TUESDAY, SEPTEMBER 20 TH 2005
Sheep pleural tissues were mounted among Ussing type chambers. Visceral pleura P3574
came from upper lobes (apical) and caudal lobes (caudal), while parietal was Aspergillus fumigatus conidia induce internalization dependent IFN-beta and
stripped over 1st – 4th rib (apical) and 8th – 12th rib (caudal). Insulin (10-5 M) was IP-10 expression in airway epithelial cells
added towards both sides of tissue. Potential Difference (PD) was measured 1, 5, Christoph Beisswenger 1 , Simone Anders 2 , Klaus Schroppel 2 , Robert Bals 1 .
1
10 and 30 min after hormonal addition. Trans-membrane Resistance (RTM ) was Internal Medicine, Hospital of the University of Marburg, Marburg, Germany;
2
calculated from Ohm’s law. Klinische Mikrobiologie, Universitaet Erlangen, Erlangen, Germany
Insulin increased RTM of visceral and parietal pleura within 1st min (insulin vs con-
trol, p0.05). heat inactivated resting A. f. conidia and swollen conidia, with living resting conidia
It is concluded that mesothelial cells appear different capability of transporting or with Pseudomonas aeruginosa for 1 and 6 hours. IFN-beta, IP-10, IL-6, IL-8
electrolytes and liquid, accordingly to which region of pleura they are situated as and hBD-2 induction was measured by RT-PCR. Internalization was blocked by
a response to hormonal agents. cytochalasin D.
Results: Resting conidia and P. a. but not swollen conidia induced IFN-beta and
IP-10 gene expression in HBEC. This induction was blocked by cytochalasin D.
In contrast to swollen conidia stimulation with resting conidia resulted in late
induction of proinflammatory cytokines. Furthermore, defensin expression was
induced by P. a. but not by A. f. conidia.
340. Airway epithelial cells in inflammatory Discussion: These results show that HBEC can recognize A. f. Stimulation with
swollen A. f. conidia only resulted in an induction of proinflammatory cytokines,
and host defence whereas resting conidia led to a strong expression of IFN-beta and IP-10 and
late induction of proinflammatory cytokines. This induction was blocked by cy-
tochalasin D showing that internalization plays a role in the activation of airway
epithelial cells by conidia. These data indicate that HBEC can discriminate between
different microorganisms and show a distinct inflammatory reaction.
P3572
Human airway epithelial cells synthesize and release epinephrine and
nor-epinephrine P3575
Sundeep Salvi 1,2 , Parimal Sheth 2 , E. Sekerel 2 , H. Yoshisue 2 , J. Holloway 2 , Cigarette smoke condensate (CSC) increases the expression of
Stephen Holgate 2 . 1 Clinical Research, Chest Research Foundation, Pune, India; beta–defensin-3 (hBD-3) in cultured primary bronchial epithelial cells
2
University Medicine, Southampton General Hospital, Southampton, United (PBEC)
Kingdom Sandra van Wetering, Dennis K. Ninaber, Marianne A.J.A. van Sterkenburg,
Klaus F. Rabe, Pieter S. Hiemstra. Dept. of Pulmonology, Leiden University
Catecholamines (epinephrine and norepinephrine) regulate important physiological Medical Center, Leiden, The Netherlands
processes in the airways and the distal lung parenchyma. Despite poor sympathetic
innervation, the lung has been shown to synthesize and release substantial amounts hBD-3 is an antimicrobial peptide produced by airway epithelial cells with both an-
of catecholamines locally. In this study, we demonstrate that human airway epithe- timicrobial and chemotactic properties. Epithelial expression of hBD-3 is inducible
lial cells (primary, secondary transformed bronchial epithelial cells [16HBE140- , by exposure to micro-organisms and pro-inflammatory cytokines. The effect of
H292] and secondary transformed alveolar epithelial cells [A549]) constitutively CSC on the expression of antimicrobial peptides by epithelial cells has not yet
expressed the tyrosine hydroxylase (TH) gene and protein using Reverse Transcrip- been reported. Studying this effect is relevant, in view of respiratory bacterial
tion Polymerase Chain Reaction (RT-PCR) and Western and Dot Blot analysis. infections in patients with chronic bronchitis, and COPD. Therefore, the aim of
