7.6) for 30 min, and immunoreactivity was demonstrated using the avidin-biotin complex immunoperoxidase system (Vector ABC peroxidase kit; Vector Laboratories, Burlingame, CA) with 3,3-diaminobenzidine tetrahydrochloride or vasoactive intestinal peptide (VIP) (for cytokeratin) as the chromogen. The expression of FasL was also determined using a rat anti-mouse FasL mAb (A11, 1/50; Alexis Biochem., San Diego, CA). A11 was localized using biotin-conjugated goat anti-rat IgM (1/1000; Jackson Immunoresearch), developed by the peroxidase technique using NovaRED, and slides were counterstained with hematoxylin. Primary Ab was replaced by an equal amount of normal rabbit IgG (negative control for N20 and M20) or rat IgM (negative control for A11). The specificity of anti-Fas or anti-FasL Ab was also confirmed by competitive inhibition experiments according to the manufacturer’s instructions. Briefly, before staining, five times weight of Fas or FasL peptide (Santa Cruz Biotechnology) was incubated with M20 or N20 for 2 h at room temperature, and slides were stained using M20 and N20, as described above. To determine whether there was any cross-reaction between N20 (anti-FasL) and M20 (anti-Fas), FasL and Fas peptide were separately incubated with M20 and N20, and respectively subjected to routine staining.
CD4/FasL and CD8/FasL dual staining
CD4 or CD8 staining was performed first on cryosections using rat anti-CD4 and CD8 mAb (GK1.5 or 53.6; American Type Culture Collection, Manassas, VA), as previously described (5). Sections were blocked in 1% BSA for 30 min and washed with PBS, and endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in PBS for 30 min. This and all subsequent washes were in PBS (0.1 M, pH 7.6). The slides were then incubated with GK1.5 or 53.6 for 30 min at room temperature. After washing, slides were incubated 30 min with a secondary biotinylated rabbit anti-rat IgG diluted 1/500. The slides were washed again, and sections were incubated for 30 min with the avidin-biotin complex immunoperoxidase system (Vector Laboratories) using SG as substrate. After blocking with avidin and biotin solution, Fas or FasL staining was performed as described above. Fas- and FasL-positive color development was obtained by incubating with VIP. Staining of frozen sections for Fas or FasL was complicated by increased background staining, which was not a problem with paraffin sections. Therefore, paraffin sections were used, except when dual staining for Fas or FasL with CD4 or CD8 was required, since CD4 and CD8 could not be stained on paraffin sections.
Localization of FasL mRNA expression by in situ hybridization
To generate a FasL-specific RNA probe (riboprobe), a 309-bp fragment of mouse FasL cDNA was subcloned into pGEM-4 plasmid (a gift from Dr. M. Estes, University of Missouri, Columbia, MO). This plasmid was either linearized with EcoRI and transcribed with T7 RNA polymerase (Promega, Madison, WI) to generate an antisense probe or linearized with HindIII and transcribed with SP6 RNA polymerase to generate a sense probe. A fluorescein-labeled hybridization probe was synthesized by in vitro transcription using fluorescein-12-UTP (Boehringer Mannheim, Indianapolis, IN) and T7 RNA polymerase. Hybridization was conducted using the manufacturer’s recommended protocol. Briefly, thyroids were removed from CBA/J recipient mice, fixed 2 h with 4% paraformaldehyde in 0.1 M PBS (pH 7.2), and embedded in paraffin. Then 7-µm sections were cut from each tissue block. Alternatively, thyroids were snap frozen in liquid nitrogen, embedded in OCT, and stored at -80°C until use. Cryostat sections (5 µm) were air dried and fixed for 15 min in 4% (w/v) paraformaldehyde in PBS. Hybridization of FasL using the ISH kit (InnoGenex, San Ramon, CA) was performed at 80°C for 5 min and then at 50°C for 16 h. The bound probe was detected by serial addition of a biotinylated anti-fluorescein Ab, streptavidin-HRP conjugate, and VIP substrate. Sections of mouse eyes were used as a positive control for staining of FasL. Omission of probe, anti-fluorescein Ab, or the streptavidin-enzyme conjugate served as negative controls.
