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Departments of
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Internal Medicine,
Molecular Microbiology & 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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gene mRNA and
production of IFN- by effector spleen cells stimulated with MTg and
IL-12 were similar for both IL-4+/+ and
IL-4-/- mice. Although IL-4 was undetectable in
IL-4-/- mice, expression of mRNA for IL-5, IL-10, and
IL-13 and production of IL-5 by both MTg-activated spleen cells and
anti-CD3-activated CD4+ T cells were comparable for
cells from IL-4+/+ and IL-4-/- mice,
indicating that the absence of IL-4 did not prevent production of other
Th2 cytokines. Production of MTg-specific IgG1 was very low or
undetectable in IL-4-/- mice. IL-4 gene mRNA and
MTg-specific IgG1 could be detected in IL-4+/+ or
IL-4-/- recipients only when they received effector cells
from IL-4+/+ donor mice, indicating that IL-4- and
IgG1-secreting cells are of donor origin. These results demonstrate
that IL-4 is not essential for development of granulomatous EAT. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAb (5, 6) develop a histologically distinct
granulomatous form of EAT in which the thyroid lesions show follicular
cell proliferation and infiltration with lymphocytes and other
mononuclear cells as well as polymorphonuclear leukocytes (PMN),
histiocytes, and multinucleated giant cells. MTg-specific
CD4+ T cells are required for induction of both lymphocytic
and granulomatous EAT (5). Based on the conditions needed to activate
cells that could transfer granulomatous EAT, we previously hypothesized
that effector cells for granulomatous EAT might be primarily Th2-like
CD4+ T cells (5, 6). More recently, however, we
demonstrated that a more severe form of granulomatous EAT can be
induced by activation of EAT effector cells in vitro in the presence of
IL-12.4 The mechanism by
which IL-12 activates effector cells to induce severe granulomatous EAT
appears to differ from the mechanism by which blocking the IL-2R
results in transfer of granulomatous EAT.4 Granulomatous responses are localized cellular reactions against an inciting agent, which can be either infectious or noninfectious in nature (7). The cellular constituents of granulomas include macrophages, lymphocytes, mast cells, epithelioid cells, and multinucleated giant cells. Recent evidence has shown that polarized Th1 or Th2 reactions can be found in granulomatous lesions of known or unknown etiology. A strong Th2 cytokine pattern has been found in pulmonary granulomas in experimental murine schistosomiasis (8, 9), whereas a Th1 cytokine pattern has been demonstrated in pulmonary granulomas induced by embolization of beads coupled to purified protein derivative of Mycobacterium tuberculosis (10, 11).
IL-4 is known to play an important role in development of Th2 cells and
humoral immune responses (12, 13). IL-4 alone, or IL-4 and IL-10 have
been shown to down-regulate inflammatory Th1 and cell-mediated immune
responses induced by IL-12 and characterized by IFN- production
(13, 14, 15, 16). The role of IL-4 in autoimmune pathogenesis is still unclear.
Although IL-4 has been shown to suppress experimental allergic
encephalomyelitis (EAE) (17) and spontaneous diabetes in NOD mice (18),
administration of IL-4 was shown to exacerbate experimental autoimmune
uveoretinitis (19) and adjuvant-induced arthritis (20). In adoptive
EAT, recipients of MTg-primed spleen cells activated with MTg in the
presence of IL-4 had a lower level of MTg-specific autoantibody, but
EAT severity was similar to that of control mice (21). To examine the
role of IL-4 in the regulation of granulomatous EAT, we generated IL-4
gene-disrupted mice on an EAT-susceptible H-2k background.
