The anticipated results differ according to the respective experimental setting. Intranasal application of substances can be useful to deliver neutralizing or agonistic antibodies, recombinant cytokines or antisense RNA molecules. In the following section we describe the results from experiments using recombinant chemicals, antibodies and cytokines. We have used this technique in the past by applying an antibody to block Th2 development via induction of regulatory T cells7 or by inducing apoptosis of Th2 cells3. When given intranasally during allergen challenge a smaller effect as compared to delivery before allergen sensitization might be the result. However, the delivery of the antibody during the allergen challenge phase mimics a more therapeutically use whereas the delivery before allergen sensitization is meant to mimic a vaccination. Figure 1a shows a typical protocol used to induce allergic asthma in mice.
When blood is sampled from the mice it should be examined for IgE content in the serum, for example with ELISA. OVA-treated mice should have significantly more IgE in their serum than PBS-treated mice (Figure 2c).
Mice are subjected to analysis of airway hyperresponsiveness (AHR) which is a typical characteristic of allergic asthma. This is typically performed at the end of experiment before the mouse is sacrificed. Mice are challenged with methacholine in order to induce bronchoconstriction. Thus, allergen treated mice should have a significantly increased AHR in comparison to PBS-treated mice (Figure 2d). Another hallmark of asthma is lung inflammation. The whole lung or parts of it can be fixed in formalin and used for histological analysis. Haematoxilin and eosin staining is typically used to analyse the intensity of inflammation. Here OVA-treated animals should show a significantly increased infiltration of inflammatory cells like eosinophils or lymphocytes compared to PBS-treated animals (Figure 2e). Performance of broncho-alveolar lavage is also a typical read-out for analysing mice in a setting of allergic asthma. The cells can be quantified and the content of eosinophils and neutrophils can be determined. This is possible with flow cytometry. CD3-CD45R-Gr1+CCR3- cells are quantified as neutrophils, while CD3-CD45R-Gr1-CCR3+ are characterised as eosinophils. Figure 3a-c and Box2 show and describe respectively a gating strategy which we apply to analyse these cells. The results shown in Fig. 3d-f demonstrate that OVA-treated mice have significantly increased neutrophils and eosinophils in the BAL in comparison to PBS-treated mice (Fig.3f)).
When analysing the role of a potential asthma therapeutics the aim is to reduce these classical hallmarks of allergic asthma in OVA-treated animals significantly.
Box2: Differential analysis of inflammatory cells in the BALF by FACS: Eosinophils and neutrophils
- Centrifuge the BALF for 5 min at 1500 rpm and 4° C. Discard the supernatant and resuspend the cell pellet in 1 ml PBS.
- To determine the number of total cells in a Neu-Bauer chamber take 10 µl of your sample and dilute it in 10 µl Trypan blue (1:2). Count the white cells of two 16 squares arranged diagonally. The blue cells are dead cells. Determine the average of alive cell number of the two 16 squares and multiply it by the dilution factor and by 104 to obtain the number of cells present per ml of BALF.
- Transfer 600-103 cells into a FACS tube. For the single staining and for the unstained cells take a mixture of cell samples out of different treated groups.
- Centrifuge the cells 5 min at 1500 rpm and 4° C and aspirate and discard the supernatant carefully.
- Stain your samples using 60 µl of a master-mix as shown in the Table below: anti- CD3-Fitc (1:200), CD45.2 Percp (1:200), Gr1-Pe (1:200), CCR3-APC (1:200) and Fc-Block (1:200) in PBS. For the single stained cells use 100 µl PBS and 1 µl of antibody. Vortex the master-mix thoroughly.
- Vortex and incubate 30 min at 4° C in the dark.
- Add 200 µl PBS and centrifuge 5 min at 1500 rpm and 4° C.
- Aspirate the supernatant carefully and resuspend the pellet in 300 µl PBS.
- Analyse the samples at the FACS machine. The unstained sample is used to adjust the forward and sideward scatter. Adjust the channels with the single stained samples. Vortex your samples before measuring.
- To analyse your samples set a wide gate around the granulocytes using the forward and sideward scatter as shown in Figure 3a.
- Gate the cells on a CD3 and CD45R scatter to distinguish the CD3 and CD45R negative granulocytes from lymphocytes (Fig. 3b).
