Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

NOTE: This research was approved by the regional Ethical Review Board in Umeå, Sweden.

1. Bronchoscopy for Sampling Endobronchial Biopsies from Human Subjects

1. Obtain informed consent from all participants.
2. Treat subjects with oral midazolam (4-8 mg) and intravenous glycopyrronium (0.2-04 mg) 30 min before the bronchoscopy. Apply topical anesthesia with lidocaine in the larynx and bronchi. Let the subject gargle with ~3 ml of lidocaine 4% and apply 3 ml to the tongue base and into the larynx via a larynx syringe. Optimize the topical anesthesia with 8-10 doses of lidocaine spray. Carry out this step with the subject sitting.
3. Insert a flexible video bronchoscope through the mouth via a plastic mouthpiece with the subject in the supine position. Use a purpose-made spray catheter to complete the topical anesthesia of the distal trachea and bronchi via the bronchoscope. Here, use a dose of approximately 5-10 ml of lidocaine 2%.
4. Take 6-9 endobronchial mucosal biopsies from the main carina and the main bronchial divisions using fenestrated forceps (Figure 1). Following the bronchoscopy and before leaving the hospital, let the subject rest for 2-4 hr and provide him/her with a light meal.

2. Embedding the Biopsies in Glycol Methylacrylate (GMA) Resin

NOTE: The GMA immunostaining protocol was originally developed at Southampton University, Histochemistry Research Unit¹⁷.

1. Fixation: For immunohistochemistry, remove the biopsies from the forceps and place them directly into glass vials containing 3 ml of ice-cold dehydrated acetone and protease inhibitors (phenylmethylsulfonyl fluoride (35 mg/100 ml acetone) and iodoacetamide (370 mg/100 ml acetone)). Fix the tissue overnight at -20 °C (Figure 2).
   NOTE: The following steps should be performed in a fume hood with appropriate nitrile gloves. The embedding kit contains components that can cause sensitization, irritation, and/or allergic skin reactions. All waste must be disposed of as hazardous waste.
2. Dehydration: Remove the acetone with inhibitors and replace with fresh dehydrated acetone (without inhibitors) for 15 min at room temperature (RT). Remove the acetone and replace it with methyl benzoate for 15 min.
3. Infiltration: Prepare a processing solution of 5% methyl benzoate solution in glycol methacrylate monomer; 10 ml is sufficient for one vial. Using plastic Pasteur pipettes, suction off the methyl benzoate solution and replace with 3 ml of processing solution. Incubate the biopsies and excess processing solution at 4 °C for 2 hr; Pasteur pipettes can be used for all subsequent solution changes, including embedding solution.
4. Exchange the processing solution every 2 hr (3x 2 hr incubations), so the total infiltration time of the biopsies in the processing solution is at least 6 hr. Insert paper labels into the top end of the embedding capsules to identify the biopsies.
5. Prepare an embedding solution of 75 mg benzoyl peroxide in 10 ml of glycol methacrylate monomer. Add 0.25 ml of N,N-dimethylaniline poly(ethylene oxide) (accelerator) to the embedding solution to begin the polymerization reaction. Remove the processing solution and place one biopsy into the relevant embedding capsule using forceps.
6. Slowly fill the embedding capsule to the top with embedding solution and close the cap. Avoid disturbing the biopsy and creating large air bubbles. Incubate the capsules at 4 °C until the resin has completely polymerized.
7. Store the embedded biopsies at -20 °C in a 50 ml tube containing approximately 5 g of silica gel.

