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

Abstract

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen 1,2 . Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response 3 .DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment 4 . Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens 5 . However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma 6,7 . After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry. Video LinkThe 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.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.

Lung dendritic cells undergo maturation and polarization towards a T helper type 2-stimulating phenotype in a mouse model of asthma: Role of nerve growth factor

“…Single-cell suspensions were prepared from the lungs, as previously described ( 16 ). Lung lavage was initially performed using ice-cold PBS with 5 mM EDTA.…”