Expression of DARC, CXCR3 and CCR5 in giant cell arteritis

H. Brühl, V. Vielhauer, M. Weiss1, M. Mack, D. Schlöndorff and S. Segerer

Medical Policlinic and 1 Institute of Pathology, University of Munich, Munich, Germany.

Correspondence to: H. Brühl, Medical Policlinic, University of Munich, Schillerstrasse 42, 80336 Munich, Germany. E-mail: hilke.bruehl{at}med.uni-muenchen.de


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Objectives. Leucocyte infiltration is the hallmark of vasculitis, chemokines being mainly responsible for leucocyte migration into inflamed tissues. The objective was to evaluate the local expression of chemokines and chemokine receptors in biopsies of patients with giant cell arteritis (GCA) compared with arteries from patients with polymyalgia rheumatica (PMR). We studied the expression of CCR5, CXCR3 and that of the Duffy antigen/receptor of chemokine (DARC), a chemokine internalizing receptor (interceptor), in parallel to the expression of the CCR5 ligand RANTES/CCL5.

Methods. Paraffin-embedded tissue sections from six patients with GCA and five patients with PMR were available for immunohistological analysis of chemokine receptor expression. RANTES/CCL5 mRNA was detected in tissue sections by in situ hybridization.

Results. In patients with biopsy-proven giant cell arteritis, CCR5 and CXCR3 were highly expressed by infiltrating leucocytes in involved tissue sections. Predominant clustering of CCR5+ and CXCR3+ leucocytes was found in the adventitia and was co-localized with the expression of CCL5/RANTES mRNA. Interestingly, we found marked expression of DARC on adventitial high endothelial venules in vasculitis lesions of patients with GCA, while in arteries from patients with PMR DARC was only expressed on a low number of vessels with flat lining endothelium.

Conclusions. The co-localization of infiltrating CCR5+ and CXCR3+ leucocytes together with CCL5/RANTES and DARC in vasculitis lesions suggests a role for these chemokine receptors in leucocyte infiltration, possibly supported by DARC-mediated vascular presentation of chemokines.

KEY WORDS: Giant cell arteritis, Chemokine, Chemokine receptor, DARC, Duffy antigen receptor for chemokines, CCR5, CXCR3, CCL5, RANTES


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The aetiology and mechanisms of vascular injury in giant cell arteritis (GCA) are only partially understood. Chemokine receptors together with adhesion molecules are critically involved in directing the migration of leucocytes into inflamed tissue. CCR5 and CXCR3 are preferentially expressed on T-helper type 1 (Th-1) cells and CD45 RO-positive memory T cells [1]. Chemokines are concentrated locally on the luminal endothelial side by binding to glucosaminoglucans to facilitate the adherence of leucocytes and their subsequent extravasation [2]. The Duffy antigen/receptor for chemokines (DARC) was recently identified as a molecule that could transcytose chemokines such as RANTES (regulated on activation, normal T cell expressed and secreted) from the subendothelial side to the endothelial surface and thus could be involved in the presentation of chemokines. DARC exhibits a broad specificity for chemokines of both the CC and CXC class. CCL5/RANTES, CXCL8/IL-8 and CCL2/MCP-1 demonstrate a particularly high-affinity binding to DARC [3]. Under physiological conditions, DARC expression is restricted to the endothelium of high endothelial venules, which are also the preferential sites of leucocyte extravasation. We therefore investigated the expression of the chemokine receptors CCR5, CXCR3 and DARC in biopsies of patients with GCA and polymyalgia rheumatica (PMR) by immunohistochemistry, and tested for presence of mRNA for the corresponding CCR5 ligand RANTES/CCL5.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Patients
Patients were classified on the basis of temporal artery biopsy and clinical signs and symptoms of PMR and GCA. Patients with PMR met the following criteria: (i) age 50 yr or older; (ii) morning stiffness lasting for 30 min or longer, and bilateral aching persisting for at least 1 month and involving at least two of the following three areas: neck or torso, shoulders or proximal regions of the arms, the hips or proximal aspects of the thighs; (iii) ESR elevated to 40 mm/h or more; (iv) exclusion of patients with any other disease that may explain the above findings (e.g. GCA). All GCA patients met the 1990 ACR criteria [4]. In addition to an age of 50 yr or older and an ESR elevated to 40 mm/h or more, all of the patients with biopsy-proven GCA presented with at least one or a combination of the following clinical symptoms: severe headache, temporal artery tenderness, jaw pain or visual loss. All of the PMR and GCA patients in the study received steroids (prednisolone 0.5–1 mg/kg) prior to the biopsy. The mean duration of steroid therapy before temporal artery biopsy was 3.2 days (range=1–7 days) for PMR patients and 3.8 days (1–7 days) for GCA patients. Blood samples for Fluorescence Activated Cell Sorter analysis were taken separately before the initiation of steroid therapy. Informed consent was given by all of the patients. The study was performed according to the institutional ethical standards and in accordance with the convention of the declaration of Helsinki.

