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Original Studies |
Division of Endocrinology, Mayo Clinic/Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Rebecca S. Bahn, M.D., Mayo Clinic, Division of Endocrinology, 200 First Street SW, Rochester, Minnesota 55905. E-mail: bahn.rebecca{at}mayo.edu
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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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Subsets of CD4+ or CD8+ T
cells, classified according to the profiles of cytokines produced, were
first described in mice and are more strictly defined in that species
than in humans. However, this classification has relevance to human
physiology and pathophysiology as well (7, 8).
CD4+ T cells that produce primarily
interferon-
(IFN), interleukin-2 (IL-2), and tumor necrosis
factor-ß (TNFß), are classified as helper-type 1
(TH1) cells and are involved primarily in
cell-mediated immune responses (9). In contrast, IL-4, IL-5, and
IL-10 are the dominant cytokines secreted by T helper-type 2
(TH2) cells involved in humoral immunity.
Similarly, CD8+ T cytotoxic cells can be divided
into TC1 and TC2 subsets
with comparable cytokine profiles. However, unlike
TH cells, TC cells of both
subsets appear to induce cell-mediated immune responses (10).
The hyperthyroidism of Graves’ disease is caused by stimulatory autoantibodies directed against the thyroid follicular cell TSH receptor (TSHr), indicating that humoral immunity plays a central role in this condition. Indeed, a cytokine profile compatible with a TH2 response has been detected in clones of intrathyroidal lymphocytes from patients with Graves’ disease (11, 12). The pathogenesis of the ocular component of this disease is not as well understood. Studies by several groups of investigators have pointed toward cellular immune mechanisms as being responsible for the eye changes characteristic of GO (3, 13, 14, 15). However, evidence exists as well for the involvement of serum autoantibodies in the disease (2, 16).
In this study we attempted to resolve some of the conflicting data in the literature by characterizing CD3+CD4+ lymphocyte clones established from the orbital adipose/connective tissues of GO patients having disease of either short or longer duration. In addition, we studied two different cloning protocols to determine the effects of these methodological differences on results.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Orbital adipose/connective tissue specimens were obtained from
six patients who underwent orbital decompression surgery for GO (Table 1
). All patients were initially diagnosed
and treated for Graves’ hyperthyroidism by their local physicians.
Each received radioactive iodine between 9–36 months before eye
surgery. One patient had a remote history of antithyroid drug
treatment. After the development of severe GO, patients were referred
to our institution for treatment. Patients 1, 2, 3, and 4 had severe
inflammatory/congestive GO, characterized by proptosis, periorbital
edema, chemosis, and eye pain. In addition, patient 3 had compressive
optic neuropathy. Patients 5 and 6 were primarily bothered by severe
extraocular dysfunction. Neither of these patients had significant
proptosis or inflammatory/congestive signs and symptoms at the time of
decompression surgery. Time from the onset of hyperthyroidism to the
time of decompression surgery ranged from 15–51 months, and the
duration of symptomatic ophthalmopathy ranged from 7–51 months. Three
patients had never received glucocorticoids. The remaining three either
were being treated with prednisone at the time of decompression surgery
or had recently discontinued this medication. Four of the six patients
had undergone orbital radiation therapy between 1–33 months earlier.
All patients were euthyroid while receiving thyroid hormone replacement
at the time of ocular surgery. The Mayo Clinic institutional review
board approved these studies.
