Download Apoptosis of Lung Epithelial Cells in Response to Meconium and

EXPRESSION OF SERINE/CYSTEINE PROTEASE INHIBITOR a1-ANTITRYPSIN IN NEWBORN LUNGS IN EXPERIMENTAL MECONIUM ASPIRATION
D. Vidyasagar1, R. Bhat1, Shan Navale2, E. Zhabotynsky2,3, Gopal Chari1, Q. Xu3, T. Keiderling3, A.Zagariya1,2
1 Pediatrics, The University of Illinois at Chicago, Chicago, IL, United States, 60612
2 Pediatrics, Michael Reese Hospital and Medical Center, Chicago, IL, United States, 60616
3 Department of Chemistry, University of Illinois at Chicago, Chicago, IL
Expression of a1-antitripsin in saline and meconium-instilled lungs.
Exp. 1 Saline Exp. 2 Saline Exp. 3 Saline Exp. 4 mec Exp. 5 mec. Exp 6 mec.
4 hrs after
Instillation
8 hrs after
Instillation
24 hrs after
Instillation
0.090
0.061
0.060
0.462
1.778
1.910
0.168
0.070
0.068
1.293
2.649
2.503
0.296
0.121
0.083
2.563
2.795
>3.001
INTRODUCTION
Meconium aspiration induces inflammation of the lungs leading to clinical Meconium
Aspiration Syndrome (MAS). Clinically MAS manifests hypoxia, hyperoxia, and acidosis
requiring oxygen therapy, assisted ventilation, and in severe cases inhaled nitric oxide
therapy and Extracorporeal Membrane Oxygenation or ECMO.
Meconium induces two forms of pathology : obstruction of small and moderate size
airways by the particulate meconium and another by way of pulmonary inflammation and
damage at cellular level. Recent literature including our own laboratory reported elevation
of cytokines, phospholipases, inflammatory mediators and nitric oxide accumulation in
meconium-instilled lungs. We analyzed the lung aspirates for various proteins including
cytokines. Lung washes after meconium instillation demonstrated excessive inflammatory
cells and proteins. The cells have been identified as polymorphonuclear cells, and
macrophages. The proteins have not been characterized in detail. It is known that cytokines
IL6, IL8 and TNFa are part of the expressed proteins. These cytokines are known to cause
apoptosis of lung epithelial cells. It is not known if there are other proteins which lead to cell
death. We hypothesized, that proteins other than cytokines are involved in meconiuminduced lung injury (MILI). To explore this hypothesis we analyzed all the proteins
expressed in lung lavage after meconium exposure. We characterized the proteins in lung
lavage to better understand their role in meconium-induced lung injury.
MATERIALS AND METHODS
Study design:
Two-week-old rabbit pups were used for the study.10% meconium solution was prepared as previously described (Zagariya A. et al
Eur. J. Ped. 2000).Pups were anesthetized by IP of 10 mg/kg Ketamine and 1 mg/kg Xylazine. 1.2 ml/kg of the 10% sterile
meconium supernatant (experimental group) or an equivalent volume of 0.9% NaCl (control group), was instilled into lungs via the
tracheotomy followed by a 5 ml bolus of air to disperse the meconium .Pups were allowed to breathe room air spontaneously. Pups
in each group were sacrificed at 0 hrs or at 8 hrs after instillation. Lungs were isolated for lung lavage. The lavage fluid was stored at
20oC.
Protein Purification:
The protein in the lung lavage was extracted by freeze-thawing procedure using dry ice at room temperature. Then, extract was
resuspended in ice-cold buffer (20 mM HEPES, pH 7.0, 50 mM NaCl and 1 mM EDTA), and centrifuged at 21,000g (4oC ) for 15
minutes. Proteins were isolated from cell extract using acetone precipitation and then purified to homogeneity on a Sephadex G100
column (2cm x 50cm), equilibrated with 0.05M sodium phosphate buffer at pH 7.0, the protein was eluted in 0.1 M sodium chloride.
