J. Biomedical Science and Engineering, 2011, 4, 609-619 JBiSE
doi:10.4236/jbise.2011.49077 Published Online September 2011 (http://www.SciRP.org/journal/jbise/).
Published Online September 2011 in SciRes. http://www.scirp.org/journal/JBiSE
IL13-induced lung fibrosis in meconium aspiration
Alexander Zagariya1*, Shangaral Navale2, Olga Zagariya3, Kenneth McClain4,
Dharmapuri Vidyasagar2
1Cancer Center at the University of Illinois at Chicago, Chicago, USA;
2Department of Cardiology, Northwestern University at Chicago, Chicago, USA;
3Department of Pediatrics, the University of Illinois at Chicago, Chicago, USA;
4Texas Children’s Cancer Center/Hematology Service to Baylor College of Medicine, Houston, USA.
Email: *zagariya@uic.edu
Received 16 May 2011; revised 26 June 2011; accepted 10 July 2011.
ABSTRACT
We demonstrated previously that inflammatory cy-
tokines TNFα, IL1β, IL6 and IL8 are significantly
expressed in meconium-instilled lungs. Captopril was
a strong inhibitor of meconium-induced lung injury,
inflammation and apoptosis and reduces lung alveo-
lar and airway epithelial cell damage. Presently we
demonstrate that IL13 expression in the meconium
aspiration syndrome (MAS). IL13 was maximally
expressed 8 hrs after meconium instillation. It was
previously described that IL13 plays a major role in
degradation of airway epithelial cells and inducing of
lung fibrosis by activating collagen production that is
a major point in identification of lung fibrosis. We
also showed that Captopril treatment significantly
inhibits IL13 expression in the lungs. We believe it
reduces meconium-induced lung injury and has a
therapeutic effect on histological and biochemical
functions of the lungs and possibly pulmonary fibro-
sis. Captopril treatment significantly reduced the
number of neuprophils and macrophages which ex-
press IL13 and levels of other inflammatory cyto-
kines after meconium instillation. Selective neutrali-
zation of IL13 ameliorated lung injury, airway hyper
responsiveness, eosinophil recruitment and mucus
overproduction.
Keywords: Meconium Aspiration Syndrome; IL13
Cytokine; New bo r n Lun gs; Fibrosis
1. INTRODUCTION
Meconium aspiration syndrome (MAS) causes inflam-
mation of airways and an inappropriate immune re-
sponse with elevated levels of TNF
, IL1
, IL6 and IL8
inflammatory cytokines expression [1]. Lung disease
severity directly correlates with progressive inflamma-
tion of the alveoli and airways which induce airway ob-
struction and cell apoptosis. We studied a meconium-
induced lung injury in isolated perfused rat lungs ex-
posed to anoxia [2]. We also demonstrated that inflam-
matory response in meconium-instilled lungs [3] resulted
in apoptotic cell death [4,5]. Lung cell response and in-
jury to inflammation is mediated via induction of cyto-
kines. Meconium in the lungs induces multiple cytokine
systems expression including TNF
, IL1
, IL6, IL8,
IL10, IL13 and other cytokines. From these IL10 and
IL13 are anti-inflammatory cytokine and others proin-
flammatory cytokines. Proinflammatory cytokines in
MAS have been studied extensively. However, the role
of anti-inflammatory agents, including IL13, have not
been studied. Its role for inducing a lung fibrosis is well
known, however, its presence in MAS was not demon-
strated before [9].
In our previous work we demonstrated that apoptotic
cell death can be effectively attenuated by treatment with
Captopril [6]. We also determined that angiotensin II
(ANG II) plays a major role in meconium-induced
apoptosis [7] and its inh ibitors like Lo sartan (inhibitor of
ANG II-AT1 receptors) dramatically decreases apoptotic
cell death [8]. Despite these findings the exact mecha-
nism of meconium-induced lung cell apoptosis remains
unclear.
