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Original Studies |
Division of Endocrinology and Metabolism, Department of Medicine (Y.H., D.Y., T.B., I.M., K.N.), Fukuoka 830-0011, Japan; Eye Division, Olympia Medical Clinic (Y.I.), Tokyo 150-0001, Japan; and Department of Endocrinology, Polish Academy of Science (T.B.), Warsaw 02-097, Poland
Address correspondence and requests for reprints to: Yuji Hiromatsu, M.D., Division of Endocrinology and Metabolism, Department of Medicine, Kurume University School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830-0011 Japan. E-mail: yuji{at}med.kurume-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN), tumor necrosis factor 
(TNF),
interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-10, CD4, CD8, and
glyceraldehyde-3-phosphate dehydrogenase. IFN, TNF, IL-1ß, and
IL-6 messenger RNA (mRNA) were mainly detected in EM tissue, whereas
IL-4 and IL-10 mRNA were detected in only one patient. On the
other hand, in OF tissue, IL-4 and IL-10 mRNA were detected in 24% and
38% of the patients, respectively, and IFN, IL-1ß, and IL-6 mRNA
were less often detected compared with EM tissue. The enlargement of EM
tissue as assessed by computed tomography correlated significantly with
TNF mRNA expression in EM tissue. The orbital volume was positively
correlated with IL-6 mRNA expression and negatively correlated with
IL-4 mRNA and IL-10 mRNA expression in OF tissue. These results suggest
that T helper (Th) 1-like cytokines predominate in EM tissue in most
patients and that the predominant cytokine profile in OF tissue varies
from patient to patient. Both Th1-like and Th2-like immune responses
may play roles in the development of two components of ophthalmopathy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three subsets of CD4+ T helper (Th) cells have
been identified—namely, the Th1, Th2, and Th0 subsets (8, 9). Th1
cells produce interferon (IFN), interleukin (IL)-2, and tumor
necrosis factor (TNF) ß, which activate macrophages and are involved
in delayed-type hypersensitivity reactions, whereas Th2 cells produce
IL-4, IL-5, IL-10, and IL-13, which are responsible for strong antibody
responses. The Th1/Th2 paradigm may play a critical role in the
development of autoimmune disorders (10, 11). In TAO, there have been a
few studies concerning cytokine profiles on retroorbital T lymphocytes
from patients with TAO, with inconsistent results (12, 13, 14, 15, 16, 17). Natt and
Bahn (18) hypothesized that Th1- and Th2-like cytokines may play roles
in the acute and late stages of TAO, respectively. However, it has not
been elucidated whether cytokine profiles are differently involved in
the two types of TAO or in the two major sites of the lesion. The aim
of the present study was to determine the cytokine profile in EM and OF
tissues from patients with TAO.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thirty-three patients with TAO, 12 men and 21 women, aged 25–76
yr (mean age, 47.5 ± 13.7 yr), underwent surgery for EM repair or
decompression. EM tissue was obtained from 14 patients, and OF tissue
was obtained from 29 patients. At the time of surgery, all patients
were euthyroid on antithyroid drugs (16 patients),
l-T4 (6 patients), or without any drugs (11
patients). One patient had received subtotal thyroidectomy, and two
patients had received radioiodine therapy. Ophthalmopathy was assessed
by full ophthalmological examination and computed tomography (CT) from
1–7 days before surgery. Proptosis, enlargement of EM and orbital
volume were measured by CT, as reported previously (19). On the basis
of the CT findings, TAO patients were divided into two groups: 1) 23
patients with enlargement of the extraocular muscles; and 2) 10
patients without evident EM enlargement. Clinical characteristics of
these two groups are shown in Table 1
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All patients had received orbital irradiation 2–21 months before the
surgery (total dose, 15–20 Gy). Seventeen patients received medication
of large doses of steroid 3–10 months before the surgery. Patients
without evident EM enlargement were younger and had longer duration of
ophthalmopathy. Informed consent after full explanation of the nature
of the study was obtained for the donation of orbital tissue from these
patients.
