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-Deficient Mice Develop Severe Granulomatous Experimental Autoimmune Thyroiditis with Eosinophil Infiltration in Thyroids1
Departments of
*
Internal Medicine,
Molecular Microbiology and Immunology, and
Pathology, University of Missouri-Columbia School of Medicine, Columbia, MO 65212
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the development of granulomatous
experimental autoimmune thyroiditis (EAT), DBA1 mice with a disrupted
IFN- gene were used for adoptive EAT induction. Effector cells from
either IFN-+/+ or IFN--/- donor
mice activated with mouse thyroglobulin and anti-IL-2R mAb induced
severe granulomatous EAT. A predominant infiltration of the thyroid by
eosinophils was observed in recipients of IFN--/-
effector cells but not in recipients of IFN-+/+ cells.
Compared with wild-type mice, thyroids of recipients of
IFN--/- effector cells had decreased expression of
mRNA for Th1 cytokines and inducible nitric oxide synthetase.
Expression of Th2 cytokine mRNA was comparable to that of
IFN-+/+ mice, and expression of eotaxin was increased in
the thyroids of recipients of IFN--/- effector cells.
Activation of cells from either IFN-+/+ or
IFN--/- donors in the presence of IL-12 also induced
severe granulomatous EAT. Eosinophil infiltration in recipients of
IFN--/- cells was unaffected when effector cells were
activated with IL-12, and thyroids expressed predominantly Th2
cytokines. The extent of fibrosis of recipient thyroids was generally
greater when donor IFN-+/+ and IFN--/-
cells were activated with IL-12. Compared with IFN-+/+
mice, IFN--/- mice produced lower levels of mouse
thyroglobulin-specific autoantibodies after immunization with MTg and
LPS. These results indicate that cells from both IFN-+/+
and IFN--/- donors can induce severe granulomatous
EAT. However, damage of thyroid follicles by IFN--/-
and that by IFN-+/+ cells appear to involve different
mediators of inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAb (5) or IL-12 (50).
Transfer of both lymphocytic and granulomatous forms of EAT by in vitro
activated spleen cells is dependent on CD4+ T cells
(4, 6).
Although the mechanism by which CD4+ T cells cause
destruction of thyroid tissue is unknown, cytokines produced by
activated CD4+ T cells are likely to play an important role
in the pathogenesis of EAT. The T cell product IFN- is involved in
many inflammatory and immune regulatory responses, such as macrophage
activation, up-regulation of both class I and class II MHC molecules
for Ag presentation, and induction of expression of adhesion molecules
such as ICAM-1 and VCAM, which are important in promoting homing of
inflammatory cells (7). Varying roles for IFN- in EAT have been
suggested. It has been shown that IFN--induced expression of MHC
class II Ag on normal thyroid tissue is responsible for rejection of
thyroid isografts (8, 9). In other studies, intrathyroidal injection of
IFN- induced thyroiditis in susceptible mice (10), and treatment of
mice in vivo with an anti-IFN- mAb suppressed active EAT
induction (11). These results suggested that IFN- may play a direct
or an indirect role in the destruction of thyroid tissue. However, in
other studies a severe granulomatous form of EAT was induced by
transfer of spleen cells activated with MTg in vitro in the presence of
mAb specific for IFN- or by treatment of recipient mice with
anti-IFN- mAb (5), and systemic injection of IFN- was shown
to suppress EAT (12). These contradictory results may reflect the
complexity of the role of IFN- in the pathogenesis of EAT.
To determine the role of IFN- in granulomatous EAT without the
limitations involved in using neutralizing mAbs, the development of
granulomatous EAT was studied in IFN- gene-disrupted mice in which
the absence of endogenous IFN- is assured. We also determined the
expression of cytokine gene mRNA in spleen effector cells and in
thyroid infiltrates. Our results indicate that severe granulomatous EAT
can be induced in mice with a disrupted IFN- gene. Compared with
wild-type mice, a decreased Th1-like and predominant Th2-like cytokine
expression was observed in the thyroids of recipients of effector cells
from IFN--/- mice. This unique cytokine profile was
accompanied by a marked infiltration of the thyroids of
IFN--/- mice by eosinophils, whereas very few
eosinophils were observed in thyroids of
IFN-+/+ mice with granulomatous EAT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice with a disrupted IFN- gene
(IFN--/-) were developed by Dalton et al. (13)
and backcrossed onto the DBA/1 (H-2q) genetic background.