The eluted protein was collected in 8 successive tubes (2.5 ml in each) and dialyzed overnight against 20 mM Tris-HCl, pH 7.5 in
refrigerator. The dialysate was concentrated with Centricon-30 (Amicon, Beverly, MA) and was stored at –80oC. The yield of the
protein was about 0.3 mg/ml in a total volume of 20 ml (8 tubes x 2.5 ml). After each purification step, protein purity was monitored
by SDS-PAGE (Fig. 1), and enriched fractions of 50 kDa protein were removed and used for purification (upto 90%).
SDS gel electrophoresis and protein sequencing:
Dialyzed protein was stored in the assay buffer (50 mM Tris-HCl, 20 mM CaCl2, 100 mM NaCl). Protein concentration was
determined with Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) using the Bradford method. Equal amounts of purified protein
from each sample (10 mg) were mixed with 2x gel loading buffer (4% SDS, 20% Glycerol, 120 mM Tris-HCl, pH 6.8, 0.01%
bromophenol blue, 2% b-mercaptoethanol), denatured at 95oC for 5 minutes and separated by 10% SDS gel electrophoresis at
constant voltage of 100 V according to the method of Laemmli. Protein bands were visualized after staining by 0.25% Coomassie
Brilliant Blue R-250, 45% methanol and 10% acetic acid.
For protein sequencing, 100 pmol of the 50 kDa protein, excised from the gel, was incubated at 37oC for 30 min, followed by the
addition of 3,4-dichloroisocoumarin or PMSF to a final concentration of 1 mM to neutralize uninhibited elastases. The reaction
mixture was subsequently desalted and washed by centrifugation on a ProSpin sample preparation cartridge (Perkin-Elmer/ABI;
Foster City, CA), and sequenced in a Beckman LF3000 protein sequencer (courtesy of Rockefeller University at New York, NY). The
lung lavages both from saline and meconium-instilled lungs were subjected to the above procedure for isolation and identification of
the protein.
Data analysis: We used ANOVA to quantities statistical significance of measurements. Results of each parameter within a rabbit
group were expressed as mean ± standard deviations. Paired evaluations were made for experimental and control groups, and the
significance was determined. Statistical significance was taken as p<0.05.
RESULTS
OBJECTIVE
1. To study the protein content differences between meconium and saline-instilled
lungs.
2. To characterize the proteins obtained from lung lavage from both groups.
Fractionation of lung lavage proteins from meconium-instilled rabbit lung lavage. Eluted protein
was seen as a one band or, possibly, two very close bands with a molecular weight of about 50 kDa
(see figure 3). This protein was excised from the gel, electro eluted, dialyzed against 10 mM Tris-HCl
(pH 7.6) in presence of protein denaturation inhibitor phenilmethylsylphonil fluoride (PMSF) 3 times per
8 hrs each at 4oC. Then the isolated protein was sequenced by the Rockefeller University sequencing
facility, and the results are presented below. Table I. Table below shows a list of proteins with
highest homology to our 50 kDa protein of interest. The closest protein to our protein is a1antitripsin. The other homological proteins are also listed.
--------------------------------------------------------------------------------------------------------------------------Protein Information and Sequence Analyze Tools:
%
kDa
--------------------------------------------------------------------------------------------------------------------------15300686 ref XP 028358.2 serine (or cysteine)
protease inhibitor, clade A (alpha-1 antiprotease,
antitrypsin
32
40.45
3183393 sp O1377 YE99 SCHPO Hypothetical 35 kDa
Protein C17A5.09C in chromosome 1
17
34.96
1077406 pir S51421 Hypothetical protein YLR176c,
Yeast (Saccharomyces cerevisiae)
13
85.65
1705613 sp P49319CAT TOBAC Catalase Isozyme
(Salicylic Acid Binding Protein)
11
56.81
15228123 ref NP 178512.1 Mutator-like transposase
(Arabidopsis thaliana)
11
88.93
6730143 pdb 1C50 A Chain A, Identification and
structural characterization of a novel allosteric
10
95.69
binding site of glycogen phosphorilase B
7662224 ref NP 055626.1 KIAA0649 gene product
(Homo sapiens)
7
127.33
Protein sequencing results.
The homology search of this protein using NIH protein sequence database showed high homology
with the predicted amino acid sequence of a1-antitrypsin (serine/cysteine inhibitor shown in red.
The presence of serine/cysteine inhibitor (SERPIN) in MAS is a new finding. We believe serpin is
released lung cells in response to meconium injury.