Determination of apoptosis
Apoptosis was determined using TUNEL (Apoptag kit; Intergen, Purchase, NY), following the manufacturer’s instruction with some modification. Briefly, sections were deparaffinized and dehydrated, and endogenous peroxidase activity was blocked in 0.3% hydrogen peroxide in PBS for 30 min. Sections were incubated at room temperature for 10 min with equilibration buffer, followed by 1-h incubation at 37°C with TdT enzyme (or reaction buffer negative controls) diluted with the reaction buffer in a humidity chamber. The TdT reaction was stopped with stop/wash buffer, and sections were washed with PBS before 30-min incubation with antidigoxigenin conjugated with HRP. Following washing, TUNEL-positive color development was obtained by incubating the section with 0.05% 3,3-diaminobenzidine tetrahydrochloride. Slides were counterstained with hematoxylin. A positive reaction for apoptosis was characterized by brown/black coloration of the nuclear or perinuclear region of the cell.
TUNEL/Fas dual staining
Fas staining was performed first, and apoptotic cells were determined by subsequent TUNEL staining, as described above. Fas-positive and TUNEL-positive cells were revealed by VIP and SG substrate (Vector Laboratories), respectively.
Statistical analysis
Statistical analysis of data was performed using an unpaired two-tailed Student’s t test, as indicated in the table legends. Values of p < 0.05 were designated by an asterisk.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fas-FasL interactions have been suggested to be important in
regulation of autoimmune thyroiditis (17, 24, 25, 26, 28, 29).
The predominant infiltrating cells, CD4 and CD8 T cells, present in
G-EAT thyroids could play a role in regulation of Fas or FasL
expression. Therefore, a kinetic study of Fas and FasL protein
expression in the thyroid during development and resolution of G-EAT
was conducted to begin to clarify the role of Fas/FasL-mediated
apoptosis in G-EAT. Although RT-PCR showed that Fas mRNA was
constitutively expressed and FasL mRNA was not detectable in normal
thyroids (5), neither Fas nor FasL protein was detected in
normal thyroids (Fig. 1, A and
B). Most recipient thyroids had 0–1+ severity scores 8 days
after cell transfer, and expression of Fas and FasL protein was either
negative (when the disease score was 0) or weakly expressed in the
inflammatory areas (when the disease score was 1+; data not shown). By
day 14, when G-EAT severity was 2+, both Fas and
FasL expression increased (Fig. 1, C and D), due
to more infiltrating cells expressing Fas (Fig. 1C) and
expression of FasL by some thyrocytes as well as some infiltrating
cells (Fig. 1D). Expression of Fas and FasL further
intensified (Fig. 1, E and F) and reached maximal
expression in 3–4+ G-EAT thyroids at days 19–21 (Fig. 1, G, H, and L, and data not shown). Fas
protein was detected on some enlarged thyrocytes, but was primarily
expressed by inflammatory cells (Fig. 1, E, G,
and J, and data not shown). Most, if not all, enlarged
thyrocytes were strongly FasL positive by days 19–21 (Fig. 1, F, H, and L), and some infiltrating
inflammatory cells also expressed FasL.
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, I and K). To exclude the possibility
that the Fas or FasL Abs may cross-react, M20 (anti-Fas) was
incubated with FasL peptide and N20 (anti-FasL) with Fas peptide,
before immunostaining of FasL or Fas. Both M20 (Fig. 1J) and
N20 (Fig. 1L) staining intensity and staining patterns were
unchanged compared with staining in the absence of peptides (data not
shown). In addition, two different anti-FasL Abs (A11, rat
anti-FasL IgM mAb; N20, rabbit anti-FasL IgG polyclonal Ab)
were used for staining of FasL. Both Abs showed a similar staining
pattern (data not shown). When primary Ab (for Fas and FasL) was
replaced with nonimmune rabbit IgG, no positive staining was observed
(data not shown).