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Orbital adipose/connective tissue was obtained during the course of transantral orbital decompression surgery, placed in a sterile container on saline-soaked gauze, and transported at room temperature to our laboratory. Tissue specimens were finely minced (without proteases) and incubated in 2-mL wells in T cell medium (RPMI 1640 containing glutamine, 10% heat-inactivated human serum, and penicillin/streptomycin). These initial cultures contained IL-2 (12.5 ng/mL; Roche Molecular Biochemicals, Indianapolis, IN) or IL-2 plus IL-4 (5 ng/mL; R&D Minneapolis, MN), but did not include any feeder cells. After 7 days at 37 C in a 5% CO2 incubator, allogeneic Epstein-Barr virus-transformed B cells (2.5 x 105 cells/well) were added to wells as feeder cells. For use as mitogenic feeders, allogeneic B cells were irradiated (10,000 rads) and incubated in serum-free RPMI with neuraminidase (0.04 U/10 million feeder cells) and galactose oxidase (0.1 U/10 million feeder cells) for 90 min at 37 C in 5% CO2 (17, 18, 19, 20, 21). Feeders were washed with B cell medium (RPMI 1640 containing glutamine, 10% heat-inactivated FBS, and penicillin/streptomycin) supplemented with 10 mmol/L galactose, then washed again and suspended in T cell medium. TCL were expanded, with IL-2 or IL-2 plus IL-4 added to new medium every 3–4 days and feeder cells added every 10–14 days. One TCL was established from each patient’s eye tissue, except in the case of tissue from patient 1 that was split into two parts to establish TCL and TCC using either IL-2 or IL-2 plus IL-4 in the medium from this point forward.
After expansion of TCL to approximately 2 x 106 cells/well, TCC were established by limiting dilution at 0.3, 1.0, and 10 cells/well in flat bottom 96-well plates. Either IL-2 alone or IL-2 plus IL-4 were added at the time of cloning and supplemented twice weekly. Irradiated allogeneic Epstein-Barr virus-transformed B cells (20,000 feeder cells/well) were added every 10–14 days. Plates showing no growth or having more than 22 positive wells at 8 weeks were discarded.
Phenotype analysis
Phenotypes of the TCC were analyzed by flow cytometry using fluorescein-conjugated monoclonal antihuman antibodies (TriTEST CD3 PerCP/CD4 FITC/CD8 PE, Becton Dickinson and Co., San Jose, CA). TCC (2.5 x 105) were transferred into round bottom tubes, washed with PBS, incubated with antibodies for 30 min at room temperature, fixed with formaldehyde (1.0%) in phosphate-buffered saline, and analyzed on a fluorescein-activated cell sorter (Excalibur, Becton Dickinson and Co.).
Quantitation of secreted cytokines
TCC were suspended in T cell medium supplemented with PHA-P (2
µg/mL) and seeded in 96-well plates coated with anti-CD3 antibodies
(P42178M, Biodesign, Kennenbunk, ME). Before the assay, TCC were
cultured for 7 days without IL-2, IL-4, or feeder cells. Supernatants
were aspirated after 24 h of incubation and immediately frozen at
-70 C. Quantitative assays for IL-4 and IFN production were
performed using commercially available enzyme-linked immunosorbent
assay (ELISA) kits (hIL-4 ELISA System, RPN 2753; human IFN ELISA
System, RPN 2757; Amersham Pharmacia Biotech, St. Louis,
MO). CD3+CD4+ clones were
assigned to TH1, TH0, and
TH2 subsets based on IL-4 to IFN ratios of
less than 0.1, 0.1–2.0, or more than 2.0, respectively (22).
Differences between the patients groups were analyzed using the
2 test with 2 x 3 contingency
tables.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 117 TCC were established from orbital
adipose/connective tissues of the 6 GO patients studied. Phenotype was
successfully determined in 77 of these clones, of which 57 of 77 were
CD3+CD4+ and 20 of 77 were
CD3+CD8+ (Table 2). Cytokine production was successfully
measured for T cell subtyping in a total of 95 clones, including all of
the CD4+ clones plus 38 clones for which no
phenotype data were obtained due to limited survival of these
clones.