MTg-sensitized spleen cells from both IL-4+/+ and
IL-4-/- mice induced severe granulomatous EAT after in
vitro activation with MTg and IL-12. These results indicate that IL-4
is not required for development of the granulomatous form of
EAT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female B10.BR mice (H-2k), 6 to 8 wk old, were obtained from The Jackson Laboratory (Bar Harbor, ME). An IL-4-/- 129Sv (H-2b) male mouse was obtained through Dr. Leonard Schultz, The Jackson Laboratory. B10.BRxIL-4-/- F2 mice that were homozygous for H-2k and for either the IL-4-negative or IL-4-positive genes were selected and backcrossed five generations to obtain H-2k homozygous mice that were either IL-4-/- or IL-4+/+ and expressed the black coat color of the B10.BR parent. Typing for expression of H-2b and H-2k was done by flow-cytometric analysis of peripheral blood, and PCR analysis of tail DNA was used to distinguish the mutant and wild-type IL-4 genes. The primer sequences were provided by Dr. Peter Schweitzer (The Jackson Laboratory) and were synthesized by the DNA Core Facility at University of Missouri (Columbia, MO). The DNA product from the uninterrupted (+/+) allele is 378 bp, and the product from the interrupted (-/-) allele is 430 bp. The B10.BR strain was selected for crossing with 129Sv IL-4-/- to attempt to minimize differences in background genes (particularly minor lymphocyte-stimulating genes) that could potentially interfere with successful transfer of EAT by transferred cells. Although B10.BR mice express the H-2k allele associated with susceptibility to EAT, we find that they develop much less severe EAT than do other H-2k strains such as AKR and CBA/J. This was also true for the IL-4 H-2k mice used for these experiments. All mice were maintained under specific-pathogen-free conditions in barrier facilities at University of Missouri. For the experiments described in this work, IL-4-/- H-2k mice were compared with age- and sex-matched IL-4+/+ H-2k mice from the same backcross generations. Mice were generally 6 to 10 wk old at the time of use.
EAT induction
EAT was induced as previously described (5). Briefly, mice were injected i.v. twice at 10-day intervals with 150 µg MTg, prepared as previously described (4), and 15 µg LPS (Escherichia coli 011:B4; Sigma Chemical Co., 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 Chemical Co.), 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 5% final concentration of culture supernatant containing anti-IL-2R mAb (M7/20) (5) and 5 ng/ml IL-12 (R&D Systems, Minneapolis, MN).4 Cells were harvested and washed twice with balanced salt solution, and 4 to 5 x 107 cells were transferred i.v. into 600 R irradiated syngeneic recipients. Recipient thyroids were collected for histologic evaluation of EAT 19 to 21 days later, the time of maximal EAT severity (5).4
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 (5, 6). 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. The thyroid inflammatory infiltrate in conventional lymphocytic EAT consists primarily of mononuclear cells with relatively few PMN. Thyroids of most mice in this study had granulomatous inflammatory lesions graded 2 to 4+ in terms of the extent of destruction of thyroid follicles. These thyroids had proliferation and enlargement of thyroid follicular cells with granulomas containing lymphocytes and other mononuclear cells, modest to moderate numbers of PMN, and large numbers of histiocytes (5). The more severely destroyed (4–5+) thyroids had more extensive granulomatous changes with more PMN, microabscess formation, necrosis, multinucleated giant cells, and large numbers of histiocytes predominating over the follicular cell proliferative changes and mononuclear infiltrates.
ELISA assay
Serum levels of MTg-specific IgG autoantibodies in individual
donor or recipient mice were determined by ELISA, as previously
described (5). The contribution of various IgG subclasses to the total
IgG autoantibody response was assessed using alkaline
phosphatase-conjugated Abs 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 with normal
mouse serum (1/100 dilution) on MTg-coated plates. Levels of IFN-
produced by cells during the 72-h culture with MTg or MTg and IL-12, or
48 h after activation with anti-CD3 mAb 145-2C11 (ATCC
CRL-1975) were evaluated by double-sandwich ELISA using mAb R46A2 as
the capture Ab, biotinylated XMG1.2 as the detection Ab, and murine
rIFN- (Biosource International, Camarillo, CA) as a standard. IL-2,
IL-4, and IL-5 were assayed using ELISA kits purchased from Endogen
(Cambridge, MA).
