An immunohistochemical analysis of folate receptor beta expression and distribution in giant cell arteritis - a pilot study

Abstract

Background: Giant cell arteritis (GCA) is a chronic vasculitis of large and medium vessels in which no targetable biomarkers exist to allow selective treatment, predict disease activity and monitor therapeutic responses. The accessibility of the temporal artery (TA) for biopsy allows morphologic studies to characterize macrophages and T cells in the microenvironment of the arterial wall. We evaluated the expression of folate receptor beta (FRB), a candidate diagnostic/therapeutic biomarker, compared its expression with key macrophage markers and correlated it with GCA severity.

Methods: Formalin-fixed paraffin-embedded tissue sections were examined from 6 patients with GCA and 2 controls. Immunohistochemistry was performed using FRB, ETB, CD68 and CD3 antibodies to evaluate for activated macrophages and T cells, assess FRB distribution along the intima, media and adventitial layers and composition of inflammatory infiltrates. We compared the expression of FRB, ETB and CD68 in GCA versus negative controls and in severe (with visual loss) versus mild (without visual loss) disease.

Results: In GCA, moderate to severe inflammation was accompanied by >90% destruction of the internal elastic lamina. Macrophages comprised 36.3 ± 4.1% while CD3+ lymphocytes accounted for 61.7 ± 4.1% of total leukocytes. FRB was selectively expressed in macrophages and localized to the adventitia. GCA patients had marginally increased median FRB (9.8 cells/hpf vs. 0; p=0.095), ETB (20.5 vs. 0; p=0.095) and CD68 (38.8 vs. 5; p=0.071) expression versus controls. ETB was found in endothelial cells, smooth muscle cells and macrophages in intima/media. FRB positively correlated with ETB (r=0.90; p-0.037) and CD68 levels (r=0.90; p=0.037). ETB expression positively correlated with CD68 (r=1.0; p<0.0001). There was no difference in FRB between severe and mild GCA.

Conclusion: FRB is a potential diagnostic and therapeutic biomarker with restricted expression in GCA macrophages. FRB+ macrophages localized to the adventitia and their expression correlated with ETB and CD68 macrophages, suggesting that they contribute to GCA pathogenesis.

Keywords: Giant cell arteritis; folate receptor beta; macrophages.

Figure 1

Comparison of normal versus GCA affected temporal arteries. Representative H and E images of a normal temporal artery with minimal immune cells (A) and GCA positive temporal arteries (B, C) with granulomatous infiltrates spanning the hyperplastic intima, media and adventitia. Arrow demonstrates fragments of internal elastic lamina. Arrowheads show 2 multinucleated giant cells. Magnification ×200 in (A); ×400 in (B); ×600 in (C).

Figure 2

FRB+, ETB+ and CD68+ macrophage expression (median) in immunohistochemical stains of temporal artery biopsies in GCA (in blue) vs. controls (in orange).

Figure 3

Differential distribution of FRB and ETB macrophage subtypes. Paraffin embedded tissue sections of temporal arteries were stained with the following antibodies: anti-FRB which localized primarily to adventita > media (A), anti-ETB to the intima and media (B) and anti-CD68 pan macrophage marker to all 3 layers (C). Magnification ×200.

Figure 4

Correlation of macrophage subtype expression in GCA patients. (A) Folate vs. CD68 (r=0.90; p=0.037) (B) Folate vs. ETB (r=0.90; p=0.037) (C) ETB vs. CD68 (r=1.0; p<0.0001).