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Analysis of cytokine production profiles in 57
CD3+CD4+ clones established
using IL-2 plus IL-4 in the T cell expansion medium revealed 25 of 57
to be TH1, 17 of 57 to be
TH0, and 15 of 57 to be TH2
(Table 2
). The 38 nonphenotyped clones were almost entirely derived
from patients 2, 4, and 6. Cytokine analysis revealed an IL-4 to IFN
ratio of more than 2.0 in 90% of these clones
(i.e.TH2/TC2
type). There was significant patient to patient variation regarding the
predominant helper T cell subset present. Patients 1 and 3, both of
whom had relatively recent onset of hyperthyroidism and GO (between
7–20 months before eye surgery), were the only subjects to have
predominantly TH1-type clones. The only other
individual to have relatively recent onset eye and thyroid disease was
patient 5. In this patient, only two clones were successfully subtyped,
and they were both TH2. However, this patient
differed from the other two in that she had no evidence of inflammatory
or active GO at the time of eye surgery. Perhaps due to this clinical
feature, we were able to establish and subtype only two
CD4+ clones from this patient, making these data
less reliable. In contrast, clones from patients 2, 4, and 6, all of
who had remote onset of Graves’ hyperthyroidism (25–51 months before
undergoing eye surgery) were predominantly of the
TH2 type. Two of these patients (no. 4 and 6)
also had the longest durations of eye disease.
For purposes of analysis, we grouped patients according to the length
of time from onset of clinical hyperthyroidism or GO until undergoing
orbital decompression surgery. Clones derived from group 1 patients
(no. 1, 3, and 5), who had onset of hyperthyroidism less than 2 yr
before undergoing eye surgery, were predominantly
TH1 type (n = 44 clones;
TH1/TH0/TH2
= 57/29/14%; Fig. 1). In contrast,
clones from group 2 patients (no. 2, 4, and 6), who had onset of
hyperthyroidism more than 2 yr before eye surgery were primarily
TH2 type (n = 13 clones;
TH1/TH0/TH2
= 0/31/69%). There was a statistically significant difference between
these two patient groups (P < 0.005, by
2 analysis).
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). Clones from
patients with GO of more than 2-yr duration were predominantly
TH2 type (n = 4;
TH1/TH0/TH2
= 0/25/75%). Statistical significance between these groups was only
borderline (P = 0.05), as relatively few clones (n
= 4) from these latter patients were successfully established and
characterized.
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Of the total 117 clones established, 29 were particularly slow growing
and took approximately twice as long to expand as did the usual clones.
Phenotyping of these slow growing clones showed nearly equal
representation between CD4+ and
CD8+ cells, unlike the 52:16 ratio seen in the
faster growing clones. Cytokine analysis of all slow growing clones
revealed an IL-4 to IFN ratio of more than 2.0 in 21 of 29
(i.e.TH2/TC2
type).
In one patient (no. 1), we established TCL and expanded TCC using two
different culture conditions. We wished to determine whether there
might be a bias toward the retrieval of TH1-type
(rather than TH2-type) clones when expansion is
carried out with feeders and IL-2 alone compared with that using
feeders and IL-2 plus IL-4 in the medium (10). We established a total
of 38 CD3+CD4+ TCC from
this patient, of which 21 were established and expanded using IL-2
alone, and 17 using IL-2 plus IL-4 (Table 3). Only 1 clone from each condition was
CD8+; the remainder were
CD4+. TH1-type TCC
predominated whether the clones were expanded in the presence of IL-2
alone (n = 20;
%TH1/TH0/TH2
= 95/5/0; Table 3), or using IL-2 plus IL-4 (n = 16;
%TH1/TH0/TH2
= 69/31/0; Table 3) in the medium. Although a slight shift toward the
TH0 phenotype was apparent in the IL-2- plus
IL-4-expanded clones, the difference between groups was not
statistically significant (P = 0.099, by
2 analysis).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF
or TNFß, but not IL-4 or IL-5)
(4, 6). Our early immunohistochemical studies are in agreement with
these findings, as we found abundant staining for IFN in Graves’
orbital tissue sections (23). In contrast, other investigators detected
the presence of messenger ribonucleic acid encoding a
TH2-dominant profile (IL-4, IL-5, and IL-10) (5),
whereas TCL that were predominantly CD8+ and
secreted cytokines characteristic of both subtypes (IFN, IL-4, and
IL-10) were identified by another group (3). These apparently
contradictory results may be explained in part by differences in the
methods used to expand the TCL and TCC, and by whether cytokines were
measured in TCL (3), TCC (4, 6), or the orbital tissues themselves (5, 23). We hypothesized that the predominant T cell subset in GO orbital
tissues might change over the course of the disease (24) and designed
the current study to determine whether the TH
subset identified might reflect the activity or stage of disease
represented in the particular tissue sample examined.