Using the CatCombi enzyme-linked immunosorbent assay (ELISA), we showed the present study was to investigate the effect of CSC on the expression of hBD-3
constitutive presence of epinephrine and norepinephrine in cell lysates as well as in in PBEC. CSC caused a dose- and time-dependent increase in the expression
culture media of human airway epithelial cells. Epinephrine was found in greater of hBD-3, as shown by RT-PCR. Preincubation of PBEC with the anti-oxidant
amounts in the culture supernatants, while norepinephrine was mainly found in cell N-acetyl-L-cysteine inhibited both the cytotoxic effects of CSC and its ability to
lysates. Incubation of epithelial cells with alpha methyl-p-tyrosine, a competitive increase IL-8 release. However, it did not prevent CSC-induced hBD-3 expression,
inhibitor of tyrosine hydroxylase markedly reduced the amounts of epinephrine but enhanced hBD-3 expression at cytotoxic concentrations of CSC (>10 AU/ml).
and norepinephrine both in the culture supernatants as well as in cell lysates, CSC-induced hBD-3 expression was inhibited by the mitogen activated protein
suggesting that epithelial cells actively synthesise and release catecholamines. To kinase (MAPK) p38 inhibitor SB203580. These results show that CSC increases
our knowledge, this is the first report to show that human airway epithelial cells hBD-3 expression by oxidant-independent mechanisms, and that this involves
actively synthesize and release epinephrine and norepinephrine constitutively. signalling through the MAPK p38 pathway. The results suggest that cigarette
smoke, like other agents that cause cellular stress, evokes an epithelial reponse
that may lead to an increase in antimicrobial defence. Supported by a grant from
P3573 the Netherlands Asthma Foundation.
Arginine levels regulate pro-inflammatory mediator responses by lung
epithelial cells
Xiao-Yun Fan 1,2 , Mieke Snoek 2 , Arjen van den Berg 2 , Laurens van der Flier 2 , P3576
Barbara S. Smids 2 , Henk M. Jansen 2 , Rong-Yu Liu 1 , Rene Lutter 2 . Secretory leukoprotease inhibitor (SLPI) is removed from media of primary
1
Pulmonology, Anhui Med. Univ., Hefei, China; 2 Pulmonology, AMC, bronchial epithelial cells (PBEC) in the presence of neutrophil elastase (NE)
Amsterdam, The Netherlands Anita Sullivan 1 , Robert Stockley 1 , Pieter Hiemstra 2 , Sandra van Wetering 2 .
1
Department of Respiratory Medicine, Queen Elizabeth Hospital, Birmingham,
Arginine plays a central role in the pathophysiology of asthma. Nitric oxide United Kingdom; 2 Department of Pulmonology, Leiden University Medical
synthases use arginine to generate the smooth muscle relaxant NO, but also, at Center, Leiden, The Netherlands
low levels of arginine, the pro-inflammatory peroxynitrite. Recent studies have
indicated that arginine availability may be reduced in airways of asthmatics, among SLPI is an important anti-elastolytic agent in the lungs, implicated in COPD
others by increased arginase activity. It was shown previously that biological poly- as it is lower in patients with frequent exacerbations and bacterial colonization.
cations, such as major basic protein, block arginine uptake. We studied the impact It exhibits a reciprocal relationship with the degree of inflammation in airway
of reduced arginine availability on lung epithelial IL-6 and IL-8 production. Ep- secretions, supported by studies of airway cells in vitro where SLPI secretion
ithelial cells (cell lines and NHBE) in medium without arginine showed markedly fell in the presence of NE. However SLPI expression increased and lysates of
enhanced IL-6 and IL-8 responses to LPS and TNF-α. This appeared to be due NE-treated cells contained excess SLPI compared to controls. This suggests that
to post-transcriptional regulation of IL-6 and IL-8 expression. At normal arginine secretion may be prevented or secreted protein may be taken up by cells in the
levels and in the presence of polycations (poly-L-arginine or major basic protein), presence of NE. We cultured PBEC in basal media for 24 hours in 12-well plates.