Methods
The monoclonal antibodies against CCR5 (MC1, MC5), DARC (2C3) and CXCR3 (1C6, BD Biosciences Pharmingen) have been described in detail previously [5, 6]. The following reagents were purchased: anti-human CXCR3 (IC6), anti-CD4 FITC (fluorescein isothiocyanate)-conjugated, anti-CD8 Cy5-conjugated and anti-CD14 APC (allophy cocyamine)-conjugated (Pharmingen, Heidelberg, Germany); isotype control mouse immunoglobulin (Ig) G-1 and IgG-2a (Sigma, Munich, Germany); PE (phycoerythin)-conjugated rabbit anti-mouse (R0439) and anti-CD68 (Dako, Hamburg, Germany), anti-CD3 (Santa Cruz Biotechnology, USA), anti-mouse biotinylated IgG (Vector, Burlingame, USA).

Immunohistochemistry was performed on sections of paraffin-embedded material as previously described, using a commercial biotin–streptavidin method [7]. DARC-positive vessels were counted in each histological specimen for each cross-section. For statistical analysis, the Mann–Whitney test was applied. In situ hybridization was performed as previously described [8]. The 359 bp RANTES probe corresponds to nucleotides 75–433 (GenBank accession number M21121) of the respective cDNA sequence. FACS analysis of peripheral blood leucocytes was performed as previously described [9].


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Expression of the chemokine receptors CCR5, CXCR3 and DARC in inflamed temporal arteries of patients with GCA
Consecutive sections of six patients with biopsy-proven GCA and five cases of PMR were available for histological analysis. Biopsies of patients with temporal arteritis were characterized by transmural inflammation and accentuated infiltration of inflammatory cells in the media and adventitial tissue (Fig. 1D–I). Strong expression of CXCR3 and CCR5 was found on these infiltrating cells. At higher magnification, this cell population consisted of small round cells consistent with lymphocytes (Fig. 1G and H). Staining for T-cell (anti-CD3) and monocyte/macrophage (anti-CD68) markers in serial sections revealed that the distribution of CXCR3+ cells correlated to the distribution of T cells. Similarly, CCR5 was mostly expressed by T cells. Only single macrophages/monocytes were considered to be CCR5+ (data not shown). A high number of DARC-positive vessels was found in the adventitia close to the inflamed arteries (Figs 1F, I and 2C). The number of DARC-positive vessels was significantly higher in patients with GCA compared with PMR patients (mean 40.5, S.E.M. 5.7 vs mean 14.9, S.E.M. 2.2, P = 0.0001). In contrast to PMR, in GCA, DARC-positive vessels were commonly lined by a prominently high endothelium. One biopsy in the GCA series contained a lymph follicle close to the inflamed artery, which consisted of a significant number of CXCR3+ cells and a lesser number of CCR5+ cells (Fig. 2A and B). Vessels with a prominently DARC-positive endothelium were localized in this area (Fig. 2C).



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FIG. 1. Immunohistochemistry performed on consecutive sections from a temporal artery biopsy of a patient with PMR (A–C) and of a patient with GCA (D–I). Slides were stained for CCR5 (A, D and G), CXCR3 (B, E and H) and DARC (C, F and I). Note the absence of colour-product-positive cells in the PMR biopsy. A high number of CCR5 (D and G) and CXCR3+ cells (E and H) were found in the vasculitis lesion. The DARC-positive vessel has a high endothelium in I. Original magnifications are 100x in A–F and 400x in G–I.

 



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FIG. 2. Staining of consecutive sections from the temporal artery of a patient with GCA for CCR5 (A), CXCR3 (B) and DARC (C). The pictures illustrate a lymph follicle next to an inflamed artery with a significant number of CXCR3+ cells and DARC-positive vessels and a lesser number of CCR5+ cells. The DARC-positive vessels are arranged around but not within the nodular infiltrate. (D–G) In situ hybridization performed on temporal artery biopsies of a patient with GCA. Hybridization with the sense probe demonstrates weak, diffuse background deposition of silver grains without accentuation of deposition over single cells (D and F). Hybridization with the CCL5/RANTES antisense probe demonstrates strong expression, particularly in the perivascular area, matching the distribution of CCR5+ cells (E and G).

 
Biopsies from patients with PMR did not contain significant numbers of infiltrating leucocytes or chemokine receptor-expressing cells (Fig. 1A–C).

