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Original Studies |
Departments of Clinical Biochemistry (A.S., J.M.B.) and Endocrinology (A.S., J.P.M., D.F.W., G.M.B., J.M.B.), St. Bartholomew’s and Royal London School of Medicine and Dentistry, London E1 1BB, United Kingdom
Address all correspondence and requests for reprints to: Dr. Ayesha Siddiqi, Medical Unit, Alexandra Wing, Royal London Hospital, Whitechapel, London E1 1BB, United Kingdom. E-mail: a.siddiqi{at}mds.qmw.ac.uk
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Abstract |
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, IL-11, and urinary deoxypyridinoline (Udpd).
Compared with controls (IL-6, 1.1 ± 0.3 ng/L; IL-8, 3.2 ±
0.8 ng/L), untreated patients with GD and TNG had elevated IL-6 (GD,
7.11 ± 0.88 ng/L; TNG, 7.30 ± 0.77 ng/L;
P < 0.001) and IL-8 (GD, 10.3 ± 1.23 ng/L;
TNG, 9.81 ± 1.27 ng/L; P < 0.001). These
levels fell after treatment and were then indistinguishable from those
in control subjects. Thyroid carcinoma patients on TSH suppressive
therapy also had significantly raised levels of IL-6 (2.5 ± 0.42
ng/L) and IL-8 (4.4 ± 0.63 ng/L). When data from all the patients
were pooled, the levels of IL-6 and IL-8 correlated with serum
T3 and free T4 but not with Udpd or b-ALP.
IL-1ß, IL-11, and tumor necrosis factor- were not raised in any
patient. The elevations in serum IL-6 and -8 that occur in hyperthyroidism seem to result from the chronic effects of thyroid hormone excess rather than the accompanying autoimmune inflammatory condition produced by Graves’ thyroid or eye disease. The site of the presumed increased production of IL-6 and -8 is most likely from bone osteoblasts, despite the inability of bone markers (such as Udpd and b-ALP) to correlate with acute changes in thyroid hormone status produced by antithyroid therapy.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Candidates for such soluble factors include the osteotropic cytokines.
We have recently demonstrated that human bone marrow stromal cell
cultures, containing osteoblast progenitor cells, release interleukin
(IL)-6 and IL-8 in response to T3 (2). IL-6 regulates
osteoclast proliferation and recruitment, and raised levels are seen in
Paget’s disease, myeloma, and estrogen deficiency (3). IL-8 receptors
are present in osteoclasts (4), and regulatory effects of IL-8 on
osteoclast development and activity have been demonstrated (5).
Cytokines, including IL-1ß, IL-11, and tumor necrosis factor
(TNF)-, are also implicated in bone resorption, particularly that
occurring in high-turnover states, such as rheumatoid arthritis and
after the menopause; evidence for their role in T3-, PTH-,
or 1,25(OH)2D3-associated bone resorption has
been lacking.
Raised levels of serum IL-6 have been demonstrated previously in hyperthyroidism (6, 7, 8), including that caused by Graves’ disease (GD) and toxic nodular goiter (TNG). These elevations have not been consistent, and it has remained difficult to differentiate the contribution of thyroid hormones to this elevation from that of the autoimmune inflammatory process present in GD. To address this question, we studied serum levels of osteotropic cytokines in patients with a hyperthyroidism of differing etiologies. To overcome the possibility that any increase in the levels of the serum cytokines might result from processes related to autoimmune inflammation, we also included a group of patients with iatrogenic subclinical thyroid excess.
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Subjects and Methods |
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Informed consent was obtained, and the study was approved by the East London and City Health Authority Research Ethics Committee. Fifteen normal subjects, 12 women and 3 men, 27–38 yr old, served as controls. Twenty-four patients with overt thyrotoxicosis were studied longitudinally. Fifteen patients (11 premenopausal women, 19–41 yr old; 4 men, 25–38 yr old) had GD, of whom 3 women and 1 man had thyroid-associated opthalmopathy (TAO). Nine patients (all premenopausal women, 25–35 yr old) had a TNG. A diagnosis of thyrotoxicosis was made on the basis of clinical examination, elevated circulating levels of free T4 (FT4) or total T3, and suppressed TSH (< 0.01 mU/L). Patients with GD were identified by the presence of one or more of the following: TAO, positive thyroid antiperoxidase antibodies (1:1600 or greater) and diffuse uptake on thyroid 99-technetium scanning. Conversely, patients with TNG had focal uptake, on 99-technetium scanning, and negative antibodies. Diagnosis of TAO was based on a combination of conjunctival congestion, periorbital edema, and proptosis. No patient had limitation of ocular movement, and none required steroid therapy for eye disease. All patients with thyrotoxicosis were treated with carbimazole (30 mg daily for 6 weeks) followed by titration of the dose of carbimazole according to the biochemical thyroid status. No patient received ß-blocker therapy. During follow-up, 2 women (patients 12 and 17) relapsed between weeks 12–24. The others remained euthyroid on carbimazole.
