SUPPLEMENTARY ONLINE MATERIAL
Keratinocyte growth factor protects against Clara cell injury
induced by naphthalene
Ali Önder Yildirim1, Martina Veith1, Tanja Rausch1, Bernd Müller1 Patrick Kilb1, Laura
S. Van Winkle2, Heinz Fehrenbach1
1Clinical
Research Group "Chronic Airway Diseases", Medical Faculty, Philipps-
University Marburg, Germany,
2Department
of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine
and Center for Health and the Environment, University of California, Davis, California,
95615, USA
MATERIALS AND METHODS
Kinetics of naphthalene (NA) induced acute airway injury
NA was purchased from Fisher (Aschaffenburg, Germany) and dissolved in corn oil.
To analyze the kinetics of NA-induced Clara cell injury in C57BL/6 mice, mice
injected with 200 mg NA per kg body weight or corn oil alone (10 µl per gram body
weight) as vehicle control were killed after 2, 3, 6, 8, 12, 16, and 24 h after injection
(n=3 per group). Distal airways were microdissected and processed for histologic
analysis. To exclude effects of circadian rhythm, all applications were performed
between 8 am and 10 am. Mice were sacrificed 12 h after NA treatment and distal
airways were isolated by microdissection.
RNA Isolation and Real-Time Reverse Transcription Polymerase Chain
Reaction (RT-PCR)
Total RNA was isolated from microdissected airways using SV Total RNA Isolation
System (Promega, Germany), and cDNA was synthesized with Omniscript Reverse
Transcription Kit (Quiagen, Hilden, Germany). Real-time RT-PCR was performed
using QuantiTect® SYBR® Green Master Mix (Abgene, Germany) and BioRad ICycler
(BioRad Laboratories, Hercules, Germany). 10 μl QuantiTect® SYBR® Green Master
Mix, 1 μl of each primer at a concentration of 50 pmol/μl, and 20 μl water were added
to 3 μl of cDNA, standard or water (negative control). Primer sequences were generated from the respective mRNA sequences obtained from the European Molecular
Biology Laboratory (EMBL) gene bank (Table E1). Primers were synthesized by
MWG Biotech (Ebersberg, Germany).
Double Immunohistochemistry
As described earlier [17], cross sections of airways 2 μm in thickness were deparaffinized in xylene, and rehydrated in ethanol and PBS. Endogenous peroxidase
activity was inactivated with 1% H2O2 in Methanol (Roth, Karlsruhe, Germany), pH
7.2, for 30 min. Antigen retrieval was performed by microwave treatment in 3% citrate
buffer at pH 6.0 (Roth, Karlsruhe, Germany). To identify ciliated cells immunostaining
was performed using mouse monoclonal antibodies against -Tubulin-IV (Bio Genex,
San Ramon,CA, USA; diluted 1:150). Antibodies were detected using a mouse-onmouse immunodetection kit (Vector MOM kit, Vector Laboratories). For the
visualizing was using 3,3′-diaminobenzidine as chromogen according to the ABC
method (Vectastain Elite ABC Kit; Vector Laboratories, USA) following the
manufacturer's instructions. For the immunostaining of Clara cells the same sections
were washed in PBS then incubated for 30 min in PBS with 1% bovine serum
albumin (Serva, Heidelberg, Germany) followed by incubation with a polyclonal rabbit
antibody (courtesy of Jörg Klug, Marburg) against Clara cell-specific protein 10 kD
(CC10) diluted 1:3000 in the same solution at +37 C for 1 h. Sections were then
incubated at room temperature for 30 min with anti-rabbit secondary antibody diluted
1:10., which was visualized using HistoGreen- Kit (Linaris, Wertheim-Bettingen,
Germany) for 8,5 min. All sections were counterstained with hematoxylin.
RESULTS
Kinetics of Acute Airway Injury induced by Naphthalene
At 2 h after NA-treatment, the bronchiolar epithelium lining the distal airways
exhibited no alterations compared to distal airway epithelium of corn oil treated
controls. However, at 3 h and 6 h post NA-injection (some) Clara cells were swollen
and slightly vacuolated. From 8 h to 24 h, alterations became increasingly severe.
Most Clara cells contained many vacuoles and were swollen until almost complete
exfoliation was observed at 24 h after NA-treatment. At this time-point, the
bronchiolar epithelium mainly consisted of squamous ciliated cells covering the basal
lamina (Fig. E1). At 12 h after NA-injection, approximately 50% of the Clara cells
were lost by exfoliation (see Fig. 3 of manuscript). Therefore, this time-point was
chosen for all experiment to define the effect of N23-KGF pre-treatment on NAinduced acute airway injury.
FIGURE LEGENDS
FIGURE E1. Hematoxilin-Eosin staining of distal airways of mice. Mice were treated
with naphthalene (200 mg/kg) and sacrificed after 2, 3, 6, 8, 12, 16, and 24 h.
Nucleus of cells are stained dark-blue. Clara cells (arrow) as well as ciliated cells
(black asterisk) are visible in the corn oil treated mice (Fig.E1 A). Mice, which were
treated for 2 h (Fig.E1 B) with naphthalene (NA), reveal no morphological alteration.
Slight vacuolation (white asterisk) and swelling (arrow) is seen at 3 h (Fig.E1 C) and
6 h (Fig.E1 D) after NA-injection. At 8 h (Fig.E1 E) Clara cells begin to exfoliate
(arrow) and severity of morphological alterations increased at 12 h (Fig.E1 F) and
16 h (Fig.E1 F). At 24 h (Fig.E1 H) most of the Clara cells are lost by exfoliation and
the basal lamina is covered by a layer of squamous epithelial cells. Scale bar 12 µm.
FIGURE E2. Double immunohistochemistry for ciliated cells and Clara cells. The
distal airway epithelium of mice was almost exclusively composed of Clara cells
(green) and ciliated cells (brown). Sections were counterstained with hematoxylin.
Scale bar 100 µm.
6
Table E1. Primers for quantitative Real-Time Reverse Transcriptase-Polymerase
Chain Reaction.
Gene
Size bp
Primer Sequence (Sense-Antisense)
TM (°C)
5mGAPDH
3mGAPDH
5`- AATGGTGAAGGTCGGTGTGAAC-3`
5`- GAAGATGGTGATGGGCTTCC-3`
53, 60
262
5mPCNA
3mPCNA
5`-CCACATTGGAGATGCTGTTG-3`
5`-CCGCCTCCTCTTCTTTATCC-3`
60
128
5mCC10
3mCC10
5`-GCTCAGCTTCTTCGGACATC-3`
5`-CTCTTGTGGGAGGGTATCCA-3`
60
167
5mCyp2F2
3mCyp2F2
5`-GTCACTCGGGACACACCTTT-3`
5`-TCCTTGATGCACATTGTGGT-3`
53
116
GAPDH: Glyceraldehyde phosphate dehydrogenaase; PCNA: Proliferating Cell
Nuclear Antigen; CC10: Clara cell-specific protein 10; Cyp2F2: cytochrome P450,
family 2, subfamily f, polypeptide 2