LPS but not TNF-α gave rise to enhanced IL-6 and IL-8 responses. This was due 25µl aliquots of media were then removed and replaced with 25µl of either
to transcriptional activity. There was no substantial contribution by arginase and basal media, or media containing sufficient NE to achieve a final concentration of
inducible nitric oxide synthase in depleting arginine. We conclude that arginine 7.5-10nM. Media were sampled sequentially. SLPI concentration fell substantially
depletion may lead to enhanced pro-inflammatory responses by lung epithelial in NE-treated cells within 10 minutes, from 1451pg/ml to 273pg/ml (p<0.05)
cells. Blocking arginine uptake by polycations also enhances pro-inflammatory (media controls 2184pg/ml to 2037pg/ml). This suggests that secreted SLPI was
responses, but this is due to another mechanism. In vivo studies are required to taken up onto the cell membrane, or into the cells, as a result of the presence of
extend these findings. NE. The concentration remained low in media of NE-treated cells, even after 24h
Financial support by Netherlands and Chinese Royal Academy of Sciences. (605pg/ml, controls 14161pg/ml, p <0.05). This could reflect prevention of further
secretion, or continuing uptake of secreted SLPI. Further studies will be necessary
to identify the exact mechanism.
554s
Thematic Poster Session Hall H-16 - 12:50-14:40
TUESDAY, SEPTEMBER 20 TH 2005
P3577 P3580
Cigarette smoke up-regulates toll like receptor 4 (TLR4) expression and Bronchial epithelial cells synthesise proteins of the extrinsic coagulation
enhances TLR4 mediated responses cascade to support fibrin formation in response to wounding
Elisabetta Pace 1 , Maria Ferraro 1 , Mario R. Melis 1 , Liboria Siena 1 , Angela Mike Perrio 1 , Mike Trevethick 2 , Gary Salmon 2 , Janis Shute 1 . 1 Institute of
M. Montalbano 1 , Giuseppina Chiappara 1 , Maria R. Bonsignore 2 , Biomedical & Biomolecular Sciences, University of Portsmouth, Portsmouth,
Vincenzo Bellia 2 , Giovanni Bonsignore 1 , Mark Gjomarkaj 1 . 1 Istituto di United Kingdom; 2 Allergy & Respiratory Research, Pfizer Global R&D,
Biomedicina Ed Immunologia Molecolare, CNR, Palermo, Italy; 2 Istituto di Sandwich, United Kingdom
Medicina e Pneumologia, Università Degli Studi di Palermo, Palermo, Italy
Animal models have indicated that bronchial epithelial repair is dependent on
Infections of the airways play a crucial role in the progression of COPD. A key exudation of plasma proteins and the formation of a provisional fibrin matrix.
component of the innate defence mechanisms is represented by the TLR family. Extravascular fibrin formation is initiated via the activity of tissue factor (TF)
We assessed TLR4 expression in surgical specimens from COPD, smokers and and cascades through FVII, FX, prothrombin and fibrinogen to form a fibrin clot
controls. TLR4 expression was higher in the epithelium of smokers and of COPDs that is stabilised by FXIII. We investigated expression of proteins of the extrinsic
than in controls. To examine the role of cigarette smoke in up-regulating TLR4 coagulation cascade by the 16HBE bronchial epithelial cell line.