Breeding pairs of heterozygous (IFN-+/-) DBA/1 mice
were obtained from Genentech (South San Francisco, CA) and subsequently
bred in the animal facilities at the University of Missouri (Columbia,
MO) to produce homozygous (+/+) and (-/-) offspring. The
IFN-+/+ and IFN--/- homozygotes were
identified by PCR analysis of tail DNA using primer sequences provided
by Genentech. All subsequent breeding was with the homozygotes. Both
IFN-+/+ and IFN--/- mice were
maintained under specific pathogen-free conditions until used for
experiments, at which time they were transferred to conventional
housing.
EAT induction
EAT was induced as previously described (4). Briefly, mice were injected i.v. twice at 10-day intervals with 150 µg of MTg and 15 µg of LPS (Escherichia coli 011:B4; Sigma, St. Louis, MO). Seven days later, donor spleen cells were cultured at 1 x 107/ml for 72 h at 37°C in RPMI 1640 containing 25 mM HEPES buffer (Cell and Immunobiology Core Facility, University of Missouri), 5% FCS (Sigma), sodium pyruvate, glutamine, nonessential amino acids, vitamins (all from Fisher Scientific, St. Louis, MO), and 5 x 10-5 M 2-ME. To induce granulomatous EAT, donor spleen cells were restimulated with 25 µg/ml MTg in the presence of a 5% final concentration of culture supernatant containing mAb specific for IL-2R (M7/20) (4). In some experiments, 5 ng/ml IL-12 was also added to culture (50). Cells were harvested and washed twice with balanced salt solution, and 3 to 3.5 x 107 cells were transferred i.v. into 600-rad-irradiated syngeneic recipients. After 19 to 21 days, the time of peak disease severity (4), recipient thyroids were collected for histologic evaluation of EAT.
Evaluation of EAT
Thyroids were scored quantitatively for EAT severity, defined as
the extent of thyroid follicle destruction, using a scale of 1+ to 5+
as described previously (4). Here, 1+ thyroiditis is defined as an
infiltrate of at least 125 cells in one or several foci, 2+ is 10 to 20
foci of cellular infiltration involving up to one-fourth of the gland,
3+ indicates that one-fourth to one-half of the gland is infiltrated,
4+ indicates that greater than one-half of the gland is destroyed, and
5+ indicates virtually complete destruction of the gland with few or no
remaining follicles. Thyroid lesions were also evaluated qualitatively.
Thyroids of most mice used in this study had severe granulomatous
inflammatory lesions (4–5+ in severity based on the extent of
destruction of thyroid follicles). These thyroids had extensive
granulomatous changes with follicular cell proliferation and large
numbers of histiocytes, numerous PMNs, lymphocytes, multinucleated
giant cells, microabscess formation, fibrin deposition, focal fibrosis,
and necrosis. The granulomatous inflammation generally extended beyond
the thyroid to involve adjacent muscle and connective tissue. A massive
infiltration of eosinophils was also observed in thyroids of recipients
of effector cells from IFN--/- donors.
ELISAs
Serum levels of MTg-specific IgG autoantibodies in individual donor or recipient mice were determined by ELISA as previously described (4). The contributions of various IgG subclasses to the total IgG autoantibody response were assessed using alkaline phosphatase-conjugated antibodies specific for IgG1, IgG2a, and IgG2b. Dilutions of the conjugated subclass-specific Abs (1/6000 to 1/8000) were determined in preliminary titrations to detect optimal Ab activity of serum on MTg-coated plates while giving minimal activity (OD = <0.05) on plates coated with an irrelevant protein (OVA) or of normal mouse serum (1/100 dilution) on MTg-coated plates.
Levels of IFN- produced by cells during the 72-h in vitro culture
with MTg or with MTg and IL-12 were evaluated by double sandwich ELISA
using mAb R46A2 (HB 170; American Type Culture Collection, Rockville,
MD) as the capture Ab and biotinylated XMG1.2 as the detection Ab with
murine rIFN- (Biosource International, Camarillo, CA) as a standard.
