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Medical Research Society
M163
NEUTROPHIL ELASTASE PARTICIPATES IN TGF-fi
ACTIVATION IN BLEOMYCIN-INDUCED PULMONARY
FIBROSIS.
F Chua, SE Dunsmore, AW Segal, J Roes, GJ Laurent.
Centre for Respiratory Research & Department of Medicine,
University College London, London WClE 655.
Neutrophil elastase is a potent serine protease implicated in the
pathogenesis of pulmonary fibrosis. However, the mechanisms by
which it acts are not known. We have previously reported that
biochemical and histological indices of pulmonary fibrosis are absent
in neutrophil elastase-deficient(NE") mice treated with bleomycin, a
profibrotic chemotherapeutic agent. Transforming growth factor-beta
(TGF-0) is a potent fibrogenic cytokine that stimulates lung
fibroblast growth and collagen production. It is present in fibrotic
human lungs and its overexpression has been shown to induce
pulmonary fibrosis in rodent lungs. We hypothesized that decreased
amounts of active TGF-P may contribute to the lack of fibrotic
response in bleomycin-treated NE'/' mice. In the present studies, WT
and NE" mice were injected intratracheally with 0.05 unit bleomycin
or saline and bronchoalveolar lavage fluid (BALF) and lung tissue
collected seven days later. Levels of active and total TGF-fl (samples
were heated to 80°C for 10 minutes to activate latent TGF-p) were
measured with a bioassay based on the ability of active TGF-P to
stimulate the plasminogen activator inhibitor-1 promoter. A multiprobe template RNase protection assay was used to quantitate TGF-fi
mRNA (Pharmingen Ltd., UK). BALF inflammatory cell profile and
total protein as well as lung tissue neutrophil burden did not differ
between bleomycin-treated WT and NE-I- mice. Active TGF-S
recovered in bleomycin-treated WT BALF averaged 0.36 t 0.02
ng/ml, two-fold higher than in saline controls. In contrast, active
TGF-b in BALF from bleomycin-treated NE-I- mice averaged 0.23 f
0.01 ng/ml (p<O.OOI vs WT) and did not differ from saline controls.
Total TGF-fl in bleomycin-treated WT BALF was also higher than in
NE-I- BALF (0.75 ? 0.14 vs. 0.36 f 0.04 ng/ml, p<0.05). TGF-fll
and TGF-P3 mRNA were similar in bleomycin-treated WT and NE'.
mice. These results indicate that neutrophil elastase plays a role in
TGF-P activation in bleomycin-induced pulmonary fibrosis and
suggest that neutrophil elastase inhibitors may provide a novel means
for inhibiting TGF-8 activation as a therapeutic intervention in
fibrotic lung disorders.
The Wellcome Trust, Medicul Reseurch Council uwl the British Luw Fouwhtion
Background: The isoprostane 8-is0 PGF, is a product of free radicalcatalysed lipid peroxidation. In vitro 8-is0 PGF, causes human
pulmonary arterial and venous constriction, platelet aggregation and
neutrophil adhesion, all processes central to lung injury. We
hypothesised that systemic arterial blood from patients with ARDS
might contain more 8-is0 PGFb than mixed venous blood due to
increased pulmonary oxidative stress, impaired 8-is0 PGF, uptake
and reduced 8-is0 PGF, metabolism by the injured lung. Methods:
Blood samples were taken simultaneously from pulmonary and
systemic arterial catheters in patients with ARDS due to direct
pulmonary insults; patients undergoing cardiopulmonary bypass
(CPB) a day previously for heart valve replacement; and patients
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M165
Cell lineage-specific surface molecular alterations associated
with neutrophil apoptosis that potentiate phagocyte recognition
Simon P. Hart, Caroline Jackson, L. MaximUhn Kremmel,
Hubertus Jersmann, James A. Ross*,and Ian Dransfield
Although a number of different phagocyte surface receptors have
been implicated in recognition of apoptotic cells, the molecular
recognition mechanism(s) that are utilised for clearance have been
assumed to be largely apoptotic cell-independent. Fwthermore,
examination of surface molecular alterations has failed to reveal cell
type-specific membrane alterations that could serve as a signal for
phagocytosis. However, specific augmentation of phagocytosis of
apoptotic neutrophils, but not lymphocytes occurs following crosslinking of macrophage CD44. Thus, phagocytes may have the
potential to recognize apoptotic cell types in a lineage-specific
manner. In the present study, we provide evidence for cell typespecific surface membrane alterations accompanying apoptosis. We
identified a novel monoclonal antibody (Bob93) that bound to the
surface of apoptotic neutrophils, but not to apoptotic lymphocytes or
eosinophils. Further investigation of the binding characteristics
revealed that Bob93 only binds to apoptotic neutrophils in the
presence of the bovine sialoglycoproteinfetuin. These data suggested
the possibility that fetuin was able to bind to surface receptors
present on neutrophils. We next examined whether FlTC-labelled
fetuin bound at high levels to annexin V positive apoptotic
neutrophils. Treatment of freshly isolated neutrophils with
neuraminidase also induced Bob93-fetuin binding, suggesting that
sialic acid may mask sites present on non-apoptotic cells. Finally, we
demonstrate that macrophage phagocytosis of apoptotic neutrophils
was augmented following addition of bovine fetuin or the human
homologue alpha2HS-glycoprotein. We propose that macrophagebound fetuin may serve to facilitate cellular interactions between the
phagocyte and the apoptotic neutrophil. Since fetuin is a negative
acute phase protein, altered plasma concentrations of fetuin
homologues during acute inflammation in vivo may provide an
additional mechanism for regulating phagocytic clearance of
apoptotic cells.