Co-localization of RANTES with infiltrating leucocytes in the adventitia of inflamed arteries
In vasculitis lesions, strong expression of the CCR5 ligand RANTES was found over cells in the adventitia by in situ hybridization, which matched the distribution of infiltrating leucocytes (Fig. 2E and G). The distribution of RANTES/CCL5 mirrored the distribution of CCR5+ cells. In addition, both RANTES RNA and CCR5+ cells were localized in close proximity to DARC-positive endothelium. Sense controls did not show significant deposition of silver grains over single cells (Fig. 2D and F).

Expression of CCR5 by monocytes and T cells in the peripheral blood of patients with PMR and GCA
To investigate whether the accumulation of CCR5+ T cells and monocytes/macrophages at the site of vasculitis is paralleled by increased expression on circulating leucocytes in the peripheral blood, we analysed the blood leucocytes of patients with GCA and PMR by flow cytometry before the initiation of treatment with steroids. No differences were found between the percentage of CCR5+CD4+ or CCR5+CD8+ T cells in GCA compared with PMR (groups of four patients each). The mean percentage of CCR5+CD4+ T cells (15% GCA, 12% PMR) and CCR5+CD8+ T cells (49% GCA, 47% PMR) fits into the expected range for healthy blood donors [10, 11]. The mean percentage of circulating CCR5+ monocytes/macrophages tended to be lower in patients with GCA (6%) compared with PMR (24%), without reaching statistical difference.


    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
We found that in biopsy-proven GCA, CCR5 and CXCR3 are highly expressed on infiltrating leucocytes in the media and adventitial tissue. We also document the co-localization of CCL5/RANTES mRNA, a ligand of CCR5, with CCR5+ cells, supporting the idea of a chemokine–receptor interaction during the process of extravasation into the temporal artery. The high expression of CXCR3 and CCR5 on infiltrating lymphocytes is in accordance with data showing a predominant Th-1 response in GCA, with activated CD4+ T cells in the adventitia expressing the Th-1 cytokines IFN-{gamma} and IL-2. In contrast, temporal arteries from patients with PMR contain transcripts for IL-2, IL-1 and TGF-ß but lack IFN-{gamma}, suggesting a critical role of the latter cytokine in the manifestation of vasculitis lesions [12, 13]. The ligands of the chemokine receptors CXCR3, CXCL9/monokine induced by {gamma}-interferon (MIG), CXCL10/{gamma}-interferon inducible protein-10 (IP10) and CXCL11/interferon induced T cell {alpha}-chemoattractant (I-TAC) are unique in that they are all induced by IFN-{gamma} in a wide variety of cell types, including endothelial cells [14]. One could therefore speculate that the observed infiltration of CXCR3+ lymphocytes in GCA could have been triggered by IFN-{gamma}-induced chemokines.

In contrast to PMR, in temporal arteritis, DARC was highly expressed on adventitial and periadventitial vessels. These vessels predominantly displayed high endothelial lining. In one involved section, we found an adventitially localized lymph follicle consisting of CXCR3+ and CCR5+ leucocytes that was surrounded by multiple DARC-positive high endothelial venules. DARC is expressed on high endothelial cells of postcapillary venules in the kidney, lung, thyroid and spleen, but not on endothelial cells of arterioles and arteries [15]. Thus, DARC is preferentially expressed in those segments of the circulation where leucocyte extravasation occurs. Recent studies demonstrated up-regulation of DARC on interstitial peritubular capillaries in the kidney during transplant rejection and crescentic glomerulonephritis and in children with HIV-associated renal diseases [7, 16, 17]. DARC-deficient mice show altered leucocyte recruitment compared with wild-type mice after challenge with lipopolysaccharide and thioglycollate [18]. DARC has no known signalling mechanism [19], but is able to facilitate the presentation of chemokines on endothelial cells and therefore appears to play an important role in inflammation [20]. In summary, the co-localization of DARC-positive high endothelial venules with CXCR3+ and CCR5+ leucocytes and CCL5/RANTES opens the possibility that chemokine presentation by DARC might favour the migration of leucocytes into the inflamed temporal artery of patients with GCA.


    Acknowledgments
 
We thank Jean Pierre Cartron and Yves Colin (Inserm, Laboratoire U76, Paris, France) for kindly providing the anti-DARC antibody (2C3), and Isabell Wenzel for excellent technical assistance. This work was supported by a grant of the Deutsche Forschungsgemeinschaft BR2139 to H.B.; S.S. is supported by the Else-Kröner Fresenius Stiftung, Bad Homburg i. d. S.

No conflict of interest has been declared by the authors.


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Submitted 5 June 2004; revised version accepted 19 October 2004.



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