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Environmental
Health
Perspectives
Vol.
85,
pp.
135-144,
1990
Detection
of
Lipocortin
1
in
Human
Lung
Lavage
Fluid:
Lipocortin
Degradation
As
a
Possible
Proteolytic
Mechanism
in
the
Control
of
Inflammatory
Mediators
and
Inflammation
by
Susan
F.
Smith,*
Teresa
D.
Tetley,*
Abraham
Guz,*
and
Roderick
J.
Flower'
Lipocortins
are
structurally
related,
glucocorticoid-inducible
proteins
that
inhibit
phopholipase
A2
(PLA2),
thereby
reducing
the
liberation
of
arachidonic
acid
from
phospholipids
and
so
limiting
the
synthesis
of
eicosanoid
inflammatory
mediators.
This
study
is
the
first
demonstration
of
one
lipocor-
tin,
lipocortin
1
(Lc
1;
37
kDa),
inhuman
lunglavage
supernatants.
In
lavage
fluid
from
healthy
volunteers,
a
higher
percentage
(>
70
%)
of
the
detected
Lc
1
was
in
its
native
form,
compared
to
that
from
patients
with
abnormal
lungs.
In
patients'
lavage
fluids,
Lc
1
was
more
likely
to
be
partially
degraded
(34
kDa).
In
abnormal
bronchoalveolar
lavage
fluid
(BALF),
the
more
polymorphonuclear
neutrophils
(PMN)/lavage,
the
lower
the
proportion
of
Lc
1
in
the
native
(37
kDa)
form
(n
=
7
pairs,
rs
=
-
0.8214,
p
<
0.05).
Furthermore,
whenBALF
cells
were
cultured
and
the
harvested
conditioned
media
incubated
with
pure
human
recombinant
Lc
1,
degradation
of
the
37
kDa
form
increased
with
the
percentage
of
PMN
(n
=
10
pairs,
s
= -
0.7200
after
1
hr;
n
=
6
pairs,
rs
=
-
0.9241
after
6
hr).
These
results
suggest
that
factors
released
from
the
PMN
are
responsible
for
Lc
1
degradation
in
man.
When
recombinant
human
Lc
1
was
incubated
with
human
neutrophil
elastase,
the
enzyme
degraded
Lc
1
in
a
dose-dependent
way,
suggesting
that
neutrophil