All scientific protocols involving the use of animals in this study have been approved by the Institution Animal Care and Usage Committee (IACUC) of Indiana University School of Medicine, and followed the guidelines established by the Panel on Euthanasia of the American Veterinary Medical Association. Protocols involving the use of recombinant DNA or biohazard materials have been approved by the Institutional Biosafety Committee and followed the guidelines established by the NIH. Animals were housed under IACUC-approved conditions in a secured animal facility at Indiana University School of Medicine. Animals were regularly screened for common pathogens (specific pathogen free, or SPF). Experiments involving animal sacrifice utilize CO2 narcosis to minimize animal discomfort.
Generation of doxycycline-controlled Api6 transgenic mice
To generate the (TetO)7
-CMV-Api6 transgenic mouse line, murine Api6 cDNA was amplified by PCR using a downstream primer (5’-AAGGAAAAAAGCGGCCGCTTATCACTTGTCATCGTCGTCCTTGTAGTCCACATCAAAGTCGTGGCA-3’ with the Not I site and the Flag sequence) and an upstream primer (5’-CGCGGATCC GCCACCATGGCTCCATTGTTCAAC-3’ with the Bam H1 site and the Kozak sequence). The PCR product was digested with BamH1/Not I and subcloned downstream of the CMV minimal promoter linked to seven Tet-responsive elements at the BamH1 and Not I sites in the pTRE2 vector (Clontech, Mountain View, CA). The expression cassette containing the CMV minimal promoter, the Api6 cDNA, and the human globin polyadenylation signaling sequence was dissected out and purified for microinjection into FVB/N mice. Founder lines were identified by a pair of primers corresponding to a pTRE2 plasmid sequence (5’-GCAGGATGATTACCAGGATGTAG-3’) and an Api6 cDNA coding region sequence (5’-GGAGAAAAGGA GGACCAAGTGG-3’). The c-fms-rtTA transgenic mice were generated and genotyped as we previously reported (14
-CMV-Api6 bitransgenic mice were obtained by crossbreeding the c-fms-rtTA transgenic mice and (TetO)7
-CMV-Api6 transgenic mice. In general, animals were treated with doxycycline at 1 month-old age after genotyping.
Bone marrow cells were flushed from femurs and resuspended in FACS buffer (PBS, 2% fetal bovine serum, 0.01% sodium azide). The spleen single-cell suspension was obtained by grinding and filtration through nylon mesh into FACS buffer. Blood mononuclear cells were obtained after red blood cell lysis with distilled water plus 10 X PBS. To prepare cells from the lung, the left atrium was opened by incision, and the right ventricle was infused with at least 2 ml of sterile PBS to remove any residual blood in the pulmonary vasculature. Lungs were cut into small pieces and placed in RPMI 1640 containing 5% FBS, collagenase (Sigma-Aldrich), and DNase (Boehringer Mannheim). After 40 minutes of collagenase digestion at 37°C, lungs were further disrupted by aspiration through an 18-gauge needle and were suspended in FACS buffer. Approximately 1 to 2 × 106 cells from various organs in FACS buffer were blocked with FcR followed by incubation with primary antibodies. Cell surface markers Anti-CD11c (N148), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-CD3 (145-2C11) and anti-B220 (RA3-6B2) antibodies were purchased from eBiosciences (San Diego, CA). Cells were analyzed on LSRII machine (BD Biosciences, San Jose, CA). Data was analyzed using the BD FACStationTM Software (BD Biosciences). The total number of positive cells was calculated as the percentage of total gated viable cells. Isotype controls IgG1, IgG2a and IgG2b were included in all experiments. Quadrants were assigned using isotype control.