Expression of IL13 receptors has been shown in vari-
ety of immune and nonimmune cells including B cells,
natural killer cells, monocytes, mast cells, endothelial
cells and fibroblasts [10-12], but not in T cells [3]. The
potent spasmogenic properties of IL13 have identified
this molecule as a potential regulator of airway hyper
reactivity in asthma and in emphysema [14]. It enhan ced
lung volumes and contributed to decreased compliance,
mucus metaplasia and inflammation. The effect of IL13
is similar to IL4. Both cytokines activate the JAK2 tyro -
sine-dependent signal transduction pathway [12]. Both
are located on human chromosome 5q25-31 [15] induce
activation of pulmonary fibroblasts [9] and mononuclear
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610
cells and macrophages [16]. Specifically, IL13 induces
fibroblast proliferation in the lungs [9,17] and liver [18].
IL13 enhances generation of other inflammatory cyto-
kines and chemokines (such as TNF
and IL6) inhibit or s
of IL13 or (IL13Ra2), markedly attenuated granuloma
development, pulmonary eosinophilia and pulmonary
fibrosis.
To induce injury IL13 should bind to its cell surface
receptor [19]. By blocking IL13 binding to its receptors
cell injury is significantly decreased. It was shown that
by blocking the IL13 receptor with IL13PE (IL13 im-
munotoxin) fibroblast proliferation decreased by about
30% [20]. Expression of IL13 induces significant colla-
gen type 1 and 2 production in fibroblast cell linesde-
monstrate a higher degree of proliferation [20]. We pro-
pose that limiting the exp osure of lung cells to IL13 will
significantly limit or reduce the degree of lung injury
and fibrosis [20].
2. MATERIALS AND METHODS
Meconium preparation. 10% meconium preparation and
cell death analysis were performed as described by us
earlier [5].
Pretreatment with Captopril. Two week old rabbits
were pretreated with 500 mg/L Captopril in their drink-
ing water 48 hrs prior to meconium instillation as de-
scribed previ o usl y [6] .
Pretreatment with Losartan. Another group of rabbits
received two days of pretreatment with Losartan (an AT1
receptor binding agent and inhibitor which inhibits pro-
duction of ANG II and consequently apoptosis) in the
rabbit chow targeted to approximate (by food consump-
tion) 5.0 mg/kg/day as described earlier [8].
Lung lavage. Rabbits were euthanized and lungs were
isolated. Lung lavage was obtained from the left lung
and the right lung was used for immunohistochemistry.
The left lung was cannulated and removed, and right
lung was instilled with 10 ml 1 × PBS and lavage was
collected immediately. The procedure was repeated 5
times and all lung lavage collections were pooled. Lav-
age cells were disrupted by freeze-thawing procedure
and then where centrifuged at 5000 RPM for 5 min at
4˚C to separate cell debris. Then supernatants were col-
lected and stored at 70˚C. The levels of IL13 were de-
termined immunologically using an ELISA kit (R&D
Systems Inc., Minneapolis, MI) according the manufac-
turers instructions. Lung lavage cell death was counted
after staining of cells by ethidium bromide and acrydine
orange as described pr eviously [5].
Lung histology. Rabbits were instilled intratracheally
with 10% meconium or normal saline and were sacri-
ficed 8 hrs afte r instilla tion. Following anes thesia-induced
euthanasia, the right lungs from meconium-instilled rab-
bits were separated and fully inflated with 4% formalin,
dissected, and placed in fresh formalin for 24 hrs. Rou-
tine histological techniques were used to paraffin-embed
the entire lung, and 2 m sections of whole lung were
stained with H&E. Each whole lung from the meconium
treatment group was examined for the presence of in-
flammatory infiltrates and interstitial fibrosis 8 hrs after
meconium instillation and graded with a previously de-
scribed scale [21]. For this purpose, 10% meconium was
instilled intratracheally into rabbit pups lungs and then
rabbits were sacrificed 8 hrs after instillation.
Immunohistochemistry. Paraffin-embedded whole
lung samples were analyzed for immunohistochemical
localization of IL13. This procedure was used for the
detection of IL13 expression in whole lung samples be-
cause of a growing concern that the ELISAs may under-
estimate IL13 actual levels in the lungs. Two micron
sections wer e de waxed with xelene, rehyd rated in gra ded
concentrations of ethanol, and blocked with normal rab-
bit serum (Vectastain ABC-AP kit; Vector Laboratories,
Burlingame, CA). A solution containing a 1/1 ratio of
water to methanol with 3% hydrogen peroxide was
added to each slide to be stained for the presence of IL13.
Goat anti rabbit IL13 and a control normal goat IgG
were diluted in PBS to a final concentration of 2 g/ml.