In a recent report, intrathyroidal TCC from patients with Hashimoto’s
thyroiditis, expanded using feeder cells and IL-2 alone, produced
primarily TH1-type cytokines (25). In contrast,
intrathyroidal TCC from patients with Graves’ disease were primarily
TH0, with rare TH2 clones
detected. However, expansion of clones using IL-2 alone (an enhancer of
IFN secretion that, in turn, inhibits the development of
TH2-type clones), without exposure to IL-4 (a
promoter of TH2-type clone development), is
thought to bias away from the retrieval of TH2
clones (26). Therefore, another group of investigators established
intrathyroidal TCC using either IL-2 alone or IL-2 plus IL-4 in the
medium (11). They found clones secreting TH0-type
cytokines to be the predominant subtype, regardless of whether IL-4 was
included in the medium. However, these investigators also found that
exposure to IL-4 in necessary to retrieve any TH2
clones, and, when present, it precluded growth of
TH1 clones.
Our study was the first to compare IL-2 with IL-2 plus IL-4 in the medium used for establishment of orbital TCC. Our results differed from those involving intrathyroidal TCC, as we found no shift toward production of TH2 clones with the addition of IL-4. The slight shift toward the TH2 phenotype apparent in the IL-2- plus IL-4-expanded clones was not statistically significant. These findings suggest that the majority of the T cells present in the ocular tissue samples, rather than being resting T cells, were probably effector T cells that had differentiated in response to prolonged antigenic stimulation (27). Although our study does not address the nature of this antigenic stimulation, the results add confidence that the observed predominance of TH1 cells in TCC from this patient (no. 1) and others accurately reflects the orbital T cell population.
These findings are in partial conflict with a recent study reporting a predominance of TH1-type TCC in GO patients’ orbital infiltrates (6). These investigators established TCL and TCC from eye tissue of three patients with GO of 6, 20, and 56 months duration, respectively. However, the TCL and TCC were established using irradiated feeders, phytohemagglutinin and IL-2, without exposure to IL-4. No TH2-type clones were retrieved, even though two of the patients had eye disease of quite long duration. These results probably reflect the culture conditions used and, as such, may not be an accurate representation of the T cells that were present in the patients’ orbits.
In conclusion, we found that the predominant subtype of CD3+CD4+ clone recovered from orbital infiltrates of patients with short duration hyperthyroidism or GO was TH1. In contrast, clones from patients with longer duration thyroid or eye disease were predominantly of the TH2 subtype. These results should be interpreted in light of the fact that the antigen specificity of the clones is unknown. Although in vivo activated clones may be selectively expanded in TCC initially established (as we did) without the use of feeder cells, the clones were not at any stage expanded in the presence of specific antigen (28). Therefore, the clones may not be tissue antigen specific and thus may not accurately reflect the intraorbital autoimmune process in GO. However, with this caveat in mind, it is interesting to speculate that T cell-mediated immune reactions by TH1 cells might predominate in the orbit in early, active GO, whereas humoral immunity and TH2 cells may play the greater role in the later stages of the disease. Future studies will be designed to address the antigenic specificity of the orbital-infiltrating T cells in GO.
Received July 30, 1999.
Revised September 22, 1999.
Accepted October 18, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 on the in
vitro development of human Th1 and Th2 clones. J Immunol. 148:2142–2147.[Abstract]
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