Staining of adjacent thyroid sections for cytokeratin (Fig. 2A) and FasL (Fig. 2B) in 3+ G-EAT thyroids demonstrated that thyrocytes
strongly expressed FasL. Since FasL exists in both membrane and soluble
forms (35), thyrocytes could bind soluble FasL produced by
other cells. To rule out the possibility that the positive staining for
FasL by thyrocytes was due to binding of soluble FasL released from
inflammatory cells, in situ hybridization was used to detect FasL mRNA.
Sections of mouse eyes were used as a positive control. FasL was not
detected in normal thyroids (data not shown), but was strongly
expressed by thyrocytes (Fig. 2C) of mice with G-EAT,
consistent with the strong expression of FasL protein by thyrocytes
(e.g., Fig. 1H). Depletion of CD8+
cells resulted in reduced FasL mRNA expression by thyrocytes (Fig. 2D). Dual staining confirmed that both
CD8+ (Fig. 2E) and some
CD4+ cells (data not shown) expressed FasL,
although at a lower level than thyrocytes. Taken together, these
results suggest that FasL is up-regulated on both infiltrating T cells
and thyrocytes during development of G-EAT.
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Up-regulation of FasL on thyrocytes following inflammatory cell
infiltration (Fig. 1, B, D, F, and H) and the
fact that FasL transcripts decreased following depletion of
CD8+ cells (5) (Fig. 2D)
suggest that CD8+ cells might regulate FasL
expression in G-EAT thyroids. To further assess the effect of
CD8+ cells on G-EAT resolution and Fas/FasL
expression in thyroids, CD8+ cells were depleted
at different times after cell transfer, and G-EAT severity and
expression of Fas and FasL in thyroids were examined. In all
experiments, thyroids of mice given anti-CD8 beginning 1, 6, or 12
days after cell transfer were almost completely depleted of
CD8+ cells, as determined both by RT-PCR for CD8
mRNA (36) and by immunohistochemical staining (Fig. 2, F, H, and data not shown). Consistent with our
previous results (3), G-EAT gradually developed and
reached peak severity at days 19–21, and resolved considerably by day
35 after cell transfer (Table I, lines 1
and 2). Administration of anti-CD8 mAb beginning 1, 6, or 12 days
after cell transfer significantly inhibited resolution at day 35 (Table I), although depletion of CD8 cells beginning at day 12 was slightly
less effective. Administration of anti-CD8 mAb beginning 20 days
after cell transfer had no effect on resolution (Table I). Although
peripheral CD8+ T cells were completely depleted,
depletion of CD8+ T cells in thyroids was not
complete at day 35, possibly because they received less anti-CD8
than other groups, or because most CD8+ cells had
migrated to the thyroid by this time and were more difficult to
deplete. Eight or 14 days after cell transfer, FasL was mainly
expressed by inflammatory cells, and depletion of
CD8+ cells beginning at 1, 6, or 12 days had
little effect on FasL expression during this time (Table II). By days 19–21, both inflammatory
cells and thyrocytes were strongly FasL immunopositive (Figs. 1H and 3A). Thyroids of mice given anti-CD8
beginning at 1, 6, or 12 days had reduced FasL expression, with fewer
thyrocytes and inflammatory cells being FasL+ at
day 19 (Table II; Figs. 2D and 3B). At day 28,
FasL expression in thyroids of anti-CD8-treated mice with
3–4+ severity scores was less intense compared
with thyroids of controls with 2–3+ severity scores that were
beginning to resolve (Table II). The intensity of FasL expression
further decreased in CD8-depleted thyroids at day 35, even though the
severity scores were similar to those at day 19 (Table II, Fig. 3C).
CD8+ cells did not appear to influence Fas
expression, since thyroids of anti-CD8-treated mice had ongoing
inflammation and still had intense Fas immunoreactivity at day 35 (Fig. 3E, Table II). Cells from
2m-/- AKR mice that
lack CD8+ cells transferred G-EAT (3–4+
severity) to 2m-/-
recipients. These lesions did not resolve by days 35–50, while G-EAT
lesions induced by transferring either wild-type AKR or
2m-/- cells to AKR
recipients did resolve (data not shown). FasL expression was
considerably reduced in thyroids of
2m-/- mice compared
with wild-type AKR mice (Fig. 3, F and G). These
results suggest that G-EAT resolution was closely associated with
regulation of FasL expression and that CD8+
cells apparently regulated FasL expression in G-EAT thyroids,
particularly from days 19 to 35 (Table II).