Specific IgG were added to histological sections for 60
min, after which each tissue section was washed thor-
oughly three times with PBS. A secondary rabbit
anti-goat biotinylated antibodies (Vector Laboratories,
Burlingame, CA) were also added to each section for 60
min, then each slide was thoroughly washed and to each
was added alkaline phosphatase (Vector Laboratories,
Burlingame, CA) conjugated to avidin. IL13 cytokine
was revealed with an alkaline substrate, 5-bromo-4-
chloro-3-indolyphosphate/nitro blue tetazolium. Cover
slips were applied to each slide using an aqueous
mounting solution. As positive IL13 control we used
human LCH tissue. As negative IL13 controlnormal
skin.
ELISA analysis. Rabbit IgG anti-IL13 monoclonal an-
tibody was coated on a 96 well plate overnight at 4˚C,
then serially diluted samples, or serial dilutions of IL13
used as a standard for 1 hr at 37˚C. Detection was per-
formed using a polyclonal biotinylated goat anti-rabbit
IL13 antibody from R&D systems (Minneapolis, MN),
according their instructions) for 1 hr at 37˚C.
The detailed technique for measurement of IL13 cyto-
kine in 50 l samples was described earlier [22]. Cell
samples were prepared from whole lung samples ho-
mogenized in 2 ml of PBS immediately before ELISA.
Each ELISA was screened to ensure antibody sp ecificity.
Recombinant cytokines were used to generate the stan-
dard curves for ELISA from which the concentrations
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611
present in the samples were calculated. The limit of
ELISA detection for each cytokine was consistently
more then 50 pg/ml. The cytokine levels in each sample
were normalized to total protein levels measured by the
Bradford Assay.
PCR analysis. IL13 gene expression was analyzed by
a real-time quantitative PCR procedure (Applied Bio-
systems, Foster City, CA). The cDNAs from lung lavage
samples before and at various times after meconium in-
stillation were analyzed for IL13 and GAPDH (an inter-
nal control). Al l primers and probes used were purc has ed
from Applied Biosystems (Foster City, CA). Cytokine
gene expression was normalized to GAPDH, and the fold
increases in IL13 gene expression was calculated via the
comparison of gene expression in all meconiuminstilled
lungs with that in lungs instilled by normal saline.
PCR conditions: PCR amplifications were conducted
on a robocycler gradient 96 (Stratagene) in a 30 l reac-
tion containing 100 ng of DNA, 10 mM Tris-HCl, pH9,
0.1% Triton X-100, 50 mM KCl, 0.2 mg/ml BSA, 1.5
mM MgCl2, 1 M f each primer, 1mM of dNTP and 1.5
U of Taq polymerase (Appligene Biosystems, Foster
City, CA). Following the initial den aturation step (94˚C,
5 min), samples were subjected to 35 cycles of PCR
consisting of 94˚C for 1 min, annealing temperature for
45 s and 72˚C for 45 s.
The PCR analysis for IL13 was previously described
by Graves et al. [42]. A 236-bp PCR fragment of IL13
cytokine was generated with use of the primers 5’-GCA-
AATAATGAGCTTTCGAAGTTTCAGTGG-3’ and 5’-
CTTCCGTGAGGACTGAATGAGACGGTC-3’. To con-
firm specificity of obtained fragment restriction analysis
were done. The NlaIV restriction enzyme analysis of the
PCR product obtained from subjects bearing the IL13
must have two bands (210 an d 26 bp) on 2% agar ose gel .
Loading volume including a loading buffer was 20 l
per well, same for all samples.
Statistical analysis. The data analyzed by ANOVA
followed. The significance of differences between ex-
perimental groups was analyzed using Student’s un-
paired t-test. Values were reported as the mean ± SEM.
Differences in mean values were considered significant
if p < 0.05.
3. RESULTS
Changes in IL13 expression after meconium-induced
lung fibrosis. We demonstrated by ELISA assays that
IL13 protein expression were increased at all times: 2, 4,
8 and 24 hrs after meconium instillation versus saline
instillation (Figure 1). The greatest, about five fold, in-
crease in IL13 protein expression compared to basal
level was observ ed 8 hrs afte r meconiu m instillation. For
a comparison, the expression of other cytokines in me-
conium-instilled lungs versus saline-instilled lungs is
also presented in the Figure 2. The meconium-saline
ratios of TNF
, IL6, and IL8 were increased signifi-
cantly in meconium-instilled lungs. TNF
reached
maximum at 2 hrs after meconium instillation and IL13,
IL6 and IL8 at 8 hrs after meconium instillation. In con-
trast, IL10 levels did not change significantly in any of
the groups studies.