The remaining 10 patients (9 premenopausal women, 24–43 yr old; 1 man, 45 yr old) had a previous history of thyroid carcinoma (TC), had no evidence of disease recurrence, and were on TSH-suppressive doses of T4. All patients with a history of TC were assessed according to the St. Bartholomew’s protocol (9) and were judged to be free of tumor by: absence of uptake on 131I scanning; serum thyroglobulin measurements of less than 0.6 ng/L, off thyroid hormone replacement; and normal chest radiographs.
Exclusion criteria for all controls and patients included previous thyrotoxicosis, amenorrhea, postmenopausal status, and any other condition of inflammatory or autoimmune etiology.
Methods
For GD and TNG patients, blood was taken just before (week zero)
and 2, 12, and 24 weeks after commencement of carbimazole. For TC
patients, samples were drawn only once. The following measurements were
made in all sera: FT4, total T3, TSH, serum bone-specific
alkaline phosphatase (b-ALP), IL-6, IL-8, IL-11, IL-1ß, and TNF-.
On each occasion, fasting, second void urine specimens were taken for
measurement of deoxypyridinoline (Udpd) and creatinine.
Thyroid function tests were assayed using the Immulite autoanalyzer (Euro/DPC Limited, Gwynedd, UK). Both FT4 and T3 were measured using a solid-phase chemiluminescent assay; and TSH, with a solid-phase two-site chemiluminescent assay. The intra- and interassay coefficients of variation (CVs) were less than 4%. The reference range was 8.8–24 pmol/L for FT4, 0.8–2.5 nmol/L for T3, and 0.4–4.0 mU/L for TSH.
Serum cytokines were assayed by competitive enzyme-linked immunosorbent
assay (Eurogenetics U.K. Ltd, Middlesex, UK) using recombinant human
cytokine as standard (10). These assays measure the total amount of
free and bound cytokine in serum. The lower limit of detection varied
with the cytokine assayed and was 0.2 ng/L for IL-1ß; 0.5 ng/L for
IL-6, IL-8, and TNF-; and 7 ng/L for IL-11. The between-batch CVs of
these assays were less than 13%.
Serum b-ALP was measured by an immunoassay (Metra Biosystems, Mountainview, CA) using a monoclonal anti-b-ALP antibody coated onto a microtiter plate to capture the bone isoform. The enzyme activity of the captured b-ALP is detected with a para-nitrophenyl phosphate substrate. The intra- and interassay CVs were 3.5% and 7.0%, respectively. The reference range was 10.0–22.0 IU/L.
Udpd was assayed by competitive immunoassay using a monoclonal antibody with selective, high affinity against free Udpd and with negligible binding to deoxypyridinoline peptides and free or bound pyridinoline (Metra Biosystems). The intra- and interassay CVs were 3.6–9.5% and 6.3–10.3%, respectively. The reference range was 2.7–6.3 nmol deoxypyridinoline/mmol creatinine.
Statistical analysis
All results are expressed as mean ± SEM. Data were compared with ANOVA, and a P value of less than 0.05 was regarded as significant. Correlations between variables within groups were tested by Spearman’s correlation test, and P less than 0.05 was accepted as significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Elevations in serum FT4 (49.2 ± 1.2 pmol/L, 43.4 ± 1.1 pmol/L) and T3 (5.6 ± 1.6 nmol/L, 5.0 ± 1.1 nmol/L) were similar in GD and TNG patients, respectively, as were elevations in Udpd (12.4 ± 1.1 nmol/mmol creatinine, 11.1 ± 1.3 nmol/mmol creatinine), and b-ALP (22.1 ± 3.5 IU/L, 20.4 ± 2.3 IU/L).