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RNA was extracted from orbital tissue using RNA zol B
(Tel-test, Inc., Friendswood, TX), and RT-PCR was performed as
reported previously (19). Briefly, the complementary DNA (cDNA)
was synthesized from 1 µg total RNA with oligo (dT) 12–18 (10
µg/mL) (Pharmacia LKB, Uppsala, Sweden), 1 mmol/L dNTPs
(Life Technologies, Inc., Gaithersburg, MD), 10
mmol/L dithiothreitol (Life Technologies, Inc.), 3 mmol/L
MgCl2, and 200 U Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Inc.) at 37 C
for 60 min. PCR amplification using a temperature control system
(Astec, Fukuoka, Japan) was performed in 50 mmol/mL Tris-HCI buffer (pH
9) containing 20 mmol/L
NH2SO4, 0.7 mmol/mL
MgCl2, 200 µmol/mL dNTPs, and 25 U/mL
Taq DNA polymerase (Life Technologies, Inc.)
with pairs of primers designed to amplify IFN (5' primer,
5'-AAYGCAGGTCATTCAGATG-3'; 3' primer, 5'-TTGGACATTCAAGTCAGTT-3'), IL-2
(5' primer, 5'-ATGTACAGGATGCAACTCCTGTCTT-3'; 3' primer,
5'-GTTAGTGTTGAGATGATGCTTTGAC-3'), IL-4 (5' primer,
5'-GTCTCACCTCCCAACTGCTTC-3'; 3' primer, 5'-CTTGTGCCTGTGGAACTGCTG-3'),
IL-6 (5' primer, 5'-CCTTCTCCACAAGCGCCTTC-3'; 3' primer,
5'-GGCAAGTCTCCTCATTGAATC), IL-10 (5' primer,
5'-ATGCCCCAAGCTGAGAACCAAGACCCA-3'; 3'- primer,
5'-TCTCAAGGGGCTGGGTCAGCTATCCCA-3'), TNF (5' primer,
5'-CAGAGGGAAGAGTT-CCCCAG-3'; 3' primer, 5'-CCTTGGTCTGGTAGGAGACG-3'),
IL-1ß (5' primer, 5'-TTGAAGCTGATGGCCCTA-3'; 3' primer,
5'-TGCTCAGGTCATTCTCCT-3'), CD4 (5' primer, 5'-TGGTGATGAGAGCCACTCAG-3';
3' primer, 5'-CATGTCTTCTGAAACCGGTG-3') and CD8 (5' primer,
5'-TTCCGGGTGTCGCCGCTGGAT-3'; 3' primer, 5'-GCTGAAGTACATGATGGAGT-3')
(20, 21, 22). Paired primers of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (5' primer, 5'-CCTCTACTGGCGCTGCCAAGGCTGT-3'; 3' primer,
5'-CTCCGAGGCCTGCTTCACCACCTTC-3') were used as controls (19). The
amplification was performed under the following conditions: 1 min at 94
C, 1 min at 55–60 C, and 1 min at 72 C, and the PCR amplification was
up to 30 cycles. The final extension was carried out for 10 min at 72
C. PCR products were applied to a 2% agarose gel and stained with 0.5
µg/mL ethidium bromide. Peripheral blood mononuclear cells obtained
from patients with TAO were cultured with PHA (0.4 µg/mL;
DIFCO Laboratories, Detroit, MI) for 18 h. Total RNA was isolated
and used as positive controls for RT-PCR.
Statistical analysis
Differences of cytokine profiles and clinical parameters between
groups were evaluated using Student’s t test, Mann Whitney
test, 
2 test, Fisher’s exact
probability test, or Kruskal-Wallis test. Correlations were assessed by
linear regression analysis or Spearman’s correlation coefficient. A
P level of less than 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cytokine cDNAs could be amplified by RT-PCR in
phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear
cells from patients with TAO, as positive controls (Fig. 1). In EM tissue from 14 patients with
TAO, IFN mRNA was detected in almost all cases, and TNF, IL-1ß,
and IL-6 mRNA were detected in 36–64% of cases (Fig. 1, Table 2). In contrast, IL-4 and IL-10 cDNA were
amplified in only one case (Table 2). On the other hand, in OF tissue
from 29 patients with TAO, IFN, TNF, IL-1ß, and IL-6 mRNA were
detected in 17–31% of cases (Fig. 1, Table 2). Moreover, IL-4 and
IL-10 mRNA were also detected in 24% and 38% of cases, respectively.