- In the last step granulocytes are classified as eosinophils and neutrophils in a Gr-1 CCR3 scatter (Fig. 3c). Eosinophils are Gr-1 negative and CCR3 positive whereas neutrophils are Gr-1 positive and CCR3 negative.
Set the gates once for one sample and apply them for all other samples. Figure 3d shows an example for three PBS and three OVA treated Balb/c mice. The percentages of the eosinophils and neutrophils are used for the statistical evaluation shown in Figure 3f. Mean, standard deviation and T-test were performed using Microsoft Excel.
Mastermix for FACS staining
Mastermix per sample
PBS 60 µl
CD3 - Fitc (1:200) 0,3 µl
CD45.2 - Percp (1:200) 0,3 µl
Gr-1 - Pe (1:200) 0,3 µl
CCR3 - APC (1:200) 0,3 µl
Fc-Block (1:200) 0,3 µl
Antibodies or antisense DNA delivered intranasally to inhibit allergic inflammation:
Both treatment strategies (vaccination and therapy) were used to block the development of Th2 cells during allergen challenge phase.
Antibodies used for intranasal application:
Antibody and control substances used for local blockade of Th2 development7:
• Anti-IL-6R antibody (rat anti-mouse; Chugai Pharmaceutical Inc., Tokio, Japan): 50µg/mouse
• Control IgG antibodies (rat anti-mouse, Sigma-Aldrich; rat IgG1 isotype control, R&D Systems): use in the same concentration as the blocking antibody
• gp130-Fc (obtained from K.J. Kallen, S. Rose-John, University of Kiel, or R&D Systems): a fusion protein that competes with gp130, the natural ligand of soluble IL-6R and the second component of membrane bound IL-6R: use in the same concentration as the actual antibody
• Anti-IL-4, anti-TGF and anti-IL-13 antibodies were also used in a setting of allergic asthma during allergen challenge to block experimental asthma13.
• Anti-IL-17A antibody
Antisense DNA Molecules given intranasally during the allergen challenge:
Antisense molecules comprise DNA oligonucleotides which are able to inhibit a specific mRNA target sequence. DNA consists of two strands, a sense and an antisense strand. Only the antisense strand codes for genes that are transcribed into mRNA. Synthetic antisense DNA molecules bind to the specific mRNA because of their complementary sequence. Then RNase H is recruited and degrades the target hybrid DNA/mRNA. This results in the inhibition of the transcription of the gene of interest. In one study we were able to block the expression of the major Th2 transcription factor GATA-3 by applying an antisense phosphorothioate oligonucleotide overlapping the translation start site of GATA-3 intranasally9,10.
Antisense molecules used for intranasal application:
• DNA antisense oligonucleotides for GATA-3 gene regulates Th2 cytokine production9,10:
a. GATA-3 antisense DNA: 5’-AGT CAC CTC CAT GTC CTC-3’
b. GATA-3 nonsense DNA, 5’-CTA TGT CAT CCG CTC CAC-3’
c. GATA-3 mismatched DNA, 5’-AGC CAC CTA CAT TTC CTA-3’
d. GATA-3 mismatched DNA 2, 5’-AGC CAC CTA GGC ATC CTC-3’
200 µg / mouse were used.
• Intranasal delivery of antisense DNA oligonucleotides against stem cell factor (SCF) were used to inhibit the maturation of mast cells in the lung or nose9:
a. SCF antisense DNA: 5´-TGT CTT CTT CAT AAG GAA-3´
b. SCF nonsense DNA: 5´-GTG TAC AGA TTA CTC ATT-3´
c. SCF mismatched DNA: 5´-TGT CTG CTC TAT AAT GAA-3´
370 µg / mouse were used.
NOTE: Phosphorothioate oligonucleotides are modified DNA molecules where the non-bridging oxygen is replaced by a sulphur atom which enhances the stability. This modification reduces the activity of nucleases thus generating DNA oligonucleotides that are resistant against enzymatic degradation. Additionally, this modification increases the ability of the oligonucleotides to cross the cellular membrane.
CAUTION: The method we describe in this protocol refers to achieve anti-inflammatory resolution of the disease in the lung. Thus, it is convenient because the therapy is delivered in close contact to the identified target cells. Both GATA-3 and SCF are molecules known to influence the development of hematopoietic stem cells which is why it is not advisable to use these substances systemically9.