Dependence of IL13 expression from Captopril pre-
tr eatment. In present study we determined whether pre-
treatment with angiotensin converting enzyme, Captopril,
will prevent meconium-induced lung fibrosis. Captopril,
similarly to Losartan described below did not change the
Figure 1. Expresison of IL13 in meconium-and saline-instilled lungs of two week old rabbit
pups. In each group of rabbits we used four pups. IL13 was measured at 0, 2, 4, 8 and 24 hr
after meconium instillation. Maximum expression was noted at 8 hr after meconium
instillation. Data are means SEM. *p < 0.001 by ANOVA when compared to saline instil-
lation alone.
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Figure 2. Ratio (meconium/saline) for different cytokines expressed. The expression of
each cytokine (pg/ml) was measured by ELISA in lung lavage obtained from meconium
and saline-instilled rabbits. Ratio meconium/saline was calculated and presented in this
figure. Ratio shows a increased expression of setrain cytokine in meconium instilled
lungs compared to saline-instilled lungs.
Figure 3. Captopril pretreatment inhibits meconium-induced IL13 and TNF
but not
IL10 cytokine expresison. Maximal expression if IL13 in meconium-instilled lungs was
noted at 8 hr after meconium instillation. Expression of IL13 was significantly decreased
by Captopril treatment. In contrast, maximal expression of TNF
cytokine was seen 2 hr
after meconium instillation and also was significantly reduced after Captopril treatment.
histological and immunological picture of the lungs (Fig-
ures 3, 7 and 8). Earlier it was demonstrated that Capto-
pril is able to redu c e lung cell inflammation, apoptosis [4 ]
and pulmonary fibrosis [24]. Pretreatment of rabbits with
Captopril significantly reduced meconium-induced ex-
pression of IL13, lung injury, lung apoptosis and fibrosis
compared to control rabbits, instilled with normal saline
(Figures 3 and 8). Captopril was also able to inhibit ex-
pression of other cytokines studied. Immunohistochemi-
cal analysis of IL13 in formalin-fixed whole lung sam-
ples from meconium-instilled rabbits showed a marked
decrease of IL13 in Captopril treated rabbits.
Description of meconium induced lung injury. Me-
conium-instilled lungs stained by Hematoxylin and Eo-
sin demonstrated a broken alveoli, accumulation of ne-
crotic cells (right side of the Figure 4), hemorrhage and
damage of bronchoalveolar alveoli. Alveolar walls of the
lungs instilled by meconium were thin, damaged and
fewer cells were observed compared to controls. The
sacules are not lined by cuboidal epithelium with apical
nuclei (progenitor alveolar epithelium). Several protu-
berances or bungles into the air spaces were noted as
well. Also, thinner secondary crests and alveoli are evi-
dent. Fewer nuclei are evident in the alveolar walls when
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compared to those of the term controls (Figure 4). Me-
conium-damaged lungs were analyzed in comparison
with saline instilled lungs, published b y us previously [4 ,
5].
Effect of Losartan on IL13 expression in MAS. We
further studied an effect of Losartan on IL13 expression.
Saline-instilled lungs with or without Losartan pretreat-
ment in immunohistochemical studies did not show
Figure 4. Meconium-induced lung injury. Hematoxylin and Eosin (H&E) staining revealed brocken
alveoli, accumulation of necrotic cells, hemorrhage and damage of bronchioles. Alveolar walls of the
lungs from meconium-instilled rabbit lungs were thin, damaged and have fewer cells compared to
controls (saline-instilled). The saccules are not lined by cuboidal epithelium with apical nuclei
(progenitor alveolar epithelium). Several protuberances or bugles into the air spaces were noted as
well. Also, thinner secondary crests and alveoli are evident. Fewer nuclei are evident in the alveolar
walls when compared to those of the term controls. Original magnification, ×40. Pictures were
analyzed side by side in comparison with normal lungs instilled with normal saline and published by
us earlier [4,5].