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G). Resolving
thyroids with 1+ severity scores at day 35 had some
CD8+ cells, but very few
CD4+ T cells (Fig. 2I). In contrast,
CD4+ T cells were predominant in thyroids of
anti-CD8 mAb-treated mice, and CD8+ cells
were markedly diminished in thyroids of anti-CD8-treated mice with
4–5+ G-EAT severity at day 19 (Fig. 2F) and day 35
(Fig. 2H), and at all other times after cell transfer (data
not shown). G-EAT resolution is dependent on CD8+ T cells rather than CD8+ DC
A few DC are detected in G-EAT thyroids at days 19–21 (data not
shown). Because some DC are CD8
+
(30), anti-CD8 Ab would deplete
CD8+ DC as well as CD8+
T cells. Therefore, it was important to determine whether G-EAT
resolution might be dependent on the activity of
CD8+ DC rather than CD8+ T
cells. Mice were given anti-CD8 mAb to deplete
CD8+ T cells and not DC (31). Mice
given anti-CD8 mAb had no detectable splenic
CD8+ T cells, as determined by flow cytometry,
and very few CD8+ cells were present in their
thyroids at day 19 or day 35 (data not shown). Results were identical
to those obtained using anti-CD8 mAb, i.e., G-EAT severity was
increased at day 19 and resolution was inhibited with ongoing
inflammation at day 39 (Table III).
Immunostaining showed that FasL expression on thyrocytes was reduced in
mice given anti-CD8, similar to that shown using anti-CD8
mAb in day 35 thyroids with 3–4+ severity scores (data not shown).
These results suggest that CD8+ T cells and not
CD8+ DC are responsible for promoting resolution
of G-EAT.
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As shown above, FasL is up-regulated on thyrocytes during
development of G-EAT, and many inflammatory cells express Fas and/or
FasL. CD8+ T cells are important for
up-regulation of FasL expression on thyrocytes and are required for
early resolution of G-EAT. These results suggest the Fas/FasL pathway
may play a role in both the development and resolution of G-EAT. To
begin to determine the role of the Fas/FasL pathway in this model, we
examined the effects of a neutralizing anti-FasL mAb (32, 33) on development and resolution of G-EAT (Table IV). Administration of anti-FasL mAb
beginning 4 days after cell transfer, i.e., before significant numbers
of cells had migrated to the thyroid, had no apparent effect on G-EAT
severity scores at day 16 (line 1 vs line 2). Thyroids of hamster Ig
controls and mice given MFL1 were similar histologically at day 16, and
the ratio of CD4:CD8+ T cells was similar (data
not shown). In the experiments in Table IV, G-EAT severity at days
16–19 was considerably greater than in the experiments shown in Tables I and III, and resolution was just beginning in controls at day 45.
Mice given anti-FasL or anti-CD8 had more severe disease than
controls at day 45 (Table IV, lines 3–5), suggesting that
neutralization of FasL might be comparable with anti-CD8 in its
ability to inhibit G-EAT resolution. In a second experiment, since mice
had very severe disease at day 19 (line 6), thyroids were removed from
the remaining mice on day 55 (lines 7–9). Although resolution was
incomplete in two of six hamster Ig-treated controls, all of the
mice given anti-FasL or anti-CD8 had severe (5+) disease with
no signs of resolution. Thyroids of all anti-FasL- and
anti-CD8-treated mice at day 45 or 55 had extensive fibrosis with
increased collagen deposition, as determined by Masson’s trichrome
staining (data not shown), whereas only 1 of 12 controls had
significant thyroid fibrosis. Thyroid lesions in all but 1 of the
controls were resolving, and had regenerating follicles, whereas
thyroids of all mAb-treated mice had very few discernible follicles. As
noted above, when thyroid lesions have resolved or are in the process
of resolving, CD8+ T cells predominate in the
thyroid infiltrate at day 35, 45, or 55. In contrast, thyroids of mice
given anti-FasL (data not shown) or anti-CD8 (Fig. 2H) had primarily CD4+ T cells at day
45 or 55. FasL expression in thyroids of anti-FasL-treated mice was
much lower at days 45–55 compared with day 16 or 19 (data not shown),
and was comparable with that of anti-CD8-treated mice (Fig. 3C). These results suggest the Fas/FasL pathway is likely to
play an important role in the resolution of G-EAT, but this pathway is
apparently not critical for thyrocyte damage that occurs during the
development of severe granulomatous thyroid lesions.
