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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 more readily. The typical findings on TAB include an infiltrate of macrophages/histiocytes and T lymphocytes found across all vascular layers of the tunica intima, media, and adventitia with concurrent destruction of the elastic lamina that normally separates these compartments². The current evidence demonstrates that GCA involves an unknown antigen that activates dendritic cells in the vascular adventitia, followed by the recruitment of helper T (Th) cells, specifically Th1 and Th17 subtypes which secrete interleukin-17 and interferon-gamma (IFG) respectively. IFG then recruits and activates macrophages to produce cytokines and proteases. These include pro-inflammatory cytokines; including IL-12, which reciprocally activates Th1 cells thus providing a positive feedback loop; tumor necrosis factor and interleukin-6, which cause arthritis and fevers and metalloproteases that damage the elastic lamina. Glucocorticoids (GC), which constitute the standard chronic therapy, partially control the cytokine pathways by attenuating the Th17/IL-17 but not the Th1/IFG arm of the immune response^3,4. Unfortunately, discontinuation of GC results in disease relapse. As an alternative modality, methotrexate has been repurposed for GCA treatment, but the desired clinical endpoints were not consistently achieved although more sensitive biomarkers have not been utilized for therapeutic monitoring^5,6. In 2017, the interleukin 6 blocker, tocilizumab, was approved for the treatment of GCA as it effectively demonstrated disease control and steroid sparing effects^7,8.

To date, there are no good biomarkers for GCA. As a result, it is difficult to monitor disease activity and titrate doses of available drugs to avoid adverse effects and reduce the burden of cost to society. Thus, it is imperative to look for a candidate biomarker that correlates with disease activity, provide novel insights on pathogenesis and aid in therapeutic decisions.

The dysregulation of macrophage activation is a critical factor in GCA pathogenesis. Thus, an effective means of treating GCA may be the selective targeting of activated macrophages. The folate receptor beta (FRB) is a glycosyl-phosphatidylinositol glycoprotein that is anchored on the cell membrane expressed in normal myelomonocytic cells, myeloid leukemia cells and activated macrophages. Its ligand, folic acid, is an essential vitamin in its reduced form, which enables cellular DNA synthesis, methylation and repair⁹. FRB expression is induced in autoimmune disease and during carcinogenesis. Its expression is restricted to the surface of monocytes or pro-inflammatory M1 macrophages in rheumatoid arthritis, or to anti-inflammatory M2 macrophages in solid organ and myeloid malignancies^10,11,12,13. In addition to its potential role as a biomarker of activated macrophages, the FRB can enable selective drug transport thru a multi-step process that starts with the receptor binding to folic acid, antifolates or folate-conjugated drugs at the cell surface, followed by internalization, release and eventual delivery of desired drug to the internal cell machinery^12,14,15,16. In contrast to FRB expressed in cancer cells and activated macrophages, FRB expressed in normal hematopoietic cells is unable to bind folates thereby ensuring that FRB/folate-mediated drug delivery is limited to FRB-positive inflammatory or cancer cells.

In our previous study, we demonstrated for the first time that FRB is expressed in GCA macrophages and its expression correlated with CD68+ and endothelin receptor beta macrophages, which contribute to GCA pathogenesis¹⁷. In this report, we describe the histopathological and immunohistochemical methods used to assess the FRB macrophage, and analyzed the total lymphocyte and macrophage counts to gain insight into the FRB's position in the GCA vascular landscape.

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Protokół

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 sections, and embedded in paraffin, paying careful attention to the proper alignment of tissue in the block which is then  stored at room temperature. Specimen selection is performed after verifying the medical records. The diagnosis is based on the American College of Rheumatology 1990 criteria which requires that subjects fulfill 3 of 5 domains: age >50 years, new headache, temporal artery tenderness, an erythrocyte sedimentation rate >50 mm/hour by Westergren method and an abnormal TAB. For safety, protective gloves must be worn at all times when handling the specimens, chemicals and antibodies.

1. From the embedded blocks, cut 4-5 µm thick sections using a microtome. and stain with Hematoxylin and Eosin (H&E). Use a control section of selected normal tissue for quality assurance.
2. Float cut sections on a warm water bath heated to 40 °C to remove wrinkles and pick up with a coated glass microscopic slide.  Place the glass slides on a warm plate in a 60 °C oven for 60 minutes to facilitate drying and adherence of section to the slide.
3. Place the glass slides on a warm plate in a 60 °C oven for 60 minutes to facilitate drying and adherence of section to the slide.
4. Mount 3 sections on microscope slides.
5. Place slides in a vertical rack and dry overnight at 37 °C for 24 h.
6. Place slides in a container and store at 4 °C.