For measurement of intracellular signaling molecules, the assays were performed according to the protocols from Cell Signaling Technology (Danvers, MA) and a previously described procedure (16
). Briefly, after surface staining, cell suspensions from bone marrow, blood, spleen and lung of doxycycline-treated or untreated c-fms-rtTA/(TetO)7
-CMV-Api6 bitransgenic mice were fixed with 2% formaldehyde. Fixation samples were resuspended in methanol at a final 90% concentration. Finally, samples were washed and resuspended in 1 x PBS containing 4% fetal bovine serum (FBS) at 106
cells in 100 μl. Cell suspensions were labeled with the primary phospho-specific antibodies for 30 min at room temperature, then washed in PBS containing 4% FBS and labeled using the secondary antibody. After washing, samples were analyzed on a LSRII machine (BD Biosciences). Data was analyzed using the BD FACStationTM
Software (BD Biosciences). Quadrants were assigned using isotype control. Anti-Phospho-P44/42 Mark antibody (T202/Y204, #4374), anti-Phospho-Stat3 antibody (Tyr 705, #4323) and anti-Phospho-P38 Mark antibody (Thr180/Tyr 182, #4551) were purchased from Cell Signaling Technology. SP-C antibody (FL-197) was from Santa Cruz Biotechnology. Monoclonal ANTI-FLAG®
M2-FITCantibody (F4049) was from Sigma (Saint Louis, MI).
Alveolar type II epithelial cell purification
Alveolar type II epithelial cells were purified from Wild-type mice (WT), doxycycline-treated or untreated bitransgenic mice as described previously (12
). After purification, alveolar type II epithelial cells were stained with anti-CD11b and Gr-1 antibodies to prove that no macrophages and neutrophils were contaminated.
Histology of lung
The lung from doxycycline-treated or untreated c-fms-rtTA/(TetO)7-CMV-Api6 bitransgenic mice was infused with a fixative solution (4% paraformaldehyde, 1 × phosphate-buffered saline) and was dissected out and stored in fixative at 4°C for ~24 h. After fixation and embedding in paraffin, lung tissue sections were cut to 5 μm thick. The adult lung slides were baked at 60°C for 2 h and washed in a series of xylene and ethanol to remove paraffin from the tissues. Multiple sections from each lung were stained with hematoxylin and eosin. Tumor incidence and multiplicity in each section was counted.
All reverse transcription reactions were set up using the Taqman Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Two μg of total RNAs from the whole lungs, alveolar macrophages or alveolar type II epithelial cells of doxycycline-treated or untreated c-fms-rtTA/(TetO)7-CMV-Api6 bitransgenic mice were used in 100 μl of reaction mixture. The reactions were carried out using a GeneAmp 9700 Thermocycler (Applied Biosystems) with a suggested cycling protocol of 25°C for 10 mins, 48°C for 30 mins, and 95°C for 5 mins. For Real-Time PCR, 2 μl of cDNA was amplified by a pair of sequence-specific DNA oligonucleotide primers for each molecule in a 50 μl of reaction mixture containing SYBR Green PCR Master Mix (Applied Biosystems). GAPDH primers were used as an endogenous control for normalizing all cDNA samples. The reactions were analyzed using the Relative Quantification Assay and 7500 System Sequence Detection Software for the 7500 Real-Time PCR System (Applied Biosystems).
Primers for Real-Time PCR:
Upstream primer: 5’-CAG TAA TGG CGT CTT GTC AGT GA – 3’
Downstream primer: 5”-CGT TGA TAT TGA GTG GCC TGA CT- 3”
Upstream primer: 5’-GGG CCC ACC AGC TCT GA- 3’
Downstream primer: 5’-TGG ATG AAA CCC TGT AGC AAA A- 3’
Upstream primer: 5’-TGC TTA CTC TAC AGC ACC TGG TTA CT- 3’
Downstream primer: 5’-TGA ACC ACG ACC CGT CCT T- 3’
Upstream primer: 5’-CCG TCC ACA ATG ACT GCT CTT- 3’
Downstream primer: 5’-GGT CCT CCT CAG AGG CTT TTC- 3’
Upstream primer: 5’-AAC GAC CTG ACT GCC AAG AAA- 3’
Downstream primer: 5’-GGT TCC GGT GTG CCA TCT C- 3’
Upstream primer: 5’-CAGCAGCCTGCAGGAACTTAT- 3’
Downstream primer: 5’-ACCAGGCCAGTTGTGATGACT- 3’
Downstream: 5’-CGGTTCCTTCGAGTGACAAAC -3’
Upstream: 5’-AAAGCTACGAGAGAATCAACAATATCC -3’