Detection and characteristics of apoptotic inflammatory cells in G-EAT thyroids
Since Fas was predominantly expressed by inflammatory cells, and
both thyrocytes and CD8+ T cells expressed FasL,
clearance of inflammatory cells by FasL+
thyrocytes and/or cytotoxic CD8+ T cells may
contribute to G-EAT resolution. Apoptosis in G-EAT thyroids was
assessed using TUNEL staining (Fig. 3, H–J). At day 14,
only a few apoptotic inflammatory cells and thyrocytes were detected in
2+ G-EAT thyroids (Fig. 3H). The greatest number of
TUNEL-positive cells was observed at days 19–21, corresponding with
the peak expression of Fas in thyroids, and both inflammatory cells and
thyrocytes were apoptotic (Fig. 3I). Although most
thyrocytes expressed FasL, many of the Fas+
thyrocytes were not TUNEL positive (data not shown). Apoptotic
inflammatory cells were frequently observed in proximity to thyrocytes
(Fig. 3J) at days 19–21 when these thyrocytes strongly
express FasL, suggesting FasL on thyrocytes could be targeted directly
to the cell surface of Fas+ inflammatory cells.
Apoptotic infiltrating CD4+ T cells were also
present in the inflammatory areas (5) (data not shown).
Combined Fas and TUNEL staining demonstrated that many apoptotic
inflammatory cells were Fas positive (data not shown), suggesting that
Fas-mediated apoptosis might contribute to the apoptotic clearance of
infiltrating cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various cells express Fas constitutively or after activation (7, 37). Although Fas mRNA is constitutively expressed in both
normal and G-EAT thyroids (5), Fas protein was detected
only after infiltration of inflammatory cells. Fas protein expression
in thyroids was maximal at days 19–21, and gradually diminished during
resolution. However, Fas protein remained high until day 35 in thyroids
of mice depleted of CD8+ T cells in which lesions
did not resolve (Fig. 3E). Fas was expressed primarily by
infiltrating cells, and was also induced on some enlarged thyrocytes.
CD4+ T cells are the primary effector cells in
G-EAT (1) and are the first cells to migrate to the
thyroid. Some CD4+ T cells express FasL (data not
shown), and presumably could bind to Fas on thyrocytes and trigger
thyrocyte apoptosis, leading to destruction of the thyroid. However,
blocking the Fas pathway did not diminish damage to the thyroid (Table IV), suggesting that apoptosis of thyrocytes, which was maximal at days
19–21 (Fig. 3I), can occur by a Fas-independent pathway.
Inflammatory cells could induce thyrocyte damage through production of
NO or cytokines such as IL-1, IFN-
, and TNF- (24, 38, 39), and others have suggested that cytokine-dependent apoptosis
may regulate thyrocyte apoptosis in Hashimoto’s thyroiditis and Graves
disease (38). Further studies will be needed to determine
the mechanisms involved in thyrocyte destruction in our G-EAT
model.
