Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

1. Sensitization of Mice with Ovalbumin

1. Prepare a solution of OVA (grade V; Sigma, MO) in sterile PBS at a concentration of 1 mg/ml (solution can be stored at -80 °C).
2. In order to prepare OVA-Alum mixture, take Alum in a tube and add OVA solution in a dropwise fashion while vortexing the tube at a ratio of 1:1. Stir the mixture for 30 minutes and use right away after mixing.
3. Using a 1 ml syringe, inject 0.2 ml of the mixture into mouse peritoneal cavity and repeat the injection again after 2 weeks.

2. Intranasal Challenge of Mice with FITC Labeled Ovalbumin

1. 7 days after the second injection of OVA-Alum, mice are ready to be challenged intranasally with OVA-FITC.
2. Prepare a solution of OVA-FITC (Sigma) using sterile PBS at a concentration of 1 mg/ml and store in aliquots at -80 °C.
3. Place a sterile gauze at the bottom of a 50 ml falcon tube and in a chemical hood, add 5 ml of Aerrane onto the gauze. In order to anesthetize mice, direct the animal into the tube for approximately 5-10 seconds.
4. Hold the anesthetized animal in an upright position and using sterile pipette tips, pipette 100 μl of the OVA-FITC solution onto the nares of mice in a drop-wise fashion.
5. Repeat intranasal challenge with OVA-FITC for the next two days.

3. Preparation of Single-cell Suspension from the Lungs and the Draining Lymph Nodes

1. Sacrifice the mice using intraperitoneal injection of Euthanyl.
2. In order to reduce macrophage contamination, which can complicate DC analysis, lung lavage is performed. To lavage the lungs, mouse tracheas are briefly exposed and cannulated with a catheter and a syringe; using ice-cold PBS with 5 mM EDTA, the lower respiratory tract is rinsed 3 times to remove cells from the alveolar spaces.
3. Perfuse the lungs with 10 ml of PBS containing 10 U/ml heparin via the right ventricle of the heart, to remove blood cells from the lung vasculature. Perform the perfusions till the lungs turn white, indicative of removal of most of the blood cells. This is especially important to remove contaminating cells from the peripheral blood.
4. Dissect the lungs out and remove mediastinal lymph nodes (MLN) and place the lymph nodes in a separate dish. (Try to minimize exposure of tissues to light to prevent quenching of FITC fluorescence.)
5. Place the lungs on a Petri dish and mince using scissors. Next digest the lungs for 25 minutes at 37 °C using 250 U/ml Collagenase D (Roche) and add EDTA (10 mM final concentration) for the next 5 minutes to stop collagenase activity.
6. Pass the fragments of digested lungs through a 100 μm cell strainer and perform hypotonic lysis to remove erythrocytes. The single cell suspension is ready for further analysis of DCs.
7. In order to prepare single cell suspension from the MLN, tease the MLN using fine needles and digest with collagenase as described above followed by straining through a cell strainer.

4. Staining for DC Markers to Assess Maturation/migration

1. In order to identify DCs in the lungs, staining is performed for CD11c and CD11b. Furthermore to assess for maturation, staining is performed for CD86 and CD80, which are upregulated as DCs undergo maturation. CD11c⁺CD11b⁺OVA-FITC⁺ cells in the MLN are identified as pulmonary DCs migrating from the lungs to the MLN in response to OVA airway challenge.
2. Suspend cells at a concentration of 10 x 10⁶ cells/ml in FACS buffer (PBS with 1% FBS and 1mM EDTA) and aliquot 150 μl of cell suspension / tube for FACS staining. For identification of lung DCs, following stains are needed: Unstained control (Lung cells from mice not exposed to OVA-FITC), OVA-FITC control, CD11c single control, CD11b single control, CD11b⁺CD11c⁺ double control, CD11b⁺ OVA-FITC⁺ double control, CD11c⁺ OVA-FITC⁺ double control, MHC II single control, CD86 single control, CD80 single control and samples stained for OVA-FITC⁺CD11c⁺CD11b⁺MHCII⁺, OVA-FITC⁺CD11c⁺CD11b⁺CD86⁺ and OVA-FITC⁺CD11c⁺CD11b⁺CD80⁺.
3. To assess DC migration, perform absolute cell counts of the single cell suspension from MLN and subsequently prepare the following tubes: Unstained control (MLN cells from mice not exposed to OVA-FITC), OVA-FITC control, CD11c⁺ single control, CD11b⁺ double control, CD11b⁺OVA-FITC⁺ double control, CD11c⁺OVA-FITC⁺ double control and CD11c⁺CD11b⁺OVA-FITC⁺ samples.

5. Representative Results

The time points required for intraperitoneal sensitization to induce airway allergic inflammation is important and should be carried out as depicted in Figure 1. Following intraperitoneal sensitization and airway OVA challenge, to confirm induction of airway allergic inflammation, some mice can be sacrificed and histological analyses can be carried out on the lung sections as shown in Figure 2. Presence of inflammatory cells can be confirmed by Hematoxylin & Eosin stain (Figure 2A) and presence of mucus production can be assessed by Periodic-acid-Schiff stain (Figure 2B). Altogether this will confirm induction of airway allergic inflammation following OVA challenge of OVA-sensitized mice⁸. In contrast, lung sections from Saline treated mice are expected to be free of any inflammation along with absence of any mucus production. Moreover, following OVA challenge, pulmonary DCs undergo maturation and subsequent migration to the draining lymph nodes. Analysis of CD11c⁺ cells in the mediastinal lymph nodes (MLN) from mice sensitized and challenged with OVA is expected to show a higher proportion of CD11c⁺ cells compared to saline-treated mice (Figure 3A). Moreover, analysis of absolute cell count of CD11c⁺CD11b⁺OVA-FITC⁺ cells in the MLN of OVA-sensitized and challenged mice, is expected to show a significantly higher (i.e. several fold higher) count that the counterparts from saline treated mice (Figure 3B).

Figure 1. Experimental protocol for induction of ovalbumin (OVA) induced allergic airway inflammation in mice along with airway challenge with FITC labeled ovalbumin (OVA-FITC).

Figure 2. OVA sensitization followed by OVA airway challenge leads to induction of airway allergic inflammation as identified by Hematoxylin & Eosin staining of lung sections shown in (A) and also leads to mucus production in the airways as identified by Periodic Acid Schiff staining of lung sections as shown in (B).

Figure 3. OVA-FITC challenge induces DC migration from the lungs to the mediastinal lymph nodes (MLN). (A) Flow cytometry plots depicting proportions of CD11c⁺ cells in the MLN of control or OVA-sensitized mice. (B) Absolute counts of CD11c⁺CD11b⁺OVA-FITC⁺ cells in the MLN of saline or OVA-sensitized mice. *p<0.05. Click here to view larger figure.