expression and activation, we stimulated, in vitro, a bronchial epithelial cell line Cells were grown to confluence in 24-well plates, quiesced overnight and scrape
(16HBE) with cigarette smoke extracts (CSE). We assessed TLR4 expression and wounded using a pipette tip. TF expression was assessed immunohistochemically.
the LPS binding by citofluorimetry and the activation of TLR4 by evaluating: 1) FVII, FX, fibrinogen, FXIII and D-dimers, a marker of fibrin formation and break-
NFkB nuclear translocation (western blot analysis), 2) IL-8 release (ELISA) and down, were quantified by immunoblot in culture supernatants 20min and 2h after
3) chemiotactic activity for neutrophils. CSE up-regulated the TLR4 expression, wounding.
the LPS binding, the NFkB nuclear translocation, the IL-8 release, and the chemio- TF was constitutively expressed and distributed through the cytoplasm and
tactic activity for neutrophils. The combined exposure to CSE and LPS further cell membrane. Levels of FVII, FX, fibrinogen, FXIII and D-dimers in super-
increased the IL-8 release and the chemiotactic activity for neutrophils without natants at baseline were negligible. However, within 20 min of wounding FVII
inducing a further increase of NFkB nuclear translocation. This study demonstrates (0.37±0.1ng/ml), FX (1.86±0.23ng/ml), fibrinogen (12.38±0.42µg/ml), FXIII
that cigarette smoke, via up-regulation and over-activation of TLR4 receptor, may (125.62±47ng/ml) and D-dimers (2.4±0.19ng/ml) levels were significantly and
promote the accumulation of neutrophils within the airways of smokers and of maximally increased to the levels shown for the most extensive degree of wounding,
COPDs. (8 scrapes).
This work is dedicated to the Memory of Maurizio Vignola. The results indicate that bronchial epithelial cells may be able to support fibrin
formation, and therefore repair, in the absence of plasma proteins.
P3578
Secretory component binds defensins and may contribute to the bactericidal P3581
activity protecting mucosal surfaces Nitric oxide induces MUC5AC mucin in A549 cells through PKC and ERK
Lindsay J. Marshall 1 , Tim Mason 2 , Janis K. Shute 2 . 1 School of Life and Health pathways
Sciences, Aston University, Birmingham, United Kingdom; 2 School of Pharmacy Jeong-Sup Song, Kyung-Sook Cho, Hyung-Kyu Yoon, Hwa-Sik Moon,
and Biomedical Sciences, Portsmouth University, Portsmouth, United Kingdom Sung-Hak Park. Pulmonary Medicine, Catholic University Medical College,
Seoul, South Korea
Classically, secretory component (SC) transcytoses IgA across mucosal epithelia,
but SC uncomplexed with IgA is anti-infective and anti-inflammatory. We recently Introduction: Nitric oxide (NO) is generally increased during inflammatory air-
demonstrated that SC binds and inhibits CXCL-8 activity [Marshall et al, J. way diseases. The primary objective of this study is to find the signal transduction
Immunol. 2001; 167:2816-23]. As the antimicrobial defensins are basic peptides pathway of NO-induced MUC5AC mucin secretion in A549 cells.
like the chemokines, we tested the hypothesis that SC binds defensins. Western Methods: NOR-1 was used as a NO donor and stimulated the A549 cells.
blotting revealed that α- and β-defensins were bound to SC in non-CF sputum, MUC5AC mucin and NO were measured by ELISA and the luciferase activity
but the absence of intact SC in CF sputum precluded detection of complexes. was measured from the MUC5AC promoter transfected A549 cells. RT-PCR was
Additionally, β-defensin-2 co-immunoprecipitated with SC from primary human done to know the MUC5AC mRNA expression. Finally, western blot for PKCα
bronchial epithelial cell culture supernatants. The antibacterial activity of SC- and MAPK were done from the subcellular fraction and cell lysates of A549 cells
defensin complexes isolated from colostrum revealed that, in water, 100 µg/ml respectively.