Losartan, Saline No Losartan, Saline
(a) (b)
Figure 5. Expression of IL13 in saline-instilled rabbits with or without Losartan pretreatment. This
figure presented as a control for IL13 staining in presence (a) and absence (b) of Losartan. Details
about Losartan pretreatment described in methods above. Note, in both cases IL13 cytokine is not
expressed.
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staining for IL13 (Figure 5). However, in meconium-
instilled lungs IL13 expression increased dramatically
with a maximum level observed at 8 hrs after meconium
instillation (Figures 1 and 6). We determined that BAL
IL13 levels were less then or equal to 75 pg/ml in me-
conium-treated lungs compared to 20 pg/ml in saline
treated. This expression was markedly attenuated by
Losartan. Expression of IL13 was always associated
with accumulation of mononuclear cells and macro-
phages in the lung lavage samples. Specifically, IL13
expression was observed in activated mononuclear cells,
airway epithelium cells and fibrotic interstitial areas.
No IL13 antibodies
(a) (b)
IL1 3 anti bodies
Losartan, Meconium Meconium
(c) (d)
Figure 6. Expression of IL13 in meconium-instilled lungs. (a)(c)—Losartan pretreament and then
meconium- instilled. (b)(d)—No Losartan, and then meconium-instilled. (a)(b)—No anti-IL13 antibodies.
(c)(d)— Anti-IL13 antibodies. Losartan pretreatment (c) abolished meconium-induced IL13 cytokine
expression (d).
Captopril
(a)
N o Capto pril
(b)
Figure 7. Absence of IL13 expression in control lungs with or without Captopril pretreatment. (a) Saline-
instilled rabbit lungs after Captopril pretreatment. (b) Saline-instilled rabbits without Captopril pretreat-
ment. Both panels shows no expression of IL13 cytokine.
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Captopril, Meconium Meconium
(a) (b)
Figure 8. Captopril pretreatment significantly inhibited meconium-induced IL13 expression. A—Captopril pretreated and
then meconium-instilled rabbit lungs. Magnification × 40. Seen much less dark brown staining. B—Maconium-instilled
lungs without Captopril pretreatment. Magnification × 40. Seen much higher dark brown staining (please note a marks on
the picture which pointed on the strong IL13 expression seen dark brown).
Histological evaluation of IL13 cytokine expression in
MAS. Newborn lungs instilled with normal saline did not
show any histological abnormalities in presence of
Losartan (Figure 5) or Captopril (Figure 7). However,
lungs instilled by meconium demonstrate strong expres-
sion of IL13 (Figures 4, 6 and 8), followed by inflam-
matory cell infiltration and lung fibrosis in rabbits. His-
tological changes were morphologically obvious. We
detected enlargement of alevoli with loss of the orderly
appearance. IL13-induced lung fibrosis was accompa-
nied with severe damage of bronchial and less damage
of vascular or alveolar epithelium. Maximum damage
was observed at 8 hrs after meconium instillation and
remained at the same level up to 24 hrs after instillation.
Inflammation consisted of increased number of macro-
phages, lymphocytes and eosinophils in peribronchial
and parenchyma tissues as well as lavage fluid.
PCR analysis of IL13 cytokine. Figure 9 shows an
PCR analysis for IL13 DNA in tracheal aspirates ob-
tained from newborn rabbits instilled intratracheally with:
lane 1—Saline, lane 2—Captopril, lane 3—Losartan,
lane 4—Meconium, lane 5—Captopril + Meconium,
lane 6—Losartan + Meconium, lane 7—NlaIV restriction
analysis of the 236 bp IL13 fragment. IL13 is absent in
non-inflamatory lungs but highly expressed in meco-
nium-instilled lungs. It’s expression can be dramatically
inhibited by pretreatment of lungs by Captopril (lane 5)
or Losartan (lane 6).