IL-2, IL-4, and IL-5 were assayed using ELISA kits supplied by Endogen
(Cambridge, MA).
Reverse transcription-PCR (RT-PCR) amplification
Total RNA was isolated from 5 x 106 spleen cells or from single thyroid lobes using TRIZOL (Life Technologies, Gaithersburg, MD) (14, 15). The dried RNA pellet was dissolved in 10 µl of sterile diethylpyrocarbonate-treated water. Total mRNA was converted to cDNA by murine leukemia virus reverse transcriptase (Perkin-Elmer/Cetus, Branchburg, NJ) and oligo(dT)12–18 primers. To determine the relative initial amounts of target cDNA, each cDNA sample was serially diluted 1/5, 1/25, and 1/125, and each dilution was amplified with cytokine-specific primers. Hypoxanthine phosphoribosyltransferase (HPRT) was used as a housekeeping gene to verify that the same amount of RNA was amplified. The cytokine gene primers used in this study were described previously (14, 15). Primer sequences for other genes were: inducible nitric oxide synthetase (iNOs): sense, TCA CGC TTG GGT CTT GTT CAC T; anti-sense, TTG TCT CTG GGT CCT CTG GTC A; and eotaxin: sense, CAG ATC TCT TTG CCC AAC CT; antisense, AGA GGC TGA GAT CCA AGC AG. All primers were designed to span genomic introns, so that any contaminating genomic DNA would be detected at a higher m.w. band. To compare relative levels of mRNA transcripts between different groups, samples were reverse transcribed and amplified at the same time using aliquots of reagent from the same master mix. PCR was performed as previously described (14). PCR products were separated by electrophoresis in 3% agarose gels and visualized by UV light following ethidium bromide staining. Densitometry analysis was performed using an IS-1000 Digital Imaging System (San Leandro, CA). Samples within the linear relationship between input cDNA and final PCR products (usually 1/25 cDNA dilution) were collected, and the densitometric units for each cytokine band were normalized to those for the corresponding HPRT band.
In some experiments, expression of mRNA for IL-5 and HPRT was semiquantitatively determined using a PCR mimic as previously described (15).
Statistical analysis
Statistical analysis of data was performed using an unpaired two-tailed Student’s t test as indicated in the figure legends. p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+/+ and
IFN--/- mice
Granulomatous EAT can be induced by transfer of MTg- and
anti-IL-2R mAb-activated spleen cells from MTg/LPS-primed donor
mice (4). To determine whether IFN- was required for the development
of granulomatous EAT, spleen cells from MTg/LPS-immunized
IFN-+/+ or IFN--/- donor mice were
cultured with MTg and anti-IL-2R mAb for 72 h before transfer
to recipient mice. In typical experiments, shown in Table I
, effector cells from both
IFN-+/+ and IFN--/- donor mice
activated with MTg and anti-IL-2R mAb induced severe granulomatous
thyroiditis. Thyroids with 5+ EAT were massively enlarged and virtually
destroyed, with almost no remaining follicles (Fig. 1, C and E).
Thyroid lesions of both IFN-+/+ and
IFN--/- mice had widespread follicular cell
proliferation with PMNs, numerous large epithelioid histiocytes,
lymphocytes, multinucleated giant cells, necrosis, and fibrosis. In
addition to these histologic changes, recipients of spleen cells from
IFN--/- mice had marked infiltration of the thyroid by
eosinophils (Fig. 1, D and F). Substantial
numbers of eosinophils were always evident in infiltrated thyroids of
recipients of IFN--/- effector cells. The extent of
eosinophil infiltration was variable, but was always extensive enough
that hematoxylin- and eosin-stained slides had a readily apparent pink
(eosin) stain when examined at low power (Fig. 1D).
Eosinophils were rarely observed in recipients of
IFN-+/+ cells even if the recipients were
IFN--/- (Fig. 1C). Eosinophil
infiltration was evident in recipient thyroids as early as 10 days
after transfer of MTg-specific cells from IFN--/-
donors (data not shown).