colostral SC, with associated defensins, significantly decreased bacterial viability Results: The transcriptional activity of MUC5AC promoter was maximal at the
to 39.7±8% and 57.2±9% of control for Escherichia coli and Staphylococcus concentration of 0.1 mM NOR-1 for 1 hr incubation. NOR-1 markedly displaced
aureus, respectively. This activity was completely abolished in 100 mM NaCl. the PKCα from the cytosol to the particulate and the PKC inhibitors, Rotillerin,
In summary, the association of defensins with SC maintains their characteristic GO6976 and Calphostin-C were all inhibit the displacement of PKCα. NOR-1
salt-sensitive activity towards Gram-negative and Gram-positive organisms. Since also markedly increased the MUC5AC mRNA expression and mucin secretion and
SC binds to mucins it may be important in anchoring defensins in the mucus PKC inhibitors inhibited the MUC5AC mRNA expression and mucin secretion by
layer, enabling efficient targeting and destruction of bacteria at mucosal sites. The NOR-1. Among the MAPKs, ERK is only phosphorylated by NOR-1 and PKC
absence of intact SC in CF airways is likely to compromise this arm of the innate inhibitors inhibited the ERK activation by NOR-1.
defence system. Conclusion: Exogenous NO induced the MUC5AC mucin gene and protein
through the PKC and ERK MAPK pathways in A549 cells. In view of our findings
that PKC inhibitors decreased the NO-induced MUC5AC mucin in vitro, PKC
P3579 inhibitors might be useful in the treatment of bronchial asthma or COPD patients
Tissue factor plays a central role in bronchial epithelial repair responses where NO is increased in the bronchial airways.
Darleen Ewen 1 , Steven Vayro 1 , Mike Trevethick 2 , Gary Salmon 2 , Janis Shute 1 .
1
Institute of Biomedical & Biomolecular Sciences, University of Portsmouth,
Portsmouth, United Kingdom; 2 Allergy & Respiratory Research, Pfizer Global P3582
R&D, Sandwich, United Kingdom Nitric oxide is involved in the physiological signaling pathway in the secretion
from tracheal gland acinar cells
Animal experiments previously suggested that repair of mechanically wounded Tsutomu Tamada, Masayuki Nara, Miyuki Nagaoka, Gen Tamura, Toshio Hattori.
bronchial epithelium is rapid and dependent on the formation of a provisional fibrin Infectious and Respiratory Diseases, Tohoku University, School of Medicine,
matrix. Extravascular fibrin formation occurs via the extrinsic coagulation cascade, Sendai, Miyagi, Japan
initiated by the activity of tissue factor (TF). We investigated the expression,
activation and role of TF in the repair of primary human bronchial epithelial cell In response to acetylcholine (ACh) or norepinephrine (NE), tracheal gland cells can
monolayers in culture. generate ionic currents, witch are activated by intracellular calcium concentration
Primary human bronchial epithelial cells were grown in submerged culture in ([Ca2+ ]i ). It is known that nitric oxide (NO) is produced in human airways, but
24-well plates until 90% confluent. Monolayers were quiesced overnight in serum the biological significance of NO in the airway secretion is hardly characterized.