The PCR analysis in 2% agarose revealed 236 bp
IL13 fragments. The NlaIV restriction analysis of the
PCR product obtained from subjects bearing the IL13
gives two bands (210 and 26 bp) on same 2% agarose
gel (Figure 9, lane 7). This analysis confirmed specific-
ity of IL13 PCR fragment obtained. The purification
procedure and PCR analysis was controlled by levels of
GAPDH in same samples. Results show, that Captopril
or Losartan has virtually no effect on IL13 expression in
lung lavage of rabbits after saline instillation. However,
meconium-induced rabbit lung demonstrate a dramatic
expression of IL13. Densitometry data showed that pre-
treatment by Captopril inhibits meconium-induced IL13
expression by 85% compared to 65% inhibition by
Losartan (Figure 9, lanes 5, 6). These data showed that
Losartan and especially Captopril are effective inhibitors
of IL13 cytokine expression and meconium-induced
lung fibrosis .
4. DISCUSSION
It has been prev iously shown that IL13, but not IL4 wa s
a major profibrotic mediator in bleomycin-induced fi-
brotic mice [16]. However expression of IL13 in me-
conium-instilled lungs was not previously reported. IL13
was produced more str ongly in the lung, th en in th e he ar t,
liver, stomach, kidney, pancreas or testis [25]. The pre-
sent study addressed two major aims: 1) to examine the
expression of IL13 during development of meconium-
induced lung injury and fibrosis. 2) To determine the
role of IL13 responsive cells in the pulmonary fibrotic
response via targeting these cells by Captopril. It was
noted that IL13 has potential activating effects on
monocytes and macrophages that include enhanced IL6,
TGF, chemokines and chemokines receptors expres-
sion [26]. Resident macrophage and recruited monocyte
activation is a well-described feature of clinical pulmo-
nary fibrosis [27,28] and these cells contribute to the
pathogenesis of pulmonary fibrosis due to bleomycin
[29]. IL13 is a potent inducer of collagen production in
vivo in the lung [30]. It also was previously shown that
IL13 inhibits proteases and degrades the extra cellular
matrix [31]. This effect of IL13 is similar to the effect of
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Figure 9. PCR analysis of IL13 cytokine in 2% agarose gel. bp—1 kb molecular
weight marker. Lane 1—Saline, lane 2—Captopril, lane 3—Losartan, lane 4—
Meconium, lane 5—Captopril + Meconium, lane 6—Losartan + Meconium. Lane
7—The NlaIV restriction analysis of the PCR product obtained from subject bearing
the IL13 cytokine gives two bands (210 and 26 bp) on 2% agarose gel. Note, IL13
cytokine is absent in non-inflamatory lungs. However significantly expressed in
meconium-instilled lungs. It’s expression can be dramatically inhibited by pretreat-
ment of lungs by Captopril (lane 5) or Losartan (lane 6). Samples preparation and
loading control was done by using GAPDH.
protease inhibitors, serpins. A balance of pro- and anti-
fibrotic proteases is critical because excess protease or
anti-protease activity has been associated with excessive
pulmonary fibrosis induced by the anti-neoplastic drug
bleomycin. This imbalance in protease regulation is
regulated by the pulmonary fibrotic factors and events.
Expression of IL13 is dependent on the activity of extra
cellular proteases [30]. Usually an increase of proteinase
activities would prevent or reverse the increased deposi-
tion of extra cellular matrix and would be associated
with lung diseases and inflammation [31].
Recently, IL13 has been identified as a potent regula-
tor of bronchoconstriction in mouse models of asthma
[14]. This cytokine is primarily secreted from T cells but
it is also expressed by mast cells, macrophages, eosino-
phils and NK cells [33-35]. IL13 shares about 30% ho-
mology with IL4 and has similar biological functions.
IL13 is a central regulator of mucus production in the
lungs.
We found increased IL13 protein in areas of active fi-
brosis in newborn lung histological sections and its ex-
pression peaked 8 hrs after meconium instillation. Im-
munohistochemistry revealed that IL13 was concen-
trated preferentially in macrophages and mononuclear
cells near or in areas of active fibrosis. This finding was
consistent with previous observations that IL13 may be
expressed by normal human alveolar macrophages as
part of a homeostatic control process, but its production
was increased in the presence of fibrotic lung disease
[36]. IL13 has significant impact on the synthetic and
proliferative properties of granuloma fibroblasts, but not
normal fibroblasts, and the IL13-responsiveness of
granuloma fibroblasts renders them targets of IL13-
based immunotoxin therapy. It also was shown that IL13
is a potent inducer of cysteine proteases production in
the lungs [25]. Protease synthesis increases in a dose
dependent manner by IL13. As proteases are major de-
structive agents in the cells. They correlate with in-
creased IL13 expression in meconium-induced lung in-
jury. The levels of mRNA-encoding cysteine proteases
have been measured. IL13 also cause an impressive in-
crease in the levels of mRNA encoding cathepsins B, H,
K, S and others [25]. Cysteine protease inhibitors such
as E-64 or leupeptin d ecreased expression of IL13. Lung
emphysema causes dramatic increases of IL13 expres-
sion and promotes an increase in lung volume and lung
alveolar size. It also induces significant histological and
morphologic changes. These studies demonstrated that
cysteine proteases play a major role in the pathogenesis
of IL13-induced lung inflammation and later fibrosis.