The importance of this finding and its role in MILI is schematically shown below.
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
1
HYPOTHESIS
We hypothesized:
1. Meconium instillation into the lungs induces expression of proteins other than the
known inflammatory cytokines.
2. These proteins act in conjunction with cytokines to induce Meconium Induced Lung
Injury (MILI).
This gel demonstrated enrichment of 50 kDa
protein in fractions 23-26 with maximal
concentration of 0.3 mg/ml in line 24.
Fractions 23-26 were excised from the gel and
sequenced (see table below).
Results of the studies are shown in figures 1-3 and table 1.
Eluted Proteinv(mg/ml)
Background: Meconium aspiration syndromes (MAS) induce inflammation, cytokine
expression and cell apoptosis. Serine proteases are activated in response to cytokines
and involved in apoptosis. Apoptotic-induced caspases are less expressed in presence
of protease inhibitors. We, hypothesize, that serpine may attenuate meconium-induced
inflammation and inhibit lung cell apoptosis. Its deficiency leads to exposure of lungs to
uncontrolled proteolytic attack from neutrophil elastase or other damaging factors
culmination in the lung destruction and cell apoptosis.
Objective: To study the protein content in the lung washes following meconiuminstillation and compare it with the proteins expressed in lung washes following salineinstillation. Second objective is to characterize expressed proteins.
Design/Methods: We used two-week-old rabbits in the study: Group I, meconiuminstilled. Group II, saline instilled. Meconium as earlier published. A small midline
incision was made to expose the trachea and 1.2 ml/kg of 10% meconium supernatant
was instilled into the lungs. Rabbits were sacrificed at 4, 8 and 24 hours after meconium
or saline instillation. Then chest was open, lungs were isolated and lung lavage was
performed. The lavage was used to study alveolar cell death apoptosis. Total protein
from Sephadex G100 column was analyzed by 10% SDS gel electrophoresis. Protein
bands were visualized after staining with Coomassie Blue. For protein sequencing 100
pmol of 50kDa protein, excited from gel, was sequenced by Backman LF3000 protein
sequencer (Rockefeller University, New York).
Results: We found that in meconium-instilled lungs expressed a 50kDa protein,
Sequencing analysis of this protein demonstrate that it is a1-antitripsin (serpin). We
purified obtained protein using chromatography on Sephadex G100, according
manufacturers instructions. Protein was relatively stable in water and saline. Heat
inactivation changed protein irreversibly.
Conclusions: We found significant expression of a1-antitripsin in meconium-instilled
lungs. It is a serpine/sycteine protease inhibitor which expressed in newborn lungs as a
protective mechanism against meconium-induced lung injury. Increase of a1-antitripsin
expression is important in preventing meconium induced lung injury.
Fig. 3. 10% SDS gel electrophoresis from
fractions 20-29 obtained from gel filtration on
Sephadex G100. As a control we loaded high
(A) and low (B) protein molecular weight
markers on the gel.
3
5 7
9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
Fraction from Sephadex G100 (2.5 ml each)
Fig. 1. The above figure shows 10% SDS gel
electrophoresis of proteins from saline and
meconium-instilled lung lavage fluid.
Accumulation of 50 kDa protein in meconiuminstilled (B) but not in saline instilled (A) lungs is
seen. Saline instilled lungs have a negligibly small
amount of protein staining in the same area.
26 kDa represents molecular weight of TNFa.
Fig. 2. Gel filtration of lavage proteins from meconium-instilled rabbit lungs using
Sephadex G100 chromatography. Column was equilibrated with a normal saline as a buffer.
The numbers 1-39 on x axis are fractions collected from the column. Y axis shows the protein
quantity in the fraction collected. Majority of protein was eluted in the fractions 21-29. These
fractions were collected and analyzed on 10% SDS gel as described in Fig. 3.
CONCLUSION AND SPECULATION
We demonstrated a 50 kDa protein in meconium instilled lungs.
We also demonstrated that this protein belongs to Serpin class of proteins as shown by
protein sequencing.
We speculate that Serpin interacts with neutrophil elastase to form an inactive protein thus
decreasing the MILI.
ACKNOWLEDGEMENTS:
This work is supported by Thrasher Foundation Grant # 02823-6.