free, ITS-supplemented, medium and mechanically wounded with a pipette tip. The To investigate the intracellular role of NO/cGMP-signaling in the airway gland
time to wound closure was significantly inhibited in the presence of 10 µg/ml neu- secretion, we examined whether swine tracheal glands can produce NO in response
tralising TF antibody (American Diagnostica). Percentage original wound width to ACh, and whether NO/cGMP signaling is involved in the ACh-triggered ionic
20h post-damage was 84.2±3.3% compared with 29±5.6% in the absence of currents. From the experiments using the newly synthesized fluorescent NO in-
antibody. Immunohistochemical analysis demonstrated constitutive expression of dicator DAF-2DA, we found that physiologically relevant low dose ACh induced
TF, present throughout the cytoplasm and in the plasma membrane. Constitutively NO-production in the acinar cells and it was almost completely suppressed when
expressed TF activity was detected on the surface of epithelial monolayers grown in cells were pre-incubated with NO synthase (NOS) inhibitors Nω-Nitro-L-Arginine
96-well plates (8.9±0.3 ng/25,000 cells). This activity was significantly increased Methyl Ester Hydrochloride (L-NAME) or 7-Nitro indazole (7-NI). Patch-clamp
15 mins after wounding and increased further to 13.2±0.5 ng/25,000 cells 2h experiments revealed that both the NOS inhibitors (L-NAME or 7-NI) and PKG
after wounding. Our results point to an important role for TF and initiation of the inhibitors (KT-5823 or Rp-8-Bromo-cGMP) partially decreased ACh-activated
extrinsic coagulation cascade in successful bronchial epithelial repair. ionic currents. These findings suggest that the cholinergic stimulation increase
the NO synthesis in tracheal gland acinar cells and that the endogenous NO may
be involved and act as a potentiator in the physiological signaling pathway in
555s
Thematic Poster Session Hall H-16 - 12:50-14:40
TUESDAY, SEPTEMBER 20 TH 2005
tracheal gland secretion. Thus, abnormalities in NO metabolism should be taken airway remodeling in COPD via TGF-β1-independent CTGF gene upregulation in
into account when considering disorders in airway secretion. airway epithelial cells.
P3583 P3586
IL-1β induced histone H3-K4 tri-methylation and RNA polymerase II Cytokine-induced EGFR protein release in BEAS-2B cells; relation with
recruitment to the SLPI gene histone acetylation and methylation status
Hiroo Wada, Kazuhiro Ito, Masahiko Kagoshima, Ian M. Adcock, Peter Marcin Kurowski, Elen Jazrawi, Peter J. Barnes, Ian M. Adcock. Thoracic
J. Barnes. Airway Disease Section, NHLI, Imperial College London, London SW3 Medicine, NHLI Imperial College, London, United Kingdom
6LY, United Kingdom
Airway remodeling results from prolonged repair of epithelium damaged by in-
Until recently histone lysine methylation was regarded as a stable modification flammation. It leads to irreversible respiratory function impairment. EGFR is a
important in epigenetic regulation of gene expression. Transcriptionally active primary regulator of epithelial repair. Its expression is increased in asthmatics
chromatin is hyper-methylated at lysine residue 4 on histone H3-K4 (H3-K4), and correlates with collagen layer thickness. We investigated the effect of his-
whereas repressed chromatin is hyper-methylated at H3-K9. In order to elucidate tone acetylation and histone/DNA methylation status on cytokine-induced EGFR
the role of histone methylation in an acute inflammatory response, A549 cells release in BEAS-2B cell line. Cells were stimulated with IL-1β or TNF-α in
were treated with IL-1β and/or the methylase inhibitor 5-azacytidine (5-aza) and presence of trichostatin A (TSA) - histone deacetylase inhibitor or 5-azacytidine
histone H3K4 methylation levels and transcription of secretory leukocyte protease (5-AZA) - methylase inhibitor. EGFR concentration was measured in supernatants
inhibitor (SLPI) measured. H3-K4 methylation levels across SLPI, a constitutively using ELISA. Both IL-1β and TNF-α significantly increased EGFR protein release,
expressed and inducible gene, were much greater than those across GM-CSF, an compared to control (p<0.05). The effect of IL-1β (206±6 pg/mL) and TNF-α
inducible gene. IL-1β stimulation enhanced histone H3K4 tri-methylation across (532±33 pg/mL) on EGFR release was further enhanced by TSA (5 ng/ml).
the SLPI coding region at 24hrs. In parallel, IL-1β enhanced recruitment of RNA Similarly, the presence of 5-AZA caused further enhancement of both IL-1β and
polymerase II to the SLPI gene. 5-aza inhibited NF-κB-associated histone methy- TNF-α induced EGFR release. Expression of EGFR after 24 hour stimulation with
lase activity and attenuated both H3-K4 tri-methylation and RNA polymerase II TNF-α in the presence of TSA or 5-AZA was significantly greater than that seen
recruitment to a similar extent, resulting in reduced SLPI mRNA and protein levels. following IL-1β stimulation in the presence of TSA or 5-AZA.