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+/+ and IFN--/- mice, MTg-primed
spleen cells from IFN-+/+ or IFN--/-
donors were cultured with MTg, anti-IL-2R mAb, and IL-12 for
72 h before transfer to IFN-+/+ and
IFN--/- recipients. Since these mice are highly
susceptible to EAT induction, activation of donor spleen cells with MTg
and anti-IL-2R mAb in the absence of IL-12 induced nearly complete
destruction of thyroid follicles (4–5+). Activation of cells from
either IFN-+/+ or IFN--/- donors in the
presence of IL-12 also induced severe 4–5+ granulomatous EAT. In most
experiments, there was little difference in the extent of thyroid
follicle destruction (severity score) in recipients of effector cells
activated in the presence or the absence of IL-12 (Table I
).
Qualitatively, however, the extent of fibrin deposition of recipient
thyroids was generally greater when donor cells were activated with
IL-12. PMNs were usually present in similar numbers, and the extent of
infiltration by eosinophils did not differ for recipients of
IFN--/- cells activated with or without IL-12.
However, as shown below, IL-12 did induce some alterations in
cytokine production and/or expression in both spleen cells
and thyroid-infiltrating cells.
Decreased production of MTg-specific autoantibodies
in IFN--/- mice
Since IFN- is a key cytokine to promote production of IgG2a by
B cells (16), we determined levels of MTg-specific autoantibodies in
IFN-+/+ and IFN--/- mice. Both
IFN-+/+ and IFN--/- donor and recipient
mice developed MTg-specific autoantibodies. The levels of total
anti-MTg IgG as well as MTg-specific IgG1 and IgG2b were usually
slightly, but not significantly, decreased in IFN--/-
donors compared with those in IFN-+/+ donors (Fig. 2). MTg-specific IgG2a was significantly
(p < 0.05) reduced in IFN--/-
donors compared with that in IFN-+/+ donors (Fig. 2).
Production of MTg-specific IgG2a was also decreased in most recipients
of effector cells from IFN--/- mice (Table I), while
total IgG (not shown) and IgG1 (Table I) were only minimally
decreased.
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Because IFN- promotes the development of Th1 responses,
in the absence of IFN-, MTg-specific responses might be skewed
toward a Th2 response. To compare Th1 and Th2 cytokines in
IFN-+/+ and IFN--/- mice,
expression of cytokine gene mRNA in pooled MTg-activated spleen cells
from 8 to 10 donor mice was examined using RT-PCR. As expected, IFN-
mRNA was undetectable in IFN--/- mice (Fig. 3). Only weak bands for IFN-, TNF-
,
and IL-5 were detected using a 1/5 cDNA dilution when spleen cells were
cultured with medium alone, and IL-2, IL-4, and IL-13 bands were
undetectable (data not shown). Expression of mRNA for IL-2, IL-4, IL-5,
and IL-13 was similar in MTg-stimulated spleen cells from both
IFN-+/+ and IFN--/- mice, while
expression of TNF- was higher for spleen cells from
IFN-+/+ mice (Fig. 3). Activation of spleen cells with
MTg in the presence of IL-12 increased IFN- gene expression in
IFN-+/+ cells and TNF- expression in
IFN--/- cells (Fig. 3), but had little or no
effect on the expression of mRNA for IL-2, IL-4, IL-5, and IL-13 (Fig. 3). The production of IL-2, IL-5, and IFN- in supernatants of spleen
cells stimulated with MTg was also determined by ELISA (Table II). Spleen cells from
IFN-+/+ and IFN--/- mice produced no
detectable IL-4 (data not shown) and low, but similar, amounts of IL-5.
IFN- was not detected in the supernatant of cells from
IFN--/- mice, while cells from IFN-+/+
mice produced high amounts of IFN-. Cells from
IFN--/- mice produced less IL-2 than cells from
IFN-+/+ mice. These results indicate that spleen cells
of both IFN-+/+ and IFN--/- mice
expressed similar mRNA for Th2 cytokines and for IL-2 after
immunization with MTg and LPS, while production of IL-2 protein and
expression of TNF- mRNA were generally lower in MTg-activated
IFN--/- cells. The reason that IL-2 mRNA expression
was unaffected while IL-2 protein was decreased in
IFN--/- cells is unknown.