CD4+ T cell purification and anti-CD3 stimulation
CD4+ T cells were isolated from spleen cells as previously described (22). CD4+ T cells were cultured at 2 x 106 cells/well (1 ml) in flat-bottom 24-well culture plates and stimulated with solid-phase-coupled anti-CD3 mAb 145-2C11 (20 µg/ml). Supernatants and cells were harvested after 48 h. Supernatants were stored at -20°C, and cells were snap frozen in liquid nitrogen and stored at -70°C until used.
Reverse-transcription PCR (RT-PCR) amplification
Total RNA was isolated from 5 x 106 spleen cells, 1 x 106 anti-CD3-activated CD4+ T cells, or single thyroid lobes using TRIZOL (Life Technologies, Gaithersburg, MD), according to the manufacturer’s instructions. The dried RNA pellet was dissolved in 10 µl of sterile diethyl pyrocarbonate-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, according to the manufacturer’s instructions. 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-guanine phosphoribosyltransferase (HPRT) was used as a housekeeping gene to verify that the equivalent amounts of RNA were amplified. Most cytokine gene primers used in this study were described previously (22). Primer sequences for IL-5 were: sense, ATG ACT GTG CCT CTG TGC CTG GAG C; antisense, CTG TTT TTC CTG GAG TAA ACT GGG G. Primer sequences for IL-13 were: sense, GAA GTG GAT CCT GAG GAC AGA TAC G; antisense, GAC CAT GGG CCA TGA GGA ACA TTC. To compare relative levels of mRNA transcripts between different samples, samples were reverse transcribed and amplified at the same time using aliquots of reagent from the same master mix.
PCR amplification was performed as previously described (22). Final 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). Dilutions of samples within the linear relationship (usually 1/25 dilution) were used for analysis, and the densitometric units for each cytokine band were normalized to those obtained for the corresponding HPRT band.
In some experiments, expression of mRNA for IL-5, IFN-, and HPRT was
determined using PCR mimics made using a PCR mimic construction kit
from Clontech Laboratories (Palo Alto, CA), according to the
manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preliminary experiments indicated that cells from
MTg/LPS-immunized IL-4+/+ or IL-4-/-
donor mice activated with MTg alone or with MTg and anti-IL-2R mAb
induced minimal or no EAT after transfer to either IL-4+/+
or IL-4-/- recipient mice (data not shown). MTg-primed
cells activated with MTg and M7/20 in the presence of IL-12 induce a
much more severe form of granulomatous EAT.4 To determine
whether granulomatous EAT can be induced in IL-4 gene-negative mice,
spleen cells from MTg/LPS-immunized IL-4+/+ and
IL-4-/- donor mice were cultured for 72 h with MTg
together with the anti-IL-2R mAb M7/20 and 5 ng/ml of IL-12. Cells
were transferred to 600-rad irradiated IL-4+/+ or
IL-4-/- recipients. The results of four representative
experiments are shown in Table I
. Cells
from both IL-4+/+ and IL-4-/- donors induced
granulomatous EAT in recipient mice. Thyroid lesions in most recipient
mice had extensive follicular cell proliferation, large numbers of
histiocytes, lymphocytes, mononuclear cells, PMN, and multinucleated
giant cells. The more severe (4–5+ severity) lesions also
had microabscess formation and focal fibrosis, and the inflammation
frequently extended beyond the thyroid into adjacent muscle and
connective tissue. Thyroids of recipients of cells from both
IL-4+/+ and IL-4-/- donors had qualitatively
similar granulomatous histopathologic features in these and several
other experiments (Fig. 1). These results
indicate that IL-4 is not necessary for development of the
granulomatous form of EAT induced by cells activated with MTg together
with anti-IL-2R mAb and IL-12. However, IL-4 may augment the
intensity of the granulomatous inflammatory response since six
recipients of IL-4+/+ cells in experiments 1 and 3
developed very severe (5+) EAT, while none of the
recipients of IL-4-/- cells had 5+ lesions
(Table I).