These data suggest that in addition to epigenetic regulation of constitutive SLPI Our data confirm that TNF-α is stronger EGFR release inducer than IL-1β and
expression, H3-K4 methylation may play a role in stimulated SLPI expression by further indicate that histone acetylation and histone/DNA methylation status play
modulating RNA polymerase II recruitment and subsequent gene transcription. a role in EGFR expression and release. Furthermore, EGFR protein expression
is influenced to a greater extent by histone/DNA methylation than by acetylation
status. Further studies are to investigate molecular regulation of EGFR synthesis
P3584 and release.
A role for PAR-1 in bronchial epithelial repair
Darleen Ewen 1 , Mike Trevethick 2 , Gary Salmon 2 , Janis Shute 1 . 1 Institute of
Biomedical & Biomolecular Sciences, University of Portsmouth, Portsmouth,
United Kingdom; 2 Allergy & Respiratory Research, Pfizer Global R&D,
Sandwich, United Kingdom
The formation of a provisional fibrin matrix has been implicated in bronchial
341. From mice to men: acute lung injury
epithelial repair. We investigated the effect of mechanical wounding and PAR-1
and -2 agonists on fibrin turnover in the 16HBE bronchial epithelial cell line.
Cells grown to 100% confluence were quiesced overnight and scrape wounded.
Experiments were carried out in the absence and presence of 400 µM PAR- P3587
1 (TFRIFD-NH2) or PAR-2 (SLIGKVD-NH2) peptide agonist, and/or 10 µM Inflammatory responses and gas exchange in a sepsis-induced acute lung
indomethacin. Fibrinogen, Factor XIII and D-dimers, a marker of both fibrin injury model
formation and breakdown, were measured in culture supernatants by immunoblot Itamar S. Oliveira-Júnior 1 , Esther Barreiro 2 , Haibo Zhang 3 , Reinaldo Salomão 1 .
1
2h after wounding. Medicine, UNIFESP, SP, SP, Brazil; 2 Unitat Recerca en Muscul i Aparell
Levels of fibrinogen, FXIII and D-dimers were low but detectable at baseline Respiratory, IMIM, Barcelona, Spain; 3 Medicine, University of Toronto, Toronto,
(1.5±0.3 mg/ml, 8.0±2.4 ng/ml, 2.3±0.9 ng/ml), but increased significantly with Canada
extent of wounding (50.9±3.4 mg/ml, 47.5±16.0 ng/ml, 16.2±0.5 ng/ml with
maximum 8 wounds in the monolayer, n=3). PAR-1, but not PAR-2, agonists Previous animal studies have shown that certain modes of sepsis can injure the
significantly increased levels of fibrinogen (30.7±11.2 mg/ml), FXIII (57.2±2.5 lungs. We examined the effects of time in the setting of a clinically relevant, in vivo
ng/ml) and D-dimers (13.9±1.4 ng/ml) at baseline and further enhanced the animal spontaneous breathing model of sepsis-induced acute lung injury. Septic
responses to wounding (52.6±23.1 mg/ml, 72.7±21.8ng/ml, 18.8±6.8 ng/ml). male Wistar rats (n=5 per group) were randomized to spontaneous breathing. Sep-
Indomethacin had no significant effect on the response to wounding, nor on the sis was induced by ip E. coli inoculum. Neutrophils, lymphocytes, macrophages,
PAR-1 mediated responses. gas exchange and respiratory rate were significantly affected in this model. PaCO2
Our data indicate that bronchial epithelial cells synthesise coagulation factors that was significantly increased, and PaO2 decreased in the times 6h and 12h.The
are released in response to epithelial damage. PAR-1 may have a protective role in groups inoculated by 107 CFU (low doses) normalized de PaO2 and PaCO2 in the
promoting fibrin formation and turnover that is not mediated via PGE2 production. time 24h. In the groups inoculated by 109 CFU (high doses) the PaCO2 were not
normalized in the time 24h. Increased lymphocytes and neutrophils, and decreased
macrophages in BAL after 6 and 12h in the septic groups.