Upstream: 5’-CTGCTCACAAACAGCGTCGTA -3’
Downstream: 5’-GTGCAGAACCAGCAGCAGTAAG -3’
Upstream: 5’-TTGACGGACCCCAAAAGATG- 3’
Downstream: 5’-CAGGACAGCCCAGGTCAAA- 3’
Downstream: 5’-GGCTGTGCCAGTTCAGAGTTG -3’
Downstream: 5’-CACAGTCCGTTTCCGGAGTT -3’
Upstream: 5’-GAGGCTGACATCATGATCAACTTT- 3’
Downstream: 5’-GCCATCAAATGGGTATCCATCT- 3’
Upstream: 5’-TGAGGACGCAGGAGTGAACTT- 3’
Downstream: 5’-CCCAGAGAGTGGCCAAATTC- 3’
Upstream: 5’-CTCAAGAGACCATGGTGACAATTC- 3’
Downstream: 5’-AAGGCATGGGCAAGGATTC - 3’
Upstream: 5’-GAGGCTGACATCATGATCAACTTT- 3’
Downstream: 5’-GCCATCAAATGGGTATCCATCT- 3’
Upstream: 5’-TGGTATTCAAGGAGATGCACATTT- 3’
Downstream: 5’-GGTTTGTGCCTTGAAAACTTTTAGT- 3’
Dual staining with fluorescein isothiocyanate-labeled-annexin V (FITC-annexinV) and propidium iodide (PI) was performed to detect cells undergoing apoptosis by using an Annexin V-FITC kit (BD Biosciences, Bedford, MA). Briefly, cells from different tissues of doxycycline-treated or untreated c-fms-rtTA/(TetO)7-CMV-Api6 bitransgenic mice were isolated as mentions above. After staining with cell surface-specific markers (e.g. CD11b, CD11c, Gr-1, CD3, B220), cells were washed twice with PBS. After resuspension of labeled cells in annexin V-binding buffer containing FITC-conjugated annexin V, PI was added into samples for 10 min incubation on ice. Cells were analyzed on a LSRII machine within one hour. At least 1 × 104 cells in each sample were analyzed. Control cells stained with annexin V-FITC or PI alone were used to compensate for the flow cytometric analysis. Annexin V and PI double-negative cells are defined as live cells. Annexin V-positive, PI-negative cells are defined as early apoptotic cells. Annexin V and PI double-positive cells are defined as late apoptotic and necrotic cells. Nonspecific binding was blocked by pre-incubating the cells with rat IgG (10 μg/ml) and anti-FcII/III.
For the cell proliferation study, littermates of doxycycline-treated or untreated c-fms-rtTA/(TetO)7-CMV-Api6 bitransgenic mice were inoculated i.p. with 1 mg BrdU/animal (Sigma-Aldrich) in PBS for 24 hrs. Cells from different tissues were harvested and stained with surface markers (e.g. CD11b, CD11c, Gr-1, CD3, B220) as mentioned above. Labeled cells were washed twice with PBS and treated with DNase I followed by staining with fluorescein–anti-BrdU using BrdU flow kit (BD BioSciences) before being analyzed on a LSR II machine.
Bone-marrow derived macrophages
For in vitro
assay, bone marrow-derived macrophages were obtained as described previously (23
). Briefly, femurs from wild-type mice were flushed using a 26-gauge needle and bone marrow cells were cultured for 3 hours. Nonadherent cells were removed and cultured in the minimum essential medium α supplemented with 10% fetal calf serum and 50 μm of 2-mercaptoethanol and 10 ng/mL of recombinant murine IL-3 and 10 ng/mL of recombinant murine GM-CSF for 14 days. As soon as the monocytes started to adhere (6–7 days), all nonadherent cells were washed away from the culture to minimize potential interactions with other cells. More than 90% of the bone marrow population was F4/80+ macrophages as determined by flow cytometry.
In vitro function analysis of Api6
Macrophages isolated above were cultured in RPMI -1640 culture medium with or without 100 ng/ml of Api6 (R&D company, Minneapolis, MN, USA) or 1 μg/ml of LPS for 24 hours. BrdU (10 μm, final concentration) was added to cells at the last hour. Cell proliferation was characterized by using the BrdU flow kit and F4/80 antibody (17–4801–82, eBioscience). In vitro apoptotic assay of Api6 was characterized by using Annexin V flow kit and F4/80 antibody.
The data shown were mean values of at least three independent experiments and expressed as Mean±SD. A paired Student’s t test or ANOVA was used to evaluate the significance of the differences. Statistical significance was set at a level of P< 0.05. Survival probability was calculated according to the Kaplan-Meier Method. Differences in survival were assessed with the log-rank test.