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After immunization with MTg/LPS, IL-4+/+ and
IL-4-/- donors and recipients of cells from either
IL-4+/+ or IL-4-/- donors produced
anti-MTg IgG autoantibody (Table I
). MTg-specific IgG levels were
lower for IL-4-/- mice in some experiments, although in
other experiments, IL-4+/+ and IL-4-/- donors
and recipients had comparable levels of MTg-specific IgG (Table I).
Since IL-4 is known to be critical for production of IgG1 (23), the
subclass distribution of the anti-MTg IgG autoantibody was also
determined. In a typical experiment, IL-4+/+ donor mice
produced primarily IgG1 and IgG2b anti-MTg and relatively low
amounts of IgG2a (Fig. 2A).
IL-4-/- donors produced amounts of MTg-specific IgG2a and
IgG2b comparable with those observed for IL-4+/+ mice, but
MTg-specific IgG1 was very low and in many sera was undetectable,
indicating that IL-4 is required for production of MTg-specific IgG1
autoantibody. Similar results were obtained with serum from recipient
mice (Fig. 2B). Recipients of cells from either
IL-4+/+ or IL-4-/- donors produced similar
amounts of MTg-specific IgG2a and IgG2b. Whereas IgG1 autoantibody was
produced in recipients of IL-4+/+ cells, recipients of
IL-4-/- donor cells produced very low amounts of
MTg-specific IgG1 autoantibody even when they were transferred into
IL-4+/+ recipients (Fig. 2B). This latter
observation demonstrates that most, if not all, of the anti-MTg
autoantibody produced by recipient mice is of donor origin. Morever,
the presence or absence of MTg-specific IgG1 autoantibody apparently
has little, if any, influence on EAT development or severity.
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To determine whether IL-4 is necessary for the generation of Th2
cytokines in vivo, cytokine gene mRNA expression by MTg-primed spleen
cells activated 72 h with MTg, M7/20, and IL-12 was analyzed by
RT-PCR, as described in Materials and Methods. As expected,
IL-4 mRNA was undetectable in cells of IL-4-/- mice (Fig. 3). Expression of mRNA for IL-2, IL-5,
IL-10, IL-13, IFN-, and TNF-
was almost equivalent in
MTg-stimulated spleen cells from both IL-4+/+ and
IL-4-/- mice in this and other experiments. Cells
cultured without MTg expressed no detectable IL-2, IL-4, or IL-13, and
only weak bands for TNF-, IL-10, and IL-5 were detected using a 1/5
cDNA dilution. IFN- bands were equally detected in cells cultured
with or without MTg in the presence of IL-12 (data not shown). These
results suggest that the absence of IL-4 did not prevent activation of
cells capable of producing other cytokines, including those normally
considered to be produced by Th2 cells (IL-13, IL-5, and IL-10).
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A, IFN- and IL-2 mRNA were
slightly up-regulated in CD4+ T cells of
IL-4-/- compared with IL-4+/+ mice, whereas
similar levels of IL-5 and IL-10 were detected in both
IL-4+/+ and IL-4-/- CD4+ T cells.
Although IL-13 mRNA was readily detected in CD4+ T cells of
IL-4-/- mice, these cells generally expressed lower
levels of IL-13 mRNA than IL-4+/+ CD4+ T
cells.
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genes in CD4+ T cells from
IL-4+/+ and IL-4-/- mice. HPRT, IL-5, and
IFN- gene mimics were prepared, and constant amounts of cDNA samples
were amplified in the presence of serial twofold dilutions of specific
gene mimics. As shown in a representative experiment in Figure 4B, HPRT mRNA expression was similar (2.5 x
10-4 attomole/µl) for both samples. IFN- mRNA
expression was higher in IL-4-/- cells (5 x
10-3 attomole/µl) compared with IL-4+/+
cells (6.25 x 10-4 attomole/µl). IL-5 mRNA
expression was similar (2.5 x 10-3 attomole/µl)
for anti-CD3 mAb-stimulated CD4+ T cells from both
IL-4+/+ and IL-4-/- mice immunized with
MTg/LPS. These results further confirmed that CD4+ T cells
from both IL-4-/- and IL-4+/+ mice expressed
similar levels of mRNA for the Th2 cytokine IL-5.