3. Sectioning of GMA-embedded Endobronchial Biopsies

1. Microscope slide coating: Wash the microscope slides in a dishwasher on a normal cycle. Cover the slides and allow them to air dry.
2. Prepare a coating solution of 10% poly-L-lysine (PLL) solution in distilled water. Submerge the slides in coating solution for 5 min, and then let them air dry. Store dry PLL-coated slides in their original boxes.
3. Wash sheet glass strips with 0.1% Tween 20 in distilled water. Rinse the glass with 70% ethanol and dry with paper towels. Cut glass blades from the glass strip using a knife maker. Store the blades in a container that prevents movement to ensure that the cutting edge does not get damaged or nicked.
4. Biopsy trimming: Place the biopsy capsule in a capsule splitter and cut down each side using a carbon-steel single-edge blade. Remove the GMA-embedded biopsy and place it firmly in a vice. Trim excess GMA from around the biopsy using the steel blade.
5. GMA sectioning.
   1. Prepare 0.05% ammonia solution with distilled water. Place the glass knife and biopsy in the microtome. Carefully align the biopsy block with the blade by adjusting the knife holder and the angle of the biopsy block so that the blade rests flat against the surface of the block.
   2. Cut 2 µm thick sections using the microtome on a slow cutting speed and use forceps to transfer and float out sections on the ammonia water. Float the sections for 45-90 sec, allowing gentle antigen retrieval and unfolding of the section.
   3. Pick up sections with microscope slide. Check the biopsy histology by quickly staining with toluidine blue.
   4. Dry the slides on a hot plate set to 50 °C and apply 500 µl of filtered toluidine blue stain to a section using a plastic Pasteur pipette. Warm the section on the hot plate until a green ring begins to emerge at the edge of the stain, and then wash it off with water.
   5. Using a light microscope, check the biopsy histology. Sections used for immunohistochemistry should contain good areas of undamaged lamina propria and epithelium, with as few glands and as little smooth muscle as possible.
   6. As in section 3.4.2, cut sections that have good histology and pick them up with PLL-coated microscope slides for immunohistochemical staining. Cut at least two sections from each biopsy for analysis. Dry slides for at least 1 hr. After drying, sections can be wrapped in tin foil and stored at -20 °C for up to 2 weeks, or they can be immediately immunostained.

4. Immunohistochemical Staining of GMA-embedded Endobronchial Biopsies

NOTE: Sodium azide is toxic. Prepare the 0.1% sodium azide solution within a fume hood and away from acids. Contact with acids liberates toxic gas.

1. Draw around sections using a diamond-tipped pen and arrange the slides onto a staining rack.
2. Perform a Peroxidase block. Prepare a peroxidase block solution of 0.3% hydrogen peroxide in 0.1% sodium azide solution. Apply 1 ml of the peroxidase block to sections and incubate for 30 min.
3. Prepare a wash solution of 0.05 M Tris-buffered saline (TBS), pH 7.6. Wash the slides in TBS, 3x 5 min.
4. Prepare a 1% BSA block solution in DMEM. Do this in advance and in large volumes, aliquot and freeze it until needed. Drain the slides and incubate the sections in block solution for 30 min to block unspecific antibody binding. If unspecific binding still occurs, include a 30 min incubation step with 5% serum block from the same species as the secondary antibody.
5. Dilute primary antibody (CD45 or CD1a) in TBS to the desired concentration (CD45 diluted at 1:1,000; CD1a diluted at 1:1,00) and apply it to slides. Coverslip the sections and incubate overnight at RT.
6. Wash slides in TBS three times. Dilute biotinylated secondary antibody (directed against the primary antibody host species, in this case rabbit anti-mouse F(ab'₂)) in TBS to the desired concentration (1:300 dilution) and apply it to slides. Incubate for 2 hr at RT.
7. Prepare a streptavidin biotin-peroxidase complex at least 30 min before use. Ensure that there is enough to cover all slides. Wash the slides in TBS three times. Apply the solution to the slides and incubate for 2 hr at RT.
8. Wash the slides in TBS three times. Prepare 3-amino-9-ethylcarbazole (AEC) peroxidase substrate solution (made per the manufacturer's instructions). Apply it to the slides and incubate for 20-30 min or until the desired stain intensity develops.
9. Rinse the slides in running tap water for 5 min. Counterstain the sections with filtered Mayer's haematoxylin solution for 2 min. Wash the slides in running tap water for 5 min.
10. Drain the slides and cover the sections with permanent aqueous mounting medium. Dry the slides at 80 °C in a drying oven. Allow the slides to cool, and then mount them with DPX and place a coverslip.

5. Staining Analysis

1. Analyze the sections at 40X magnification using a light microscope with a mounted camera connected to a computer.
   NOTE: For cellular analysis, count positively stained, nucleated cells within the bronchial lamina propria and intact epithelium, excluding areas of smooth muscle, glands, large blood vessels, and mismatched or damaged tissue.
2. Average the cell counts and correct the average cell count for the area of the lamina propria and the length of the epithelium. The area of the lamina propria and the length of the epithelium can be calculated using an image analysis program.