Recombinant cytokines used for intranasal application:
Murine recombinant IL-28A (PeproTech, Hamburg, Germany; cat. no.: 250-33)
We recently reported that intranasal treatment with Interferon (IL-28A) led to amelioration of allergic asthma. In this paper, intranasal treatment of mice with recombinant IL-28 causes a reduction of Th2 cytokines and lung inflammation in a murine model of allergic asthma4.
Other therapeutical substances used for intranasal application:
Dexamethasone (Sigma Chemical Co, St Louis, Mo; Dexamethasone (water-soluble), cat. no. D2915): 1 mg/50µl sterile saline per intranasal application per mouse should be used. Local delivery of steroids is the golden standard treatment for allergic asthma. This treatment results in the resolution of all the hallmarks of the allergic asthmatic phenotype9.
Galiellalactone (gift from T. Anke, University of Kaiserslautern, Germany): 50-100 µg/mouse applied in PBS. It is an inhibitor of both pSTAT3 and pSTAT5. This compound was given intranasally both during the allergen challenge and before the allergen sensitization6.
Taken together, intranasal application of substances is a useful way to administer drugs or allergens to the mouse. It is the easiest way to access the lungs and deliver substances to the airways.
Box 3: RNA extraction and quantitative real-time PCR
For the RNA extraction from the lung, PeqGoldRNAPure (PeqLab Biotechnology, Erlangen, Germany) was used according the manufacturer´s protocol.
To isolate RNA from lung tissues, add 1 ml of PeqGoldRNAPure to the frozen tissue and homogenize. Cells can be lysed directly on the cell culture well in 1 ml of PeqGoldRNAPure.
- Incubate the samples for 5 min at room temperature.
- Transfer the lysed cells into autoclaved eppendorf tubes and add 200 µl chlorophorm to the samples and mix well for 10 sec.
- Incubate for 3 min at 4°C.
- Centrifuge for 5 min, 12.000 g at 4°C. After centrifugation a you should see three different phases. The lower phase and interphase comprises DNA and proteins while the upper phase contains the RNA and is transferred to a new autoclaved eppendorf tube.
- Add 400 µl chlorophorm and mix well for 10 sec.
- Centrifuge for 3 min, 12.000 g at 4°C. Again a partition of two phases should be seen. Transfer the upper phase into a new autoclaved eppendorf tube.
- Add 3 µl of glycogen (10 mg/ml) and 350 µl Isopropanol to the samples and mix well for 10 sec.
- Incubate for 15 min at 4°C.
- Centrifuge for 10 min, 12.000 g at 4°C.
- Discard supernatant and wash RNA pellet two times with 70% ethanol.
- Centrifuge 5 min, 12000 g at 4°C.
- Discard ethanol completely and let the RNA dry for 5-10 min. The RNA is ready for elution when the pellet is no longer visible and no droplets of ethanol can be seen.
- Elute the RNA in 20 µl sterile nuclease free water. Alternatively it is possible to use DEPC-treated water.
- Incubate for 5 min at 65°C with gentle shaking.
The concentration of RNA can be measured and then stored at -80°C. It is now possible to create cDNA from the RNA by reverse transcription.
The template-cDNA was amplified by quantitative real-time PCR (qPCR) using SsoFast EvaGreen Supermix (Bio-Rad Laboratories, München, Germany). qPCR was performed with one cycle of 2 min 98°C, 50 cycles at 5 s 95°C, 10 s 60°C, followed by 5 s 65°C and 5 s 95°C in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). The mRNA of the genes of interest were normalized using the mRNA levels of the housekeeping gene HPRT. See Table III for examples of primers and their sequence used.
TableIII: primers used for real time PCR
HPRT 5´-GCC CCA AAA TGG TTA AGG TT-3´
5´-TTG CGC TCA TCT TAG GCT TT-3´
GATA3 5´-GTC ATC CCT GAG CCA CAT CT-3´
5´-TAG AAG GGG TCG GAG GAA CT-3´
RORgT 5´- GTG TGC TGT CCT GGG CTA CC-3´
5´-AGC CCT TGC ACC CCT CAC AG-3´