Production of selected cytokines in the supernatants from
CD4+ T cells and spleen cells stimulated with anti-CD3
mAb and MTg was also determined by ELISA. As shown in Table II, CD4+ T cells or spleen
cells from IL-4+/+ and IL-4-/- mice produced
comparable amounts of IL-5 and IFN- after stimulation with
anti-CD3 mAb, while IL-2 production was increased in
CD4+ T cells, but not in spleen cells from
IL-4-/- mice. IL-4 was produced by
anti-CD3-stimulated T cells of IL-4+/+ mice (Table II),
but was not detected in MTg-activated IL-4+/+ T cells (data
not shown). Spleen cells from both IL-4+/+ and
IL-4-/- mice produced similar, but very low, amounts of
IL-2 and IL-5 after activation by MTg, M7/20, and IL-12 (data not
shown). IFN- production was also similar for both
IL-4+/+ and IL-4-/- cells (data not shown).
Together, these results indicate that IL-4-/- mice are
capable of producing Th2-associated cytokines after immunization with
MTg/LPS. Production of Th1 cytokines is either unchanged or slightly
up-regulated in IL-4-/- mice compared with
IL-4+/+ mice.
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After receiving MTg- and IL-12-activated spleen cells, both
IL-4+/+ and IL-4-/- mice developed
granulomatous EAT. To determine whether cytokine gene expression in the
target organ might differ in IL-4+/+ and
IL-4-/- mice, expression of cytokine gene mRNA in
individual thyroid lobes of recipient mice was determined by RT-PCR 19
days after cell transfer. Since the total amount of RNA differs for
thyroids with 1+ EAT compared with the larger thyroids with
4+ EAT, cytokine gene mRNA expression was expressed as a
relative value to a housekeeping gene HPRT. As expected, IL-4 mRNA was
undetectable in thyroids of IL-4-/- mice even using 50
cycles of amplification (Fig. 5).
However, mRNA for other Th2 cytokines such as IL-5, IL-10, and IL-13
was present in the thyroid infiltrates of both IL-4+/+ and
IL-4-/- mice. Th1 cytokines IFN- and IL-2 and TNF-
were also expressed in the thyroids from both IL-4+/+ and
IL-4-/- mice. Although the levels of cytokine gene
expression in IL-4+/+ and IL-4-/- mice varied
in different experiments, levels of cytokine gene mRNA expression
generally correlated with the severity of disease, i.e., thyroids
graded 1+ expressed less mRNA for all cytokines than did
thyroids graded 3 to 4+. Thyroids from normal mice or mice
with no EAT expressed mRNA for HPRT, but not for any cytokines (data
not shown). Taken together, these findings indicated that the intrinsic
absence of IL-4 did not alter the relative expression of mRNA for
either Th1 or Th2 cytokines in thyroid infiltrates of mice with
granulomatous EAT. IL-4 mRNA was expressed in thyroids only when donors
were IL-4+/+. IL-4 was not detected in thyroids of
IL-4+/+ recipients when they received cells from
IL-4-/- donors (data not shown), indicating that
intrathyroidal IL-4-secreting cells are of donor origin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAb (5, 6). Since activation of
granulomatous EAT effector cells is promoted when IL-2 or IFN- is
neutralized or when IL-2-induced cell growth is blocked, we initially
suggested that these effector cells may be primarily Th2-like