6. Enzymatic Digestion of Endobronchial Biopsies

1. Washing biopsies: Using sterile forceps, place intact biopsies in a 15 ml conical tube containing 10 ml of Hank's buffered saline solution (HBSS) (Figure 3). Incubate for 5 min at RT on a rocking platform at 30 rpm. Transfer the biopsies to a sterile dish by decanting the content of the 15 ml tube with HBSS.
2. Disrupting mucus with DTT: Replace the HBSS in the tube with 10 ml of HBSS containing 5 mM 1,4-dithiothreitol (DTT). Transfer the biopsies back into the 15 ml tube using sterile forceps. Incubate for 15 min at RT on a rocking platform at 30 rpm.
3. Vortex the tube gently for 15 sec. Transfer the biopsies onto a sterile dish by decanting the content of the 15 ml tube with HBSS and DTT.
4. Transferring the biopsies to the culture medium: Replace the HBSS and DTT in the tube with 10 ml of RPMI 1640 culture medium. Transfer the biopsies back into the 15 ml tube using sterile forceps. Incubate for 10 min at RT on a rocking platform at 30 rpm.
5. Digesting biopsies with collagenase and DNase.
   1. Prepare a digestion solution of collagenase II (0.25 mg/ml) and DNase (0.2 mg/ml) in pre-warmed RPMI containing 1 M HEPES solution. Add 500 µL of digestion solution per well in a 48-well tissue culture plate.
   2. Place 1 biopsy per well in digestion solution. Incubate the plate on a shaking platform for 60 min at 37 °C at 220 rpm. Pipette up and down after 30 min to re-disperse the biopsies, and after 60 min, to completely disaggregate the tissue.
6. Preparing single cell suspensions.
   1. Pool together the digested biopsies from the wells into a 50 ml tube by passing it through a 40 µm cell strainer. Collect the remaining cells by washing the wells with ice-cold FACS buffer (phosphate-buffered saline (PBS) containing 2% fetal bovine serum).
   2. Squeeze the remaining tissue on the cell strainer using the back of the plunger of a syringe. Centrifuge the digested cells at 400 x g for 5 min at 4 °C. Carefully remove the supernatant and resuspend the pellet in 5 ml of ice-cold FACS buffer. Count the cells manually using a hemocytometer and Trypan blue stain to assess the viability of the cells.

7. Flow Cytometric Analysis of Single Cells of Endobronchial Biopsies after Enzymatic Digestion

1. Resuspend the cell pellet to approximately 1 x 10⁶ cells in 200 µl of FACS buffer.
2. Add a cell viability dye for dead cell exclusion according to the manufacturer's protocol.
3. Add 5 µl of FcR blocking reagent and incubate for 5 min at 4 °C.
4. Add cell surface antibodies against CD45 (2 µl), lineage (CD3, CD20, CD56, CD66abce, and CD14; 2 µl each), CD16 (0.5 µl), HLA-DR (3 µl), CD11c (5 µl), CD123 (5 µl), CD1c (3 µl), and CD103 (2 µl) and incubate for 15 min at 4 °C. Details on antibodies can be found in the methods section, but titrate and optimize the antibodies to the precise flow cytometer in use. Wash off excess antibodies with PBS for 5 min at 400 x g. Acquire the samples fresh on a flow cytometer or fix the cells in 1% paraformaldehyde prior to later acquisition.
5. Identify human DCs in lung biopsies using a flow cytometry assay¹⁸ (Figure 4).
   NOTE: Using a flow cytometry analysis software, immune cells are distinguished from other lung cells by gating on CD45. Dead cells can be excluded as cells that are positive for the LIVE/DEAD dye. Of all live CD45⁺ cells, lineage cells (B cells, T cells, NK cells, neutrophils, and monocytes) can be excluded using a cocktail of antibodies against CD20, CD3, CD56, CD66abce, CD14, and CD16 in one channel. Following that, HLA-DR⁺ cells will allow the identification of all human DCs. MDCs can be distinguished from PDCs based on the expression of CD11c or the lack of CD11c, respectively. MDCs can be further divided into CD1c⁺ MDCs or CD141⁺.