Overall, 8 of 29 patients showed both Th2 cytokine profiles in OF
tissue, although 3 patients showed both Th1 and Th2 cytokine profiles
and 5 patients showed Th1 predominance. There were significant
differences in IFN, IL-1ß, and IL-6 mRNA expression between EM
tissue and OF tissue. IFN, IL-1ß, and IL-6 were predominantly
expressed in EM tissue, whereas IL-4 and IL-10 tended to be
predominantly expressed in OF tissue.
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). Although there were significant differences in age, duration
of ophthalmopathy between two subtypes of TAO (Table 1), IL-10 mRNA was
more dominantly detected in OF tissue from patients without evident EM
enlargement than the other groups (Table 2).
We further compared the cytokine mRNA profiles between EM tissue and OF
tissue obtained, at the same time, from 11 patients with TAO. As shown
in Table 3, the cytokine profile was
different between EM tissue and OF tissue. IFN mRNA was detected in
significantly greater number of EM specimens compared with OF tissue
samples (11 of 11 vs. 3 of 11, Fisher’s exact probability
test, P < 0.001, Table 3). In contrast, IL-10 mRNA
tended to be predominantly expressed in OF tissue compared with EM
tissue (5 of 11 vs. 1 of 11, Fisher’s exact probability
test, P = 0.08, Table 3).
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The EM enlargement assessed by CT was greater in patients with
positive expression of TNF mRNA in EM tissue than in those with
negative expression (Fig. 2A). However, either IL-1ß
or IL-6 did not significantly relate to the EM enlargement (data not
shown). There was a significant positive correlation between the number
of positive amplifications of cytokine cDNA in eye muscle tissue and
eye muscle enlargement (Fig. 2B). Other parameters, such as proptosis,
orbital volume, free T3, free
T4, thyrotropin receptor antibody titer,
antithyroglobulin antibody titer, and antimicrosomal antibody titer,
did not correlate with any cytokine gene expression in EM tissue (data
not shown).
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The OF volume, assessed by CT, was greater in patients with
positive IL-6 mRNA expression than in those with negative expression
(Fig. 3B). In contrast, the volume was smaller in patients with
positive IL-4 mRNA expression or positive IL-10 expression compared
with those with negative expression (Fig. 3, A and C). Neither IFN
nor TNF correlated with the OF volume (data not shown). The mean
(±SD) duration of ophthalmopathy tended to be short in
patients with Th1 and Th2 profiles and in those with Th1 predominance
compared with those with Th2 predominance (11 ± 3.1, 19 ±
6.7, 32 ± 26 months, respectively; Kruskal-Wallis test,
P = 0.14). The patients, whose EM tissue and OF tissue
were obtained, were significantly older, and their TSH receptor
antibody activity was greater than that in patients whose OF tissue
alone was obtained. However, there was no difference in cytokine gene
expression in OF tissue between these two groups (data not shown). The
other parameters did not correlate with any cytokine mRNA expression in
OF tissue (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA expression in EM tissue; and 2) the
predominant cytokine profile in OF tissue varied from patient to
patient. However, the orbital volume assessed by CT was positively
correlated with IL-6 mRNA expression and negatively correlated with
IL-4 mRNA and IL-10 mRNA expression in OF tissue. The other parameters,
such as duration of ophthalmopathy or antithyroid antibodies, did not
correlate with the cytokine gene expression.
Because of the difficulties in obtaining tissue specimens from patients
with TAO and the technical problems related to small specimen size and
the patchy distribution of the lesion, in vivo studies of
cytokine profiles in TAO have been limited. Most of the studies were
confined to T cell lines or T cell clones derived from orbital tissues,
and the results have been controversial. (13, 15, 16, 17). Pappa et
al. (16) reported the predominance of CD4+ T
cell lines derived from EM tissue from patients with TAO. In their
study, both Th1- and Th2-like cytokine profiles were present in their T
cell lines. The other reports showed the predominance of
CD8+ T cells in the orbit, and the reported
cytokine profiles were inconsistent (13, 15). Whereas De Carli et
al. (13) reported the predominance of Th1-like T cell clones
derived from T cell lines from TAO OF tissue, Grubeck-Loebenstein
et al. (15) reported a predominance of
CD8+ T cells in T cell lines derived from OF
tissue and showed the production of both Th1- and Th2-like cytokines in
their T cell lines. It is well known that the presence of IL-2 and PHA
might induce selective pressure away from Th2-type T cells (23). The
successful expansion of Th2-type T cells requires both IL-2 and IL-4 in
culture (24). IL-2 enhances IFN production, whereas IL-4
down-regulates IFN production (10). Therefore, the culture condition
might affect the results on cytokine profiles of T cell lines from
orbital tissues.