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+/+ and IFN--/- mice
To determine cytokine gene expression in the target organ,
expression of cytokine gene mRNA in individual thyroid lobes was
determined by RT-PCR 21 days after cell transfer, at the peak of
disease. HPRT was used as a housekeeping gene to normalize cytokine
gene expression in thyroids with different degrees of EAT severity. As
expected, IFN- mRNA was undetectable in the thyroids of recipients
of MTg- and anti-IL-2R mAb-activated
IFN--/- effector cells (Fig. 4A). IL-2 and TNF- mRNA
were expressed in thyroids from IFN--/- mice. TNF-
expression was decreased (p < 0.05) compared with that in
thyroids of IFN-+/+ mice, while IL-2 expression was not
significantly decreased. Expression of mRNA for IL-4, IL-5, and IL-13
was similar in the thyroids from recipients of MTg- and anti-IL-2R
mAb-activated IFN-+/+ or IFN--/-
effector cells (Fig. 4B). In addition, IFN- mRNA was
expressed in thyroids only when donors were
IFN-+/+. IFN- was not detected in thyroids
of IFN-+/+ recipients when they received cells from
IFN--/- donors (data not shown), indicating that all
detectable intrathyroidal IFN--secreting cells are of donor
origin.
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+/+ and IFN--/- mice,
intrathyroidal cytokine gene mRNA expression was analyzed in recipients
of cells activated in the presence of IL-12. Higher levels of TNF-
mRNA (p < 0.05) were detected in thyroids of recipients of
IFN-+/+ or IFN--/- donor cells
activated with IL-12 than in those of recipients of cells activated
with no IL-12 (Fig. 4C). IFN- expression in recipients of
IFN-+/+ cells was also increased when cells were
activated in the presence of IL-12. A significant (p <
0.05) up-regulated expression of mRNA for IL-4, IL-5, and IL-13 was
observed in the thyroids of mice receiving IFN--/-
effector cells activated with IL-12, while IL-4 and IL-13 expression
was unaffected in thyroids of IFN-+/+ mice (Fig. 4D). IL-5 expression was significantly decreased in thyroids
of IFN-+/+ recipients of effector cells activated with
IL-12 (Fig. 4D), although the decrease in IL-5 was less
pronounced in some other experiments (data not shown).
Competitive PCR was used to semiquantitatively analyze the expression
of IL-5 and HPRT genes in thyroid infiltrates from
IFN-+/+ and IFN--/- mice. HPRT and IL-5
gene mimics were prepared as previously described (15). Constant
amounts of cDNA samples were amplified in the presence of serial
twofold dilutions of specific gene mimics. As shown in Figure 5, the levels of IL-5 mRNA were similar
in thyroid infiltrates from recipients of effector cells from
IFN-+/+ and IFN--/- mice activated with
MTg and anti-IL-2R mAb. However, intrathyroidal expression of IL-5
mRNA was significantly higher (p < 0.05) in recipient of
cells from IFN--/- donors compared with
IFN-+/+ donors when cells were activated with IL-12.
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-/- donors had a
marked accumulation of eosinophils in their thyroids (Fig. 1), eotaxin
mRNA expression was examined in thyroids of
IFN-+/+ and IFN--/- mice. As
shown in Figure 6A, low levels
of eotaxin were detected in normal thyroids. Eotaxin expression was
significantly higher in granulomatous EAT thyroids from
IFN--/- mice than in those from
IFN-+/+ mice. This increased expression of eotaxin is
consistent with the accumulation of large numbers of eosinophils in
thyroids of IFN--/- mice.
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+/+ and IFN--/-
mice
The free radical NO is considered a major mediator of inflammatory
responses (18). There is evidence that the accumulation of NO mediates
damage in inflammatory tissues in autoimmune responses (19). Production
of NO in macrophages is controlled by iNOs. Since proinflammatory
cytokines such as IFN- and TNF- induce the transcription of iNOS
in macrophages and in various somatic cells (20), RT-PCR was used to
determine expression of iNOs mRNA in thyroids from
IFN-+/+ and IFN--/- mice. As shown in
Figure 6B, mRNA for iNOs was readily detected in thyroids of
recipients of effector cells from IFN-+/+ mice. In
contrast, iNOs mRNA in the thyroids of IFN--/- mice
was barely detectable. This result suggests that NO is not required for
inducing damage to the thyroid in this granulomatous EAT model.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is not required
for induction or expression of granulomatous EAT. Effector cells from
both IFN-+/+ and IFN--/- mice
induced severe granulomatous EAT, with extensive destruction of thyroid
follicles. Compared with IFN-+/+ mice,
IFN--/- mice developed lower levels of MTg-specific
autoantibodies after immunization with MTg and LPS. Together with
decreased Th1 cytokine gene expression, decreased iNOs expression, and
massive eosinophil infiltration in the thyroids of recipients of
effector cells from IFN--/- mice, these results
indicate that cells from IFN--/- donors induce EAT,
which is as destructive as that induced by cells from
IFN-+/+ donors but appears to involve different
mediators of inflammation.