IL13 induced lung enlargement was significantly de-
creased by treatment with cysteine protease inhibitors
and antagonists [25]. To understand the balance of pro-
teases and antiproteases in the lungs the levels of IL13
must be examined. We believe, that IL13 is an important
regulator of pulmonary protease and antiprotease bal-
ance that can contribute to the patophysiology of MAS.
Previous studies suggest that the deregulation of apop-
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617
totic pathways may explain the pathogenesis of a number
of chronic lung diseases [37]. Our interest in IL13 in-
duction of genes responsible for an apoptotic process in
the lung stemmed from preliminary studies, where
IL13-induced cytotoxicity in tumor cell lines via its abil-
ity to up-regulate proapoptotic and down-regulate anti-
apoptotic factors in these cells [15]. The TNF
related
proapoptotic factor TRAIL is a potent inducer of fibro-
blast apoptosis by induction of a number of proapoptotic
factors, especially caspase 9 [38].
IL13 is a pleiotropic cytokine with remarkable effects
in the newborn lungs. Overexpression of IL13 causes
macrophage, lymphocyte and eosinophILrich pulmonary
inflammatory response, mucus metaplasia, crystal depo-
sition, subepitelial fibrosis and alveolar enlargement in
MAS. The majority of alveoli are formed by develop-
mental process that occurred at birth and we not sure
whether alveolar enlargement is due to alterations in
alveolar development or due to the destr uction of alr eady
normally formed alveoli. However, results with adult
rabbits also showed alveolar enlargement. These results
demonstrate, that alveolar enlargement is a short term
lung response to injury but lung fibrosis require more
extensive periods of time. We believe that injuries effect
of IL13 involved a production of ANG II and ACE in-
hibitor Captopril should significantly decrease its inju-
ries effect. Captopril inhibits conversion of ANGEN to
ANG II and consequently apoptosis formation. IL13 is
also is a strong activator of tissue proteolysis [2 5].
Blockage of serine or cysteine proteases has remarka-
bly ameliorated the effects of IL13 induced lung in-
flammation and apoptosis. The effect of cysteine prote-
ase inhibitors, serpins, on IL13 effect was also evaluated.
Serpins abrogated the IL13-induced lung inflammatory
response [25]. Serpins also were able to significantly
reduce IL13 induced increase in lung volume and alveo-
lar size. They reduce IL13 induced lung and lavage cell
inflammation.
Our findings demonstrate the impact of IL13 on the
alveolar cells, lung injury and potentially on lung fibro-
sis. The responses of alveolar cells to the expression of
IL13 suggest that these cells could be targeted with an
anti-IL13 antibody [39]. It is very possible that its anti-
bodies and other inhibitors may be important in pre-
venting a MAS and consequent lung fibrosis.
5. CONCLUSIONS
We have demonstrated elevated expression of the in-
flammatory cytokine IL13 in meconium-instilled two
week old rabbit pups’ lungs. We also showed that pre-
treatment with Captopril significantly decreased me-
conium-induced expression of IL13 cytokine as well as
meconium-induced lung injury and apoptosis. This find-
ing suggests that the mechanism of IL13 induced lung
injury is similar to that caused by TNF
, IL6 and IL8
cytokine mechanisms.
6. ACKNOWLEDGEMENTS
We are grateful for a support of Dr. Garry Kruh, UIC Cancer Center
Director, who unexpectedly left us earlier this year as a result of a
tragic death.
We also thank Dr. Beck and Dr. Ozer who secured an opportunity to
complete a several projects af ter this happens.
We thank Huali Dong, Islam Shariful and Derrik Jordan for a won-
derful technical and supportive assistance.
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