Acid-induced acute lung injury in mice associates with p44/42 and c-Jun Nterminal kinase activation and requires the function of Tumor Necrosis Factorα Receptor I
Nikolaos A. Maniatis1,3, Aggeliki Sfika1, Ioanna Nikitopoulou1, Alice G. Vassiliou1,
Christina Magkou5, Apostolos Armaganidis3, Charalambos Roussos1,2, George
Kollias4, Stylianos E. Orfanos1,3, Anastasia Kotanidou, MD1,2
”Marianthi Simou” and Experimental Surgery Laboratories, 1st Dept. of Critical Care,
1
“Evangelismos” Hospital, National and Kapodistrian University of Athens Medical
School, Athens, Greece
2 st
1
Dept. of Critical Care, “Evangelismos” Hospital, National and Kapodistrian
University of Athens Medical School, Athens, Greece
3 nd
2
Dept. of Critical Care, “Attikon” Hospital, National and Kapodistrian University of
Athens Medical School, Athens, Greece
Institute of Immunology, “Alexander Fleming” Biomedical Sciences Research
4
Center, Vari, Greece
Dept. of Surgical Pathology, “Evangelismos” Hospital, Athens, Greece
5
Detailed methodology
Reagents: Antibodies: anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204),
anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-p44/42 MAP Kinase, anti-SAPK/JNK
and anti-Caspase-3 were purchased from Cell Signaling Technology (Danvers, MA,
USA). Etanenercept (Enbrel) was from Pfizer (NY, USA), Dynamo SYBR Green and
PCR Master Mix from Finnzymes (Thermo Scientific, Waltham, MA, USA), Vectastain
ABC Kit and DAB peroxidase staining kit from Vector Laboratories (Burlingame, CA),
1
Supersignal ECL detection system was from Pierce Biotechnology (Rockford, IL,
USA). All other reagents were obtained from Sigma (St Louis, MO, USA).
Quantitative Real-time PCR: RNA extraction from lung tissue samples using
the Trizol reagent and the PureLink RNA Mini kit (Invitrogen Corporation, Carlsbad,
CA, USA) and first-strand cDNA synthesis using the M-MuLV Reverse Transcriptase
(NEB, Ipswich, MA, USA) and Oligo(dT) primers were performed according to
manufacturer instructions. A highly sensitive quantitative real-time PCR (qPCR)
method was developed for the quantification of both GAPDH and TNFRI mRNAs,
with the use of SYBR® Green Dye detection systems. The NCBI Sequence database
and the advanced software of the Primer Express programme were used for design
of gene-specific primers, as follows: for GAPDH (endogenous reference gene) the
forward primer, consisting of 21 nt, 5′-AGGTCGGTGTGAACGGATTTG-3′ and the
reverse primer of 23 nt 5′-TGTAGACCATGTAGTTGAGGTCA-3′, gave rise to a 136
bp
amplicon;
for
the
TNFRI
CCGGGAGAAGAGGGATAGCTT-3′
as
gene,
well
both
as
the
the
forward
5′-
reverse
5′-
TCGGACAGTCACTCACCAAGT-3′ primers, each with a length of 21 nt, produced an
amplicon of 113 bp.
Quantitative real-time PCR analysis was performed in 96-well plates on a
PTC-200 Thermal Cycler (MJ Research Inc., Waltham, Massachusetts MA, USA).
The 25 μL reaction mixture contained 10 ng cDNA, 0.3 μM primers and 1× SYBR®
Green PCR Master Mix, in which Thermus brockianus DNA polymerase was
included. All samples were amplified in triplicates and the average Cycle Threshold
(CT) values were calculated for their subsequent expression analysis. Following
amplification, a dissociation curve was generated to distinguish the PCR products of
interest from the non-specific ones or any primer-dimers, through their particular
melting temperatures (Tm), recorded in the software. Using the comparative CT
method 2−DDCT and saline-treated mice as a calibrator, the relative quantification of
the expression analysis of all mice samples treated with HCl was carried out. GAPDH
2
expression was used for the normalization of TNFRI expression levels between the
different samples.
Immunohistochemistry. De-paraffinized 5 μm-thick sections of formalin-fixed,
paraffin-embedded mouse lung tissue samples were incubated for 10 min with 3%
H2O2 to quench the endogenous peroxidase activity. After heat-mediated antigen
retrieval in sodium citrate buffer (pH 6.0) for 15 min, tissues were incubated with 5%
fetal bovine serum in PBS for 30 min at ambient temperature, followed by incubation
with primary antibody at 40C overnight. Sections were washed in PBS-Tween 20 and
antibody binding was detected using Vectastain ABC Kit according to manufacturer
instructions. After washing the slides, visualization using 3,3-diaminobenzidine as
chromogen was performed with DAB peroxidase substrate kit. Slides were
counterstained with hematoxylin, mounted and observed under an Olympus
(BX50F4) microscope.
Immunoblotting. Lung tissue samples were homogenized in ice-cold lysis
buffer (containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% Sodium
Dodecyl-Sulfate, 50 mM Tris pH 8.0 and protease inhibitor cocktail) using the Tissue
Tearor by Biospec Products and centrifuged for 15 min at 40C and 13,000 x g. SDSPAGE electrophoresis of supernantants was performed on a ‘‘Biorad Mini Protean II’’
apparatus (Bio-rad, Hercules, CA, USA), using 10% polyacrylamide gels. Following
electrophoresis, samples were transferred onto an Immobilon-P PVDF membrane
(Millipore, 0.45 μl pore size, Millipore Corporation, Billerica, MA, USA). Western
transfer was performed on a wet transfer apparatus (Bio-rad) and immunological
detection was performed using primary and horseradish-peroxidase-conjugated
secondary antibodies. Band quantification was performed with densitometry using
the Image J analysis software (National Institutes of Health, Bethesda, MD, USA).
Supplemental results
3
Release of TNF in BAL of mice challenged with HCl. To ascertain the
presence and kinetics
of TNF in the rodent
TNF in BAL
300
*
model
aspiration,
of
acid
we
200
challenged mice with
it HCL and measured
100
TNF levels in the BAL
six and 24 hours post-
0
HCL 6hr
HCl 24 hrs
HCL
challenge.
Levels of TNF were determined by ELISA (Peprotech, Rocky Hill, NJ) according to
manufacturer’s
instructions. As shown in “Supplemental Figure 1”, intratracheal
njection of HCl induces a time-dependent increase in TNF levels in BAL fluid, which
peaks at 6 hours post-injection and returns to baseline by 24 hours.
Supplemental Figure 1: Bronchoalveolar lavage fluid levels of TNF in mice
challenged with intratracheal injection of HCl. Mice were sacrificed at the indicated
time-points and TNF was determined by ELISA. * denotes p<0.05 by Student’s t-test.
Activation of p44/42 by HCl in TNF-/- . We tested the activation status of p44/42 in
mice lacking TNF expression in response to it HCl by immunoblotting for activated
(phosphorylated) p44/42 (“Supplemental figure 2”). In these mice, we observed
increased levels of p44/42 (n=2) upon HCl exposure, compared to mice treated with
placebo.
4
p-p44/42
total p44/42
Supplemental Figure 2: Immunoblotting study of activated (phosphorylated) p44/42
(top) in whole-lung homogenate of TNF-disrupted mice treated with intratracheal
normal saline (NS) or HCl for 24 hours. Total p44/42 (bottom) is used as loading
control.
5