An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Summary

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

Abstract

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.

Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.

Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.

Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.

This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

Introduction

Giant cell arteritis (GCA) is an inflammatory disease of medium-to-large arteries, targeting the aorta and its branches and affecting older adults. It presents with mild to severe ischemic complications such as headaches, jaw pain, vision loss, stroke and tissue gangrene. The diagnosis is confirmed by high inflammatory markers like erythrocyte sedimentation rate (ESR) and a distinct histopathologic pattern on the temporal artery biopsy (TAB)¹. GCA is the most common adult vasculitis, and the accessibility of the temporal artery obtained for diagnosis presents an advantage over other vasculopathies, thus enabling one to study its pathogenesis mo....

Protocol

All methods described were approved by the Penn State Institutional Review Board.

1. Histopathological Preparation, and Processing After Obtaining Temporal Artery Biopsy

NOTE: The temporal artery biopsy (TAB) is performed under local anesthesia and sterile conditions by a certified surgeon. After surgically obtaining a 3 cm arterial section on the more affected side, the specimen is fixed in formalin immediately for 24 h, divided into 3 to 4 mm s.......

Discussion

GCA is the most common vasculitis in adults, and its pathologic hallmark exemplifies a potent combination of T helper lymphocytes and activated macrophages that can also be found in other granulomatous diseases or vasculopathies like sarcoidosis and granulomatous polyangiitis^2,3. In GCA, the temporal artery provides a good representation of a medium-to-large sized artery and the TA biopsy gives a sizeable sample that can be stored in paraffin blocks for years, th.......

Materials

Name	Company	Catalog Number	Comments
Microtome	Reichert-Jung
plain and frosted microscope slides and cover slips	Fisher Scientific
Vertical rack	Fisher Scientific
Light microscope	Olympus BX60  microscope
Xylene
Acetone in distilled water			concentrations 100%, 90%, 70%
Tris-buffered saline solution (TBS)	Dako Wash BufferS3006
3% hydrogen peroxide in TBS
Diaminobenzidine (DAB)	Sigma Fast Tablet set
Chemical Permount  Mounting Medium	Fisher Scientific	SP-15-100
Harleco Gill's III Hematoxylin	Fiisher Scientific 23-750-019
Harleco Eosin Y 1% Alcoholic Stock Solution	Fisher Scientific	23-749-977
10 mM citric acid monohydrate (pH 6.0)
Polyclonal rabbit anti-folate receptor beta	Manohar Ratnam		optimized at 1:800
Monoclonal mouse anti-human CD68	Dako	M071801	optimized at 1:200
Polyclonal rabbit anti-CD3	Agilent Dako	A0452	optimized at 1:100
Polymer goat anti- rabbit/mouse secondary antibody	Dako
Placental tissue			for FRB positive control