The FasL protein expression in G-EAT thyroids was consistent with its
mRNA level, as FasL mRNA and protein are undetectable in normal
thyroids, and are up-regulated during G-EAT development
(5) (Table I; Fig. 1, B, D,
F, and H). Because thyroid FasL mRNA levels
correlated well with G-EAT severity scores, we previously suggested
that FasL was expressed primarily by infiltrating inflammatory cells
(5). However, although some infiltrating cells expressed
FasL, FasL was also expressed by most of the enlarged thyrocytes at
days 19–21, as demonstrated by FasL/cytokeratin staining (Fig. 2, A and B) and in situ hybridization (Fig. 2C). Because expression of FasL on thyrocytes decreased
after depletion of CD8+ T cells (Table II; Figs. 2D and 3B), one function of
CD8+ T cells in this model may be to induce FasL
on thyrocytes. This would be consistent with results of others
demonstrating induction of FasL on nonlymphoid cells by activated T
cells (14).
FasL was expressed by both CD4+ and
CD8+ T cells in G-EAT thyroids. Apoptosis of
inflammatory cells was maximal at days 19–21 when Fas expression by
inflammatory cells was high and was reduced after depletion of
CD8+ T cells (5). Apoptotic
inflammatory cells were frequently observed in proximity to thyrocytes
(Fig. 3J), suggesting that CD8+ T
cells might induce up-regulation of FasL on thyrocytes to kill
Fas+ inflammatory cells, thus limiting thyrocyte
destruction and promoting resolution. A role for the Fas/FasL pathway
in G-EAT resolution is suggested by the finding that a neutralizing
anti-FasL mAb inhibited resolution as effectively as did CD8
depletion (Table IV). We are currently generating lpr and
gld mice on an EAT-susceptible background to examine more
directly the role of this pathway in G-EAT resolution.
FasL expression by thyrocytes has been shown to be functional (28, 40, 41), and T cells that approach thyroid follicles in Hashimoto’s thyroiditis can be killed by FasL+ thyrocytes (40). Expression of FasL in particular tissues may also contribute to survival of tissue cells (42), and host-derived FasL was shown to be important in the recovery from experimental allergic encephalomyelitis (21, 22, 23). Dayan et al. (43) suggested that FasL expression by thyrocytes can be protective, and transgenic expression of FasL by thyrocytes was shown to prevent EAT (28) and rejection of thyroid allografts (41). It was suggested that Fas and FasL induced on thyrocytes following IL-1 stimulation could result in destruction of the thyroid in both Hashimoto’s thyroiditis and Grave’s disease (24, 44). However, other reports suggest that thyrocytes are resistant to Fas-mediated cell death through expression of antiapoptotic proteins (25, 38). These latter results are consistent with our preliminary observation that the antiapoptotic protein Flip increases on thyrocytes during G-EAT development (Y. Wei, unpublished results). Our study provides additional evidence that nonlymphoid tissue cells can be induced to express FasL (14, 21) and suggests that induction of FasL on nonlymphoid tissue may aid in apoptotic clearance of inflammatory cells.
Many components required to initiate apoptosis have been identified and
shown to preexist in most cells. Such ubiquity requires that the
propensity of cells to trigger apoptotic death be strictly regulated
(25). Fas and FasL expression in the thyroid is strictly
regulated, since both proteins are expressed only by specific cells and
at specific times during G-EAT. Although irradiation can induce FasL
expression (45, 46), the fact that normal thyroids and
thyroids with 0–1+ EAT express little or no FasL (Fig. 1A
and Table II) indicates that FasL up-regulation in G-EAT is not a
result of irradiation of recipient mice. Since expression of Fas and
FasL is increased on thyrocytes following inflammatory cell
infiltration, inflammatory cells may be responsible for the expression
and differential regulation of these molecules in G-EAT. In addition to
effector CD4+ T cells, CD8+
T cells are major thyroid-infiltrating cells, and they outnumber
CD4+ T cells at the peak of disease and during
resolution (Fig. 2, G and I). CD8 depletion led
to diminished FasL expression by thyrocytes (Table II) and decreased
apoptosis of inflammatory cells (5), suggesting that CD8
cells may regulate FasL expression in the thyroid. Depletion of
CD8+ T cells within the first 12 days after cell
transfer, i.e., before most CD8+ T cells had
migrated to the thyroid, prevented G-EAT resolution (Table I). This
suggests that early activation and expansion of
CD8+ T cells, which are derived from the naive
recipients (5), may be necessary for them to promote FasL
expression by thyrocytes. CD8+ T cells might
promote G-EAT resolution by inducing up-regulation of FasL expression
on thyrocytes and through their own expression of FasL, resulting in
apoptosis of effector CD4+ T cells. Although
CD8+ DC can kill CD4+ T
cells through Fas-FasL interaction (30, 47, 48),
resolution was inhibited as effectively in thyroids of mice depleted of
only CD8+ T cells as in mice in which both
CD8+ T and DC were depleted (Table III). These
results indicate that CD8+ T cells
(3) and not CD8+ DC are required for
resolution of G-EAT.