Serum IL-1ß, IL-11, and TNF- were undetectable in both patients
and controls. Mean serum levels of IL-6 (GD, 7.1 ± 1.0 ng/L,
P < 0.01; TNG, 7.3 ± 0.9 ng/L, P
< 0.01) and IL-8 (GD, 10.4 ± 1.0 ng/L, P <
0.01; TNG, 9.8 ± 1.4 ng/L, P < 0.01) were
significantly elevated in both GD and TNG patients, compared with the
controls. Figure 1
shows individual values
from each subject.
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To ensure that GD patients with TAO did not introduce any bias in our calculations, we compared GD patients with and without TAO and found no significant difference in IL-6 (with TAO, 7.0 ± 0.9 ng/L; without TAO, 7.2 ± 0.86 ng/L) and IL-8 (with TAO, 10.5 ± 1.2 ng/L; without TAO, 10.3 ± 1.2 ng/L) levels between them. Similarly, the correlation between FT4, T3 and serum cytokines was maintained if the four patients with TAO were excluded from the analyses.
Changes during treatment of thyrotoxicosis
During the 6 months of treatment of thyrotoxicosis (Fig. 2), b-ALP levels rose, as expected (11), to
peak at 12 weeks (GD, 25.9 ± 1.1, not significant vs.
week 0; TNG, 28.1 ± 1.3, P < 0.01 vs.
week 0) before falling towards control levels. Udpd levels fell in
parallel to serum FT4 and T3 and, coincident with the
attainment of euthyroidism, were within the normal range after 6–8
weeks. Serum IL-6 and IL-8 fell more slowly with treatment, remaining
significantly higher than controls until week 12. In patients 12 and
17, both of whom relapsed between weeks 12–24, serum IL-6 and IL-8
rose significantly, and measurements from week 12 in these two patients
were omitted in subsequent analyses.
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Mean serum levels of FT4 and T3 in TC patients on
T4 were significantly higher than those in controls
(P < 0.05) but significantly lower (P
< 0.05) than those with thyrotoxicosis caused by GD and TNG. These
patients provided an intermediate group between controls and the
overtly thyrotoxic patients, not only in terms of their thyroid hormone
concentrations but also their IL-6 and IL-8 levels (Fig. 1
). Levels of
both cytokines were significantly raised (IL-6, 2.5 ± 0.42 ng/L;
IL-8, 4.4 ± 0.63 ng/L; P < 0.01) above the
control mean while remaining significantly lower (P <
0.001) than the levels seen in GD and TNG patients. Serum IL-1ß,
IL-11, and TNF- were undetectable. As with the other patient groups,
IL-6 and IL-8 levels in TC patients were significantly correlated with
FT4 and T3: IL-6 (FT4 r = 0.74, P <
0.01; T3 r = 0.70, P < 0.05) and IL-8
(FT4 r = 0.71, P < 0.05; T3 r =
0.69, P < 0.05). Despite the raised serum thyroid
hormone and cytokine levels, Udpd (6.6 ± 0.9 nmol/mmol
creatinine) and b-ALP (19.8 ± 1.0 IU/L) concentrations were not
significantly increased, relative to controls, and did not correlate
with either IL-6 or IL-8.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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but have found no abnormalities. The
patient group with exogenous subclinical hyperthyroidism also shows
elevated IL-6 and IL-8, but levels are not as high as in overt
thyrotoxicosis.
Raised levels of IL-6, usually higher than those found in GD and TNG,
have been reported in thyroiditis (12), amiodarone-induced
thyrotoxicosis (13), and after fine-needle aspirations and radioiodine
treatment (14). Conversely, two studies have failed to show a rise in
serum IL-6 in patients with postpartum thyroiditis (15) and
interferon- thyroiditis (16); but nevertheless, serum IL-6 may be
elevated in a variety of thyroid destructive disorders. Previous
studies have tried to address the contribution of autoimmune
inflammation to the rise seen in cytokine levels. Lakatos et
al. (7) found no difference in IL-6 levels in patients with TNGs,
compared with patients with GD; and Salvi et al. (6) found
no difference in the elevations of serum IL-6 seen in patients with and
without TAO. In direct contrast, Molnar et al. (17), who
studied a greater number of patients with more severe eye disease,
found that the presence of TAO significantly increased serum IL-6. Our
work has demonstrated elevated serum levels of IL-6 in clinical and
subclinical hyperthyroidism, with no significant differences seen
between levels in the autoimmune GD or the nonautoimmune TNG. There is
some evidence from Salvi et al. (6) that serum sIL-6R, the
soluble receptor regulating the biological activity of IL-6, correlates
well with serum levels of thyroid hormones and may prove a better
determinant for thyrotoxic bone resorption. Further studies are needed
to confirm this.