CD4+ T cells (5, 6). Since IL-4 is critical for
development and differentiation of Th2 cells (12, 13, 24), this
hypothesis would predict that IL-4-/- mice would be
unable to develop Th2 responses, and therefore would not develop
granulomatous EAT. IL-4 gene knockout mice expressing the
EAT-susceptible H-2k haplotype on the B10.BR background
were generated to address the potential role of IL-4 in granulomatous
EAT. Since these IL-4+/+ and IL-4-/- mice
were relatively EAT resistant compared with other H-2k strains used in
our previous studies (5, 6), it was necessary to activate MTg-primed
donor cells with MTg in the presence of IL-12, which strongly promotes
activation of CD4+ T cells to induce granulomatous
EAT.4 In these experiments, both IL-4+/+ and
IL-4-/- donor cells induced granulomatous EAT when
effector cells were activated with MTg, anti-IL-2R mAb, and IL-12
(Table I). IL-4-/- donors and either IL-4+/+
or IL-4-/- recipients of cells from IL-4-/-
donors produced little, if any, detectable MTg-specific IgG1 Ab (Fig. 2), consistent with previous studies demonstrating that IL-4 is
critical for generation of IgG1 responses (23). These results indicate
that neither IL-4 nor IgG1 autoantibody is essential for development of
the granulomatous form of EAT.
Although the thyroids of IL-4+/+ and IL-4-/-
mice had similar granulomatous histopathologic features (Fig. 1), none
of the recipients of cells from IL-4-/- donors had
thyroid follicle destruction graded 5+ in severity (Table I). This suggests that IL-4 might potentiate the intensity of
granulomatous EAT, as shown recently for both Th2 and Th1 granulomas
(26). In addition, since the mechanism by which IL-12 activates
effector cells for severe granulomatous differs from that by which
blocking the IL-2R results in granulomatous EAT,4 IL-4
might have a more profound role if granulomatous EAT were induced in
the absence of IL-12. We are currently generating IL-4-/-
mice on a more EAT-susceptible background to address this issue.
While cells from IL-4-/- mice expressed no detectable
mRNA for IL-4 even after activation with anti-CD3, these cells did
express mRNA for other cytokines normally produced by Th2 cells, i.e.,
IL-5, IL-10, and IL-13 (Figs. 3 and 4A). These Th2
cytokines were also expressed by anti-CD3-activated
CD4+ T cells (Fig. 4A), and similar
amounts of IL-5 were produced by both spleen cells and CD4+
T cells of IL-4+/+ and IL-4-/- donors (Table II). IL-5, IL-10, and IL-13, as well as Th1 cytokines such as IL-2,
IFN-, and TNF- were present in the thyroid infiltrates of both
IL-4+/+ and IL-4-/- mice (Fig. 5).
Quantitation of IL-5 and IFN- expression by competitive PCR
indicated that IL-5 mRNA was expressed at similar levels in
anti-CD3-stimulated CD4+ T cells of both
IL-4+/+ and IL-4-/- mice, while
CD4+ T cells of IL-4-/- mice expressed more
IFN- than CD4+ T cells of IL-4+/+ mice (Fig. 5). Since the absence of IL-4 did not prevent development of
CD4+ T cells expressing other Th2 cytokines, these results
do not exclude the possibility that CD4+ T cells producing
non-IL-4 Th2 cytokines might have a role in development of
granulomatous EAT.
