Thyroid associated ophthalmopathy: evidence for CD4(+) gammadelta T cells; de novo differentiation of RFD7(+) macrophages, but not of RFD1(+) dendritic cells; and loss of gammadelta and alphabeta T cell receptor expression

Abstract

Aim: To characterise periorbital immune cells (stages, kinetics) in active and inactive thyroid associated ophthalmopathy (A-TAO; I-TAO).

Methods: In orbital tissue cryosections of patients with A-TAO (n = 15), I-TAO (n = 11), and healthy controls (n = 14), adipose and fibrovascular areas were evaluated for MHC II(+) cells, CD45(+) total leukocytes, myeloid cells (CD33(+) monocytes; CD14(+) macrophages; mature RFD7(+) macrophages; RFD1(+) dendritic cells (DCs)), and lymphoid cells (CD4(+) T cells; alphabeta and gammadelta T cells; CD20(+) B cells). Results are expressed as medians and 5% confidence intervals.

Results: In fibrovascular septae, a surge of CD33(+) immigrants clearly correlating with disease activity generated significantly increased (p<0.05) percentages of CD14(+) and RFD7(+) macrophages. Intriguingly, CD4(+) cells were mostly gammadelta T cells, while alphabeta T helper cells were much less frequent. Successful treatment rendering TAO inactive apparently downregulates monocyte influx, macrophage differentiation, and T cell receptor expression. Similar trends were recorded for adipose tissue. Interestingly, RFD1(+) DCs were completely absent from all conditions examined.

Conclusion: A-TAO coincides with periorbital monocyte infiltration and de novo differentiation of macrophages, but not DCs. The authors discuss a novel potential role for inflammatory CD4(+) gammadelta T cells in TAO. Successful treatment apparently downregulates orbital monocyte recruitment and effects functional T cell knockout.

Figure 1

Representative immunohistochemistry of the leukocyte infiltrate in fibrovascular septae of a patient with active TAO after staining for (A) MHC class II; (B) CD14; (C) RFD7, and (D) CD33. Note the intense staining for MHC class II (covering HLA-DR, DP, DQ, and DX). Areas revealing faint expression suggest that these molecules are increasingly shed in active disease and attach to neighbouring structures. Also note the scattered monocyte and macrophage morphologies displaying CD33, CD14, and RFD7. Original magnification ×400.

Figure 2

Fibrovascular septae in periocular control tissue (see legend to fig 1). Control tissue harboured almost none of the cells detected in A-TAO. Also note that the fibrovascular tissue is much less voluminous than in A-TAO. Original magnification ×400.

Figure 3

Quantification of leukocytes in periorbital fibrovascular septae of (A) control subjects, (B) patients with active TAO, and (C) patients with inactive TAO. Specimens were stained for MHC class II molecules and leukocyte common antigen CD45 (first set of bars); myeloid marker CD33 indicating recently infiltrated monocytes, monocyte and early tissue macrophage marker CD14, and the RFD7 antigen expressed by mature and inflammatory macrophages (second set of bars); as well as CD4⁺ and CD8⁺ T cells, and the T cell receptor variants discriminating αβ and γδ T cells (third set of bars). Dendritic cells (RFD1) or B lymphocytes (CD20) were not detected and have, therefore, been omitted from the graphs. Note that the increase in numbers of cells expressing CD33, CD14 and/or RFD7 correlates significantly with disease activity. This increase is paralleled by a higher percentage of MHC class II⁺ cells. Also note that decreased disease activity, most interestingly, is accompanied by an even higher number of CD4⁺ cells, yet a virtually complete loss of γδ and αβ T cell receptor expression. Data are given as medians with 95% confidence intervals.

Figure 4

Cells in periorbital adipose tissue of (A) controls, (B) patients with A-TAO, and (C) patients with I-TAO. For markers and statistics notes, see legend to figure 3. Control adipose tissue revealed lower leukocyte numbers than control fibrovascular tissue. This is in line with a much less pronounced infiltration of adipose tissue by disease associated myeloid and lymphoid cell types. Nevertheless, TAO associated alterations were qualitatively similar to those observed in the fibrovascular septae, with the important exception of freshly invading CD33⁺ blood monocytes. Also note that, in stark contrast to fibrovascular tissue, decreased disease activity was accompanied by a concomitant decrease in CD4⁺ cells.