We have demonstrated, for the first time, elevated serum IL-8 concentrations in hyperthyroid patients. The concentrations in these subjects were significantly higher than those measured in control subjects, a result consistent with our in vitro findings of elevated IL-8 in the media of T3-stimulated osteoblasts (2). As with IL-6, there seemed to be no difference in serum concentrations of IL-8 between patients with GD and TNG. Previous reports have found elevated IL-8 in the serum of patients with hematological malignancies (18) and in the synovial fluid of patients with rheumatoid arthritis (19), but there is a paucity of data linking it to disorders of thyroid metabolism.
We also screened our patients’ sera for TNF-, IL-1ß, and IL-11
(all cytokines with established inflammatory and osteotropic
properties) but found no evidence, either in this current study or in
our in vitro work (2), linking them to thyroid hormones.
Celik et al. (8) found raised serum TNF- in patients with
GD; but we, like others (20), have been unable to confirm this. Our
patients were premenopausal, with a duration of disease estimated to be
less than 12 months, and discrepancies between reports may lie in
patient selection and chronicity of autoimmune disease.
Serum IL-6 and IL-8 have a number of sources, which include blood mononuclear cells and bone tissue. IL-6 and IL-8 mRNA is also present in thyroid follicles, and increased levels are seen in patients with GD (21). Our study demonstrates elevated levels of IL-6 and IL-8 in TC patients with ablated thyroid glands and therefore suggests that the origin of these elevations is extrathyroidal. The correlation with FT4 further suggests that these elevations are dependent on the severity of hyperthyroidism, rather than its etiology. Though the precise significance of IL-6 and IL-8 in thyrotoxicosis remains unclear, both cytokines are involved in bone metabolism and are released from osteoblasts in vitro after T3 stimulation (2). It is possible that the mechanism for T3-induced osteolysis is similar to postmenopausal osteolysis and closely associated with serum IL-6. Supporting this, there is some suggestion, from previous reports, of an inverse relationship between IL-6 and BMC (7). Although we have been unable to find any correlation between serum cytokines and Udpd in this study, the interaction of T3 with IL-6 and other cytokines should continue to be a likely focus for future investigations.
The normalization of Udpd, which precedes the normalization in serum cytokine concentrations, together with the absence of raised Udpd in TC patients suggests that higher thyroid hormone levels are required to effect a rise in Udpd (and perhaps bone resorption) than in serum cytokines. The continuing elevation of serum cytokines and serum b-ALP, in the absence of raised Udpd levels, suggests on-going bone remodeling after attainment of the euthyroid state. Our work is consistent with several reports that have found no significant elevations in urinary pyridinium cross-links in premenopausal women with primary autoimmune hypothyroidism replaced with L-T4 (22, 23, 24). This is in contrast to earlier work that had suggested increased bone loss (25, 26), but these early studies included postmenopausal women possessing greater susceptibility to osteoporosis.
In summary, we have shown a rise in IL-6 and IL-8 but not in IL-1ß,
IL-11, or TNF- in thyrotoxicosis. We have further shown that their
levels fall as the patients become euthyroid on antithyroid treatment.
There is a close association seen in all patient groups between IL-6,
IL-8, and FT4 and T3; and the elevations which occur in
these cytokines seem independent of autoimmune inflammation. We have
confirmed bone resorption, judged by Udpd, in thyrotoxicosis but have
found no evidence of increased bone resorption in the TC group. We have
found no evidence to show a direct relationship between these
osteotropic cytokines and markers of bone resorption.
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Footnotes |
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Received September 26, 1998.
Revised October 21, 1998.
Accepted October 26, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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therapy. J Invest Med. 44:370–374.[Medline]
in thyroidal and non-thyroidal
illnesses. J Clin Endocrinol Metab. 72:1113–1116.[Abstract]
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, Mean ±
SEM; **, P < 0.001; *,
P < 0.01 vs. C.
) and TNG (•) after treatment of thyrotoxicosis. Upper limit of
normal range (—) in control subjects is shown.