IL-4 is considered to play a key role in the differentiation of Th2
cells from naive CD4+ T cells (12, 13, 14, 15, 24, 25). Initial
studies showed that CD4+ T cells of IL-4-/-
mice produced low or undetectable amounts of Th2 cytokines such as IL-5
and IL-10 (27), and had low IgG1 and IgE responses to
Nippostrongylus brasiliensis (28). CD4+ T cells
of IL-4-deficient mice infected with Leishmania
major or Brugia malayi also had a reduced ability
to produce the Th2-associated cytokines IL-5, IL-10, and IL-13 (29, 30). In contrast, our results indicated that mRNA for IL-5, IL-10, and
IL-13 was expressed in spleen cells, CD4+ T cells, and
thyroid infiltrates of IL-4-/- mice. These results are
consistent with those of von der Weid et al., who showed that mRNA for
IL-5, IL-6, and IL-10 was expressed at comparable levels in both
IL-4+/+ and IL-4-/- mice at the later stages
of Plasmodium chabaudi infection, and Th1 function was
sustained (31). Th1 (IL-2 and IFN-) and Th2 (IL-5 and IL-10)
cytokine transcripts were also comparable in granulomatous livers from
IL-4+/+ and IL-4-/- mice infected with
schistosome eggs (32). The production of IL-5 in IL-4 gene-disrupted
mice was also reported in several other studies (33, 34). The reasons
for these apparent discrepancies are unknown, but may be due to
differences in stimulation protocols, or adjuvants (34) and mouse
strains used. Taken together, these data suggest the existence of an
IL-4-independent pathway for the generation of Th2-like
CD4+ T cell responses.
Recent evidence indicates that polarized Th1 or Th2 reactions can be found in granulomatous lesions caused by known or unknown etiology. For example, pulmonary sarcoidosis is characterized by a dominant Th1 immune response associated with enhanced production of IL-12 by lung macrophages (35). In animal models, a predominant Th1 cytokine pattern was demonstrated in pulmonary granulomas induced by embolization of beads coupled to purified protein derivative of Mycobacterium tuberculosis (10, 11), while a strong Th2 cytokine pattern was found in pulmonary granulomas in experimental murine schistosomiasis (8, 9). The role of IL-4 in schistosome egg-induced granulomas is still controversial, since there were no significant differences in granuloma formation between IL-4+/+ and IL-4-/- mice (32), while IL-4 adenovirus-transfected mice had increased granulomatous lesions after schistosome egg infection (26).
Granulomatous EAT can be induced in certain strains of mice by
immunization with MTg and CFA (36, 37). The mechanism involved in this
granuloma formation is unknown, although MTg-specific CD4+
T cells are essential for development of granulomatous EAT (5).
Activation of granulomatous EAT effector cells in the
IL-4+/+ and IL-4-/- mice used in this study
required restimulation with MTg together with IL-12, a cytokine known
to promote IFN- production and activation of Th1 cells (24). While
this may suggest that Th1-like cells are likely to be the primary
effector cells for granulomatous EAT, IL-12 did not promote obvious
differentiation of MTg-sensitized cells to a Th1 phenotype, since cells
restimulated with MTg and IL-12 produced both Th1 and Th2 cytokines
(Fig. 3). A mixed Th1 and Th2 cytokine profile was also present in
thyroid infiltrates (Fig. 5), and in other strains of mice such as
CBA/J, the ratio of Th1 to Th2 cytokines in granulomatous thyroid
infiltrates is similar whether donor cells are activated in the
presence or absence of IL-12 (Tang and Braley-Mullen, in preparation).
These observations all suggest that both Th1 and Th2 cells may
contribute to the pathogenesis of granulomatous EAT. Although it is
possible that either Th1 or Th2 cells alone could be sufficient for
inducing granulomatous EAT, we have been unable to generate
MTg-specific Th1 or Th2 clones that would be needed to directly address
this possibility. Alternatively, analysis of cytokine gene expression
during earlier stages of thyroid infiltration may be more informative
for understanding the mechanisms involved in development of
granulomatous EAT. These studies are currently in progress.
In conclusion, these studies demonstrate that IL-4 is not essential for induction of granulomatous EAT induced by donor cells activated in the presence of IL-12. Granulomatous inflammatory lesions are a major pathologic feature of several human diseases, such as Wegener’s granulomatosis, allergic granulomatosis, giant cell arteritis, and sarcoidosis. Since the precise mechanisms involved in induction of these lesions are poorly understood, studies of the mechanisms by which specific cells and cytokines regulate the development of granulomatous lesions in animal models may ultimately provide important information for development of strategies for treatment of these human diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Helen Braley-Mullen, Division of Immunology, Department of Medicine, M450 Medical Sciences, University of Missouri, Columbia, MO 65212.