2. Immunohistochemical Preparation, Dewaxing, Antigen Retrieval and Staining^15,18,19,20

1. Load slides into a slide rack. Run the rack through dishes as follows: Twice in 100% xylene for 5 minutes with 10 seconds of gentle agitation every 30 seconds, once in 100% ethanol with 10 seconds agitation, once in 90% ethanol with 10 seconds agitation, once in 70% ethanol with 10 seconds agitation, and twice in double distilled H2O with 10 seconds agitation. 
   Note: Handling of xylene and acetone should be done in ventilated hoods to avoid inhalation and respiratory toxicity.
2. Retrieve the antigen by transferring the rack into a glass container with 200 mL of 10 mmol/L, pH 6.0 citrate buffer prewarmed to 95 °C. Set the timer for 30 minutes in the water bath, then cool in room temperature for 20 minutes then rinse with running water for 5 minutes.
3. Remove endogenous peroxidase activity by incubating in 200 mL of 3% hydrogen peroxide for 10 minutes. Wash slides 3 times in 200 µL of Tris-buffered saline solution (TBSS).
4. Remove the slides from the rack and dab each one gently to wipe excess buffer. For staining, add 200 µL of the following primary antibodies, add cover slips on slides and incubate in a humid chamber for 1 hour at room temperature:
   Anti-FRB antibody diluted 1:800
   Monoclonal mouse anti-human CD68, 1:200 dilution
   Polyclonal rabbit anti-CD3 1:100 dilution
   Note: Formalin-fixed paraffin-embedded sections of placenta were also processed as outlined in Steps 2.1 to 2.9 and incubated with anti-FRB18 and used as positive control. Staining results were obtained by finding the optimal antibody concentration and was performed by titrating the antibody in double dilutions (e.g. 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600).
5. Remove cover slip after incubation and discard. Rinse slides 3 times for 2 minutes each with TBSS, drain and wipe excess buffer.
6. Add 200 µL of secondary antibody solution containing biotinylated Anti-Rabbit/Mouse secondary antibody to react with unconjugated primary antibody bound to tissue antigen. Place coverslips on slides and incubate in a humid chamber for 45 minutes at room temperature.
7. Remove cover slip after incubation and discard. Rinse slides 3 times for 2 minutes each with TBS, drain and wipe excess buffer.
8. Add 200 µL of diaminobenzidine (DAB)+substrate buffer (Imidazole HCl) -chromogen system and incubate for 10 minutes to visualize staining pattern. Wash with TBSS then in running tap water for 10 minutes.
9. Counterstain with hematoxylin for 3 minutes.
10. Mount the slides by applying 70 µL of mounting medium to the surface of the slide. Slowly tip the coverslip onto the mounting medium and avoid creating bubbles as you lower it into place. Wait 24 hours to dry. 

3. Histopathologic and Immunohistochemical (IHC) Analysis

Examine the H&E and IHC stained sections from GCA positive and normal subjects using a light microscope.

1. For the H&E stained sections:
   1. Assess the vascular architecture and identify endothelial cells in the tunica intima, smooth muscle cells in the tunica media, and fibroblasts and vasa vasorum in the tunica adventitia^21,22.
   2. Identify GCA features, which include lymphocyte and macrophage/histiocyte infiltration, cellular hyperplasia, and internal elastic lamina degradation.
   3. Analyze the lymphohistiocytic infiltrate by identifying and quantifying the number of macrophages relative to the lymphocytes. Assess the extent of internal elastic lamina disruption and hyperplastic changes in resident cells and compare with controls.
2. For the IHC stained sections:
   1. Examine the staining pattern of FRB, taking note of its expression by a particular type or types of cells and its distribution within the vascular layers and its relationship to the total immune infiltrate, the total macrophage marker, anti-CD68, and the pan lymphocyte marker anti CD3.
   2. Quantify the expression of FRB, CD68 and CD3 cells by counting the positively stained cells on 10 multiple randomly selected high power fields (hpf) and record the means.
      NOTE: Identification of these normal and pathologic cells and structures were performed by a certified cardiovascular pathologist (J.M.) and rheumatologist (S.A.A.) and the results were recorded for all cases.
   3. Capture representative images using a digital camera.
      NOTE: After these steps, remaining aliquots may be stored at -80 °C.

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Wyniki

Histopathologic findings

The H&E stains in normal specimens demonstrated normal arterial anatomy with endothelial cells in the tunica intima, smooth muscle cells in the tunica media, and a heterogenous collagen matrix which include fibroblasts and feeder vessels called vasa vasorum in the tunica adventitia. There is no inflammatory infiltrate identified. GCA positive temporal arteries demonstrated moderate t...

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Dyskusje

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...

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Materiały

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