High expression of IP-10/CXCL10 and Mig/CXCL9 mRNA in thyroids of patients with recent-onset GD. A: High expression of IP-10/CXCL10 mRNA levels in the thyroid of patients with GD in comparison with patients with TA (P < 0.05) or normal thyroids (P < 0.01). B: High expression of Mig/CXCL9 mRNA levels in the thyroid of patients with GD in comparison with patients with TAs (P < 0.05) or normal thyroids (P < 0.005). The difference in the levels of both IP-10/CXCL10 and Mig/CXCL9 mRNA expression between normal thyroids and TAs, as determined by quantitative image analysis, was not significant. C: Significant differences in IP-10/CXCL10 and Mig/CXCL9 mRNA expression in GD-affected patients in relation to recent or late onset (P < 0.05 and P < 0.005 for IP-10/CXCL10 and Mig/CXCL9, respectively). Quantitative evaluation of IP-10/CXCL10 and Mig/CXCL9 mRNA was performed by means of a computerized video image analysis system. Results are expressed as medians and percentiles.

IP-10/CXCL10 and Mig/CXCL9 protein expression and localization in thyroid tissue specimens from patients with GD. A: High immunoreactivity for IP-10/CXCL10 protein in the thyroid tissue specimen from the same patient with GD shown in Figure 1 ▶ (red color). Inset: High-power magnification of a portion of the same section shown in A. B: Absence of staining for IP-10/CXCL10 when immunohistochemical staining was performed after preadsorption with 100 μg/ml of IP-10/CXCL10 in the same tissue specimen shown in A. C: Double immunostaining for IP-10/CXCL10 (red) and TSH receptor (bluish gray) in the thyroid follicular epithelium from the same patient with GD. No counterstain was applied. D: Double immunostaining for IP-10/CXCL10 (red) and CD3 (bluish gray) in many mononuclear infiltrating cells in the thyroid tissue specimen from the same patient with GD. No counterstain was applied. Arrows point to cells expressing only IP-10/CXCL10. E: Double immunostaining for IP-10 (red) and CD68 (bluish gray) in many mononuclear infiltrating cells in the thyroid tissue specimen from the same patient with GD. No counterstain was applied. Arrows point to cells expressing only IP-10/CXCL10. F: High immunoreactivity for Mig/CXCL9 protein in the thyroid tissue specimen from the same patient with GD (red). Inset: High-power magnification of a portion of the same section shown in F. G: Double immunostaining for Mig/CXCL9 (red) and TSH receptor (bluish gray) in the thyroid follicular epithelium from the same patient with GD (arrows). No counterstain was applied. H: A section from the same patient was stained with the anti-TSHr and revealed using vector SG as a peroxidase substrate. I: Double immunostaining for Mig/CXCL9 (red) and CD3 (bluish gray) in many mononuclear infiltrating cells in the thyroid tissue specimen from the same patient with GD. No counterstain was applied. Arrows point to cells expressing only Mig/CXCL9. J: Double immunostaining for Mig/CXCL9 (red) and CD68 (bluish gray) in many mononuclear infiltrating cells in the thyroid tissue specimen from the same patient with GD. No counterstain was applied. Arrows point to cells expressing only Mig/CXCL9. Original magnifications: ×400 (A–J); ×1000 (insets in A and F).

IP-10/CXCL10 serum levels are related with disease duration in patients affected by GD. A: Mean ± SD. IP-10/CXCL10 serum levels in GD are significantly higher (P < 0.05) in comparison with age- and sex-matched healthy patients, although in a large number of GD IP-10/CXCL10 levels overlaps with controls with high SD. B: Analysis of IP-10/CXCL10 serum levels in patients with different duration of GD. IP-10/CXCL10 serum levels are inversely correlated with the duration of the disease, the highest levels being a property of patients with recent-onset GD (R² = 0.146, P < 0.05). C: Analysis of MDC/IP-10 ratio serum levels in patients with different duration of GD. The ratio between MDC/CCL22 and IP-10/CXCL10 serum levels is strictly correlated with the duration of the disease, the highest levels being a property of patients with late-onset GD (R² = 0.2198, P < 0.005).

IP-10/CXCL10 and Mig/CXCL9 expression in thyroid tissue specimens from patients with GD. A: Section of normal thyroid hybridized with anti-sense IP-10/CXCL10 mRNA probe showing virtually no signal. B: Section of a TA hybridized with anti-sense IP-10/CXCL10 mRNA probe showing virtually no signal. C: High signal in the thyroid specimen from a patient with GD hybridized with anti-sense IP-10/CXCL10 mRNA probe. D: Absence of signal in a consecutive section hybridized with a sense IP-10/CXCL10 mRNA probe. E: High-power magnification of the thyroid follicular epithelium from the same patient with GD hybridized with anti-sense IP-10/CXCL10 mRNA probe. F: Section of normal thyroid hybridized with anti-sense Mig/CXCL9 mRNA probe showing virtually no signal. G: Section of a TA hybridized with anti-sense Mig/CXCL9 mRNA probe showing virtually no signal. H: High signal in the thyroid specimen from a patient with GD hybridized with anti-sense Mig/CXCL9 mRNA probe. I: High-power magnification of the thyroid follicular epithelium from the same patient with GD hybridized with anti-sense Mig/CXCL9 mRNA probe. Dark-field original magnifications: ×100 (A–D, F–H); ×1000 (E, I).

IFN-γ mRNA levels are related to IP-10/CXCL10, Mig/CXCL9, and disease duration in thyroids of patients affected by GD. IFN-γ mRNA levels are strictly associated with IP-10/CXCL10 (A) and Mig/CXCL9 (B) mRNA expression (R² = 0715, P < 0.001; R² = 0376, P < 0.01). C: Statistically significant difference in IFN-γ mRNA levels in GD in relation to recent or late onset (P < 0.01).

CXCR3 expression and distribution in thyroid tissue specimens from patients affected by GD. A: High CXCR3 expression in the thyroid of the same patient shown in Figures 1 and 2 ▶ ▶ affected by GD (red color). Section was counterstained with Gill’s hematoxylin. B: Absence of CXCR3 signal in an adjacent section immunostained with an isotype control Ab with irrelevant specificity. C: Higher power magnification showing CXCR3 expression in infiltrating inflammatory cells (red color) in the thyroid of the same patient affected by GD. Section was counterstained with Gill’s hematoxylin. D: Higher power magnification showing CXCR3 expression in endothelial cells (red color) in the thyroid of the same patient affected by GD. Section was counterstained with Gill’s hematoxylin. E: Double immunostaining for CXCR3 (red color) and CD4 (blue color) in the thyroid of the same patient affected by GD demonstrating CXCR3 expression by infiltrating T cells of the CD4 phenotype. No counterstain was applied. F: Double immunostaining for CXCR3 (red color) and CD8 (blue color) in the thyroid of the same patient affected by GD demonstrating CXCR3 expression by infiltrating T cells of the CD8 phenotype. No counterstain was applied. G: Double immunostaining for CXCR3 (red color) and CD68 (blue color) in the thyroid of the same patient affected by GD demonstrating CXCR3 expression by infiltrating monocytes/macrophages. No counterstain was applied. H: Double immunostaining for CXCR3 (red color) and CD11c (blue color) in the thyroid of the same patient affected by GD demonstrating CXCR3 expression by infiltrating dendritic cells. No counterstain was applied. I: Double immunostaining for CXCR3 (red color) and CD83 (blue color) in the thyroid of the same patient affected by GD demonstrating CXCR3 expression by infiltrating dendritic cells. No counterstain was applied. Original magnifications: ×100 (A–B); ×400 (C–I).