3 Abbreviations used in this paper: EAT, experimental autoimmune thyroiditis; EAE, experimental allergic encephalomyelitis; HPRT, hypoxanthine-guanine phosphoribosyltransferase; MTg, mouse thyroglobulin; PMN, polymorphonuclear leukocytes; RT-PCR, reverse-transcription polymerase chain reaction.
4 H. Braley-Mullen, G. C. Sharp, H. Tang, M. Kyriakos, and J. T. Bickel. 1997. IL-12 promotes activation of effector cells that induce a severe destructive granulomatous form of experimental autoimmune thyroiditis. Submitted for publication.
Received for publication July 7, 1997. Accepted for publication September 22, 1997.
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antibody. J. Immunol. 149:2219.[Abstract]
ß T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065.This article has been cited by other articles:
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  K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Decreasing TNF-{alpha} results in less fibrosis and earlier resolution of granulomatous experimental autoimmune thyroiditis J. Leukoc. Biol., January 1, 2007; 81(1): 306 - 314. [Abstract] [Full Text] [PDF]
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C. Vasu, R.-N. E. Dogan, M. J. Holterman, and B. S. Prabhakar Selective Induction of Dendritic Cells Using Granulocyte Macrophage-Colony Stimulating Factor, But Not fms-Like Tyrosine Kinase Receptor 3-Ligand, Activates Thyroglobulin-Specific CD4+/CD25+ T Cells and Suppresses Experimental Autoimmune Thyroiditis J. Immunol., June 1, 2003; 170(11): 5511 - 5522. [Abstract] [Full Text] [PDF]
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S. Yu, G. C. Sharp, and H. Braley-Mullen Dual Roles for IFN-{gamma}, But Not for IL-4, in Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice J. Immunol., October 1, 2002; 169(7): 3999 - 4007. [Abstract] [Full Text] [PDF]
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K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Induction of Experimental Autoimmune Thyroiditis in IL-12-/- Mice J. Immunol., August 1, 2001; 167(3): 1720 - 1727. [Abstract] [Full Text] [PDF]
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K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Characterization of thyroid fibrosis in a murine model of granulomatous experimental autoimmune thyroiditis J. Leukoc. Biol., December 1, 2000; 68(6): 828 - 835. [Abstract] [Full Text]
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 J. George, Y. Shoenfeld, B. Gilburd, A. Afek, A. Shaish, and D. Harats Requisite Role for Interleukin-4 in the Acceleration of Fatty Streaks Induced by Heat Shock Protein 65 or Mycobacterium tuberculosis Circ. Res., June 23, 2000; 86(12): 1203 - 1210. [Abstract] [Full Text] [PDF]
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K. E. Peterson, G. C. Sharp, H. Tang, and H. Braley-Mullen B7.2 Has Opposing Roles During the Activation Versus Effector Stages of Experimental Autoimmune Thyroiditis J. Immunol., February 1, 1999; 162(3): 1859 - 1867. [Abstract] [Full Text] [PDF]
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B. Balasa, C. Deng, J. Lee, P. Christadoss, and N. Sarvetnick The Th2 Cytokine IL-4 Is Not Required for the Progression of Antibody-Dependent Autoimmune Myasthenia Gravis J. Immunol., September 15, 1998; 161(6): 2856 - 2862. [Abstract] [Full Text] [PDF]
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H. Tang, G. C. Sharp, K. P. Peterson, and H. Braley-Mullen IFN-{gamma}-Deficient Mice Develop Severe Granulomatous Experimental Autoimmune Thyroiditis with Eosinophil Infiltration in Thyroids J. Immunol., May 15, 1998; 160(10): 5105 - 5112. [Abstract] [Full Text] [PDF]
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