P3585
Regulation of connective tissue growth factor gene by lipopolysaccharide in
bronchial epithelial cells PaO2 (mmHg) Lymphocytes Neutrophils Macrophages Respiratory Rate
Michiyoshi Nishioka, Emiko Ogawa, Toyohiro Hirai, Shigeo Muro, G1 6h 105±4.3 13±1.8 24±2.6 64±3.6 95±1.9
Michiaki Mishima. Respiratory Medicine, Kyoto University Graduate School of G1 12h 101±5.4 11±0.5 24±1.9 65±1.9 96±2.1
Medicine, Kyoto, Japan G2 6h 68±2.1 24±0.5 48±4.5 29±4.9 113±4.6
G2 12h 63±4.4 23±0.8 43±1.3 34±0.8 112±2.9
Chronic obstructive pulmonary disease (COPD) is defined by progressive and G3 6h 65±4.2 23±0.7 43±3.5 34±3.5 117±2.4
irreversible airflow limitation primarily due to airway remodeling. The cigarette G3 12h 55±1.1 28±0.7 44±2.1 28±2.2 121±1.3
smoking habit itself and repeated bacterial infections during the acute exacerbation m ± SD; G1 6 and 12h=control group; G2 6 and 12h=low septic group;G3 6 and 12h=high septic
in COPD may cause lipopolysaccharide (LPS) exposure to the airway epithelial group; G1 6h vs G2 6h vs G3 6h p<.05 and G1 12h vs G2 12h vs G3 12h p<.05
cells. Previous studies showed that the expressions of connective tissue growth
factor (CTGF) and transforming growth factor (TGF) -β1 were upregulated in These results show the substantial differences in the balance of alveolar macro-
COPD patients. In this report we hypothesized that LPS could induce CTGF, a phages and neutrophils influx.
prime candidate regulator of collagen and matrix deposition, in airway epithe-
lial cells. Experiments were performed on cultured human bronchoepithelial cell
line BEAS-2B. CTGF and TGF-β1 mRNA were quantified by real-time reverse P3588
transcription polymerase chain reaction (RT-PCR) and CTGF protein expression The influence of mechanical ventilation during general anesthesia on the state
was analyzed by Western blotting. BEAS-2B cells were stimulated by 10 µg/ml of metabolism markers in exhaled breath condensate
LPS. After 2 hour-stimulation by LPS, CTGF mRNA was significantly upregu- Aliaksandr S. Smirnou 1 , Anatoli D. Tahanovich 1 , Aliaksandr E. Skrahin 2 .
1
lated in BEAS-2B cells, however TGF-β1 mRNA wasn’t. The inhibition of c-jun Biochemistry, Belarusian State Medical University, Minsk, Belarus; 2 Intensive
NH2 -terminal kinase (JNK) or p38 pathway blocked the effect of LPS on CTGF Care, Belarusian Institute of Pulmonology and Tuberculosis, Minsk, Belarus
mRNA expression. We also confirmed that the expression of the LPS receptor,
Toll-like receptor 4 (TLR4), was not affected by LPS by Western blotting. We Mechanical ventilation causes lung damage through inflammation and oxidative
conclude that smoking and repeated bacterial infections could contribute to the stress. We studied the influence of volume-controlled mechanical ventilation during
556s
Thematic Poster Session Hall H-16 - 12:50-14:40