RT-PCR is a highly sensitive and specific method to detect RNA, even in
small tissue samples, and is expected to directly reflect the in
vivo cytokine profile. However, this technique has its
limitations; it is not quantitative and may not reflect the expression
of protein levels. With respect to the cytokine profile in EM tissue
from TAO, there are only two reports up to date. McLachlan et
al. (14) studied one EM specimen from a patient with TAO and
reported the predominance of Th2-like cytokine expression in the
tissue. Pappa et al. (16) studied 12 EM specimens from five
patients with TAO and found both Th1- and Th2-like cytokine mRNA
expressions. In contrast, we have studied a relatively large number of
patients and showed the predominance of Th1-like profile and a
pro-inflammatory cytokine gene expression in EM tissue. We further
showed that those cytokine gene expressions, including TNF in EM
tissue, were significantly correlated with the degree of enlargement of
EM. IFN mRNA was detected in EM tissue from almost all patients with
TAO. IFN is known to stimulate glycosaminoglycan production (5),
human histocompatibility leukocyte antigen (HLA)-DR expression
(25), and may augment an autoimmune response in the orbit. Taken
together, the present study supports the notion that Th1-like cytokines
play an important role in initiating and maintaining the autoimmune
response in TAO (6, 18). The discrepancy of cytokine profiles in EM
between the previous reports and our study may reflect different
patient characteristics, such as disease stage and disease activity,
patchy distribution of the inflammatory lesion, and possibly
differences in the RT-PCR methods used.
With respect to the cytokine profiles in OF tissue, McLachlan et
al. (14) studied five specimens from five patients with TAO and
showed a predominance of Th2-like cytokine profiles. The present study
confirmed these findings. However, 3 of 29 (10%) patients showed both
Th1 and Th2 profiles, and 5 of 29 (17%) patients showed Th1
predominance. In those patients the duration of ophthalmopathy tended
to be shorter compared with the patients with Th2 predominance. IL-4
stimulates proliferation of activated B cells and antibody production
(10). Thus, the number of target cells may be decreased by
antibody-dependent cell-mediated cytotoxicity. Both IL-4 and IL-10
inhibit Th1 cell proliferation and IFN production (10). Therefore,
these cytokines may ameliorate the inflammatory lesion. Natt and Bahn
(18) postulated that Th1-like cytokines may play a role in the acute
stage and Th2-like cytokines may play a role in the chronic stage of
TAO, mediating the late fibrosis. Most of the specimens we studied were
obtained at the chronic stage of the disease. Thus, the present
demonstration of a negative relationship between IL-4 and IL-10 mRNA
expression and retroorbital volume suggests that Th2-like cytokines may
play a role in the chronic stage of TAO. Additional studies are needed
to clarify these issues, using a more quantitative method.
In the present study, IL-10 mRNA was more dominantly detected in patients without EM enlargement. However, we could not conclude whether cytokine gene expression is different between the subtypes of TAO because the duration of ophthalmopathy, age, and prior steroid treatment were different between the subtypes of TAO.
There is considerable evidence of the presence of autoantibodies against various orbital antigens, but their roles in the pathogenesis of TAO remain unclear (26). Therefore, it would have been interesting to compare the cytokine gene expression with serum antibody profiles of patients, sensitivity of their T cells to a panel of orbital muscle and connective tissue antigens. This could be focus of a future study.
In conclusion, Th1-like cytokine profiles are predominant in EM tissue in most patients and related to the extent of EM enlargement. The predominant cytokine profile in OF tissue varies from patient to patient, and Th2-like cytokine profiles are negatively related to orbital volume. These results suggest that both Th1-like and Th2-like immune responses may play roles in the development of the two components of ophthalmopathy. Additional large-scale studies using quantitative methods and/or studies using animal models are indicated to clarify the difference of cytokine profiles between EM tissue and OF tissue and its role in the pathogenesis of TAO.
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Footnotes |
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Received October 20, 1998.
Revised June 15, 1999.
Accepted November 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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