Requirement for TNF-Α Receptor I for P44/42 and C-Jun N-Terminal Kinase Activation And

Acid-induced acute lung injury in mice associates with p44/42 and c-Jun N-terminal kinase activation and requires the function of Tumor Necrosis Factor-α 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

1”Marianthi Simou” and Experimental Surgery Laboratories, 1st Dept. of Critical Care, “Evangelismos” Hospital, National and Kapodistrian University of Athens Medical School, Athens, Greece

21st Dept. of Critical Care, “Evangelismos” Hospital, National and Kapodistrian University of Athens Medical School, Athens, Greece

32nd Dept. of Critical Care, “Attikon” Hospital, National and Kapodistrian University of Athens Medical School, Athens, Greece

4Institute of Immunology, “Alexander Fleming” Biomedical Sciences Research Center, Vari, Greece

5Dept. of Surgical Pathology, “Evangelismos” Hospital, Athens, Greece

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), 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 gene, both the forward 5′-CCGGGAGAAGAGGGATAGCTT-3′ as well as the 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 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. SDS-PAGE 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

Release of TNF in BAL of mice challenged with HCl. To ascertain the presence and kinetics of TNF in the rodent model of acid aspiration, we challenged mice with it HCL and measured TNF levels in the BAL six and 24 hours post-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.

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.