IFN- plays a pivotal role in immune and inflammatory responses,
including promoting Th1 cell development and suppression of Th2 cell
development (21). Earlier studies showed that IFN- was correlated
with disease severity and relapses in experimental allergic
encephalomyelitis (EAE) and was implicated in EAE induction (22).
IFN--producing Th1 cell clones or lines could transfer EAE, whereas
Th2 cells of identical MHC peptide specificity were ineffective (23).
However, studies of the role of IFN- in autoimmune diseases using
neutralizing mAbs or administration of IFN- yielded conflicting
results. The timing of manipulation of the levels of IFN- using
neutralizing mAb apparently determined the different outcomes: either
suppression or promotion of EAE, EAT, and adjuvant arthritis (5, 11, 24, 25, 26, 27). In addition, systemic injections of IFN- suppressed
collagen-induced arthritis or EAT (12, 27), whereas local injection of
IFN- exacerbated these diseases (10, 28).
Recent studies using IFN- or IFN- receptor gene-disrupted mice
clearly indicate that IFN- is not required for the induction of EAE
(29, 30, 31), collagen-induced arthritis (32, 33), and experimental
autoimmune uveitis (EAU) (34), all of which are considered to be
induced primarily by Th1 CD4+ T cells. Higher mortality in
IFN--/- animals after EAE induction (30, 31), earlier
onset of arthritis (32, 33), and greater ocular infiltration in EAU
(34) suggest that IFN- may play a down-regulatory role in these
autoimmune responses. In the current study, thyroids from both
IFN-+/+ and IFN--/- mice had similar
destruction of thyroid follicles, with extensive follicular cell
proliferation, large numbers of histiocytes, multinucleated giant
cells, lymphocytes, PMN infiltration, and fibrosis (Fig. 1). However, a
predominant infiltration of thyroids by eosinophils (Fig. 1) was only
observed in recipients of IFN--/- effector cells.
To begin to determine the mechanisms involved in the development of
this destructive thyroid histopathology in IFN--/-
mice, we asked whether granulomatous EAT in IFN--/-
mice might be associated with a different pattern of cytokine or
chemokine production. Our previous results showed that both Th1 and Th2
cytokines were present in the thyroids of mice with granulomatous EAT
at the time of peak disease severity (15) (our manuscript in
preparation). Consistent with these findings, mRNA transcripts for both
Th1- and Th2-associated cytokines were detected in the thyroids from
IFN-+/+ mice (Fig. 4, A andB). Lower levels of IL-2 and TNF-, and no IFN- mRNA
were detected in the thyroids from IFN--/- mice, while
they expressed levels of IL-4, IL-5, and IL-13 comparable to those in
IFN-+/+ mice (Fig. 4, A andB). Eotaxin expression was also increased in
IFN--/- thyroids with eosinophil infiltration (Fig. 6A). These results indicate that in the absence of
IFN-, mice developed granulomatous EAT with a decreased expression
of Th1 cytokines in the thyroid infiltrates.
IL-12 is known to promote the differentiation of Th1 cells and to
suppress the development of Th2 responses (35). Since cells activated
with IL-12 induce very severe granulomatous EAT in recipient mice (see
Footnote 4), it was of interest to determine whether IL-12 could
regulate the development of granulomatous EAT or eosinophil
accumulation in thyroids of IFN--/- mice. Since the
mice used in this study were highly susceptible to adoptive EAT
induction, effector cells activated with MTg and anti-IL-2R mAb in
the absence or the presence of IL-12-induced EAT in both
IFN-+/+ and IFN--/- mice with similar
severity (4–5+) and histopathologic features. However, cells activated
in the presence of IL-12 did induce more severe granulomatous EAT when
low numbers of cells were transferred to recipients (data not shown),
and there was increased fibrosis in the thyroids of recipients of both
IFN-+/+ and IFN--/- cells activated in
the presence of IL-12. Interestingly, an increased expression of mRNA
for IL-5 and IL-13 and, in some experiments, IL-4, was observed in
thyroids from recipients of IFN--/- cells activated in
the presence of IL-12 (Fig. 4, C and D).
