Glucocorticoid Effects on Tissue Residing Immune Cells in Giant Cell Arteritis: Importance of GM-CSF

Abstract

Giant cell arteritis (GCA) is a systemic granulomatous vasculitis clinically characterized by a prompt response to glucocorticoid therapy. Dendritic cells (DCs) play a central role in the pathogenesis of the disease and are increased in temporal arteries from GCA patients. The aim of this study was to determine the effects of glucocorticoid therapy on granulomatous infiltrates and on peripheral DCs of GCA patients. Immunohistochemical staining of temporal artery specimens from 41 GCA patients revealed a rapid reduction of the number of DCs after initiation of glucocorticoid treatment. TUNEL staining was performed to quantify apoptotic S100+ DC, CD3+ T cells, and CD68+ macrophages in the granulomatous infiltrates. An increase of apoptotic cells up to 9 ± 2% after 4-5 days of glucocorticoid therapy and up to 27 ± 5% (p < 0.001, compared to earlier timepoints) after 6-10 days was detected. A decrease of CCL19 and CCL21 expression was observed after starting glucocorticoid therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression also significantly decreased under glucocorticoid therapy. No GM-CSF expression was detected in the control specimens. Glucocorticoid therapy leads to a rapid, time-dependent reduction of DCs in temporal arteries from GCA patients and reduction of mediators for cell migration. Our data suggest GM-CSF as a novel therapeutic target of GCA.

Keywords: GM-CSF; apoptosis; dendritic cells; giant cell arteritis; glucocorticoids.

Figure 1

Reduction of DC under glucocorticoid therapy. (A) Temporal artery biopsy sections from three individual patients with GCA. Cross sections from a newly diagnosed untreated GCA patient are shown in column I, sections of a GCA patient after 2 days of glucocorticoid therapy are depicted in column II and tissue sections of a GCA patient 5 days after the initiation of glucocorticoid treatment are demonstrated in column III. Cross sections were stained with anti-MHC II mAb (row 1, 2), anti-S100 Ab (row 3), and anti-CD163 mAb (row 4). Cells positive for the particular epitope are indicated by red stain. Nuclei were stained with haematoxylin and eosin. Scale bar = 500 μm in row 1, scale bar = 50 μm in row 2–4. (B) Number of S100+ DC in 75 paraffin-embedded consecutive temporal artery sections from 41 GCA patients and 11 control patients. For each patient and control patient, S100+ cells were counted in three different temporal artery sections at 40 × magnification. Patients were grouped depending on the duration of treatment (0, 1, 2–4, 5–10, >10, controls).

Figure 2

Glucocorticoid therapy induces apoptosis. (A) DNA fragmentation of apoptotic cells (brown stain) is demonstrated by applying TUNEL assay on temporal artery sections of five GCA patients. Nuclei were stained with haematoxylin and eosin. Scale bar = 50 μm. (B) The number of TUNEL-positive cells was quantified in 31 paraffin-embedded consecutive temporal artery specimens. For each patient and control patient, TUNEL-positive cells and nuclei of all cells in four random fields (magnification 100 × ) of each specimen were counted. The percentages of TUNEL-positive cells of all cells in the samples were calculated. (C) Demonstration of intracellular DNA fragmentation in CD3+ cells (i) and CD68+ cells (ii) in a temporal artery biopsy specimen from a patient with GCA 5 days after glucocorticoid treatment. T cells and macrophages are stained with anti-CD3 mAb and anti-CD68 mAb (green fluorescene), respectively. Applying the ApopTag Red Kit, rhodamine-labeled DNA fragmentation (red fluorescene) was identified. Scale bar = 20 μm. (D) Demonstration of intracellular DNA fragmentation in tissue-residing DC in a temporal artery biopsy specimen from a patient with GCA 5 days after glucocorticoid treatment. Confocal microscopy reveals rhodamine-labeled DNA fragmentation (red fluorescence) within DC, that is stained with anti-S100 Ab (green fluorescence). Scale bar = 50 μm.

Figure 3

Reduction of CCL19 and CCL21 expression under glucocorticoid therapy. Temporal artery sections of patients were stained with (A) anti-CCL19 mAb and (B) anti-CCL21 mAB, respectively, and numbers of positive cells were determined in three sections. For each patient and control patient, positive cells were counted in three different temporal artery sections.

Figure 4

GM-CSF expression is down-regulated under glucocorticoid therapy. GM-CSF expression was determined on mRNA level by in situ hybridization (black bars) and on protein level by immunohistochemistry using anti-GM-CSF mAb (gray bars). Temporal artery biopsy specimens were obtained from GCA patients without glucocorticoid treatment (0 days; n = 3), or after 1 day of therapy (n = 10), or after 2 days of therapy (n = 9). For each patient GM-CSF+ cells were counted in two different temporal artery sections at 40 × magnification.