FasL is induced on some thyrocytes before day 19 (Fig. 1D),
but depletion of CD8+ cells has little effect on
FasL expression before day 19. Since FasL was also expressed, albeit at
very low levels, on thyrocytes of
2m-/- mice (Fig. 3G), some non-CD8 cells can apparently induce FasL
expression on thyrocytes. It is difficult to address the role of
CD4+ T cells in regulation of FasL in the thyroid
since G-EAT does not develop after depletion of
CD4+ T cells (1, 6). However,
because CD4+ T cells were abundant in thyroids of
mice depleted of CD8+ T cells, and these thyroids
had reduced FasL expression, CD4+ T cells are
unlikely to play a major role in up-regulation of FasL on thyrocytes.
Our results do suggest that even though Fas and FasL expression in the
thyroid increase as disease severity increases over the first 3 wk
after cell transfer (Fig. 1 and Table II), the Fas pathway is not
essential for damage to the thyroid. Thus, mice given anti-FasL mAb
developed G-EAT that was similar both in severity and histologic
features to that of control mice 16 days after cell transfer
(Table IV).
Apoptosis can also develop through other pathways, including cytokine
withdrawal, perforin-dependent lysis of target cells, or following
interaction of TNF- and TNF- receptors (7). Thyroids
of mice depleted of CD8+ T cells produce lower
amounts of cytokines at day 35 than at days 19–21 (36),
and apoptosis is reduced. This suggests apoptosis due to cytokine
withdrawal may not be important for G-EAT resolution. Perforin-induced
apoptosis is unlikely to be involved in G-EAT since
CD8+ T cells do not appear to contribute to
thyroid damage in this model (3, 5). However, further
studies will be needed to clarify the role of TNF- and perforin in
G-EAT development and resolution.
An important characteristic of our model is that G-EAT undergoes spontaneous resolution, and this may be related to up-regulation of FasL on thyrocytes. It may therefore be possible to promote G-EAT resolution by FasL gene delivery, which has been shown to be effective in treating EAT and other autoimmune diseases (16, 18, 28). These observations suggest that the immune response that leads to autoimmune damage can be controlled by induction of apoptosis and by a subsequent regulatory immune reaction. It will be important to focus on the regulatory role of various inflammatory cells, cytokines, and pro- and antiapoptotic proteins on FasL up-regulation by thyrocytes. Inducible nonlymphoid expression of FasL has been reported to be responsible for peripheral deletion of T cells and amelioration of some autoimmune diseases (14, 21). Our data suggest that nonlymphoid FasL may be capable of inducing apoptosis in infiltrating T cells, thereby minimizing tissue damage following inflammation. Investigation of the cell type or cytokine-specific roles of FasL may potentially lead to a better understanding of the pathogenesis of autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Y.W. and K.C. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Helen Braley-Mullen, Department of Medicine, Division of Immunology and Rheumatology, University of Missouri, M450 Medical Sciences, Columbia, MO 65212. E-mail address: mullenh{at}health.missouri.edu
4 Abbreviations used in this paper: G-EAT, granulomatous experimental autoimmune thyroiditis; 2m, 2-microglobulin; DC, dendritic cell; FasL, Fas ligand; MTg, mouse thyroglobulin.
Received for publication May 1, 2001. Accepted for publication October 2, 2001.
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References |
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