This observation is consistent with the report of Wynn et al. who
showed that in vivo administration of IL-12 increased Th2-associated
cytokine expression in the absence of endogenous IFN- (36). A
similar result was reported in a related in vitro model (37). These
data suggest that in the absence of IFN-, IL-12 can promote, rather
than suppress, Th2 responses.
Evidence from several studies suggests that NO is an important mediator
of tissue damage in experimental autoimmune diseases such as diabetes
(38), inflammatory arthritis (39), and EAE (40). NO has been shown to
be an effector in microglial cytotoxicity toward oligodendrocytes in
vitro (41). In addition, inhibition of NOs could prevent induction of
EAE (40). However, in contrast to the high level of expression of iNOs
mRNA in IFN-+/+ thyroid infiltrates, iNOs mRNA was
barely detectable in IFN--/- thyroid infiltrates,
suggesting that NO is not required for tissue destruction in
granulomatous EAT. Similar results have recently been described by
Jones et al. in EAU using IFN--/- mice (34).
Another important observation in our study is that both
IFN-+/+ and IFN--/- recipient
mice developed massive eosinophil infiltration in the thyroids after
receiving effector cells from IFN--/- donor
mice, suggesting that IFN- plays an inhibitory role in eosinophil
accumulation. An accumulation of eosinophils is a feature of the
inflammatory reactions that occur in parasitic infections, allergic
asthma, and eczema (42). A number of factors have been described as
being chemotactic for eosinophils, such as eotaxin, RANTES, and
monocyte chemoattractant protein-3 (43, 44, 45). IL-5 specifically
regulates eosinophil growth and differentiation (46). In addition,
mucosal IFN- gene transfer inhibited both Ag- and Th2 cell-induced
pulmonary eosinophilia and airway hypersensitivity (47), and mice
lacking the IFN-R had an impaired ability to resolve an eosinophilic
response in the lung associated with a Th2 cytokine profile (48).
Increased infiltration by eosinophils in the eye in EAU (34) and in
type 1 and type 2 granulomas (49) was also observed in
IFN--/- mice. These data suggest that IFN- acts as
an inhibitory factor in the development of eosinophilia. Whether
eosinophils play a role in the pathogenesis of granulomatous EAT is not
known. Preliminary results suggest that neutralization of IL-5 in vivo
prevented eosinophil infiltration in the thyroids, but did not affect
the extent of damage to thyroid follicles (our unpublished
observations).
In conclusion, our results clearly demonstrate that IFN- is not
essential for the induction of granulomatous EAT.
IFN--/- effector cells were capable of inducing severe
granulomatous thyroid lesions with a distinct cytokine pattern and
eosinophil infiltration, indicating that IFN- may play a regulatory
role in EAT induction. These data are consistent with recent evidence
(34, 49) which demonstrated that damage to the eye in EAU or type 1 and
type 2 granuloma size were not diminished in IFN--/-
mice, but there was an alteration in the expression of cytokines,
eotaxin, and iNOs in the inflammatory sites. These results indicate
that disruption of a single cytokine gene can lead to alteration of
inflammatory mediators in autoimmune or immune responses without
apparently influencing the extent of damage to the organ.
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Acknowledgments |
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+/- mice and the primer sequences for analysis
of tail DNA. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Helen Braley-Mullen, Division of Immunology, Department of Medicine, M450 Medical Science, University of Missouri, Columbia, MO 65212.
3 Abbreviations used in this paper: EAT, experimental autoimmune thyroiditis; MTg, mouse thyroglobulin; PMN, polymorphonuclear leukocyte; RT-PCR, reverse transcription-polymerase chain reaction; HPRT, hypoxanthine phosphoribosyltransferase; iNOs, inducible nitric oxide synthase; NO, nitric oxide; EAE, experimental allergic encephalomyelitis; EAU, experimental autoimmune uveitis.
Received for publication November 21, 1997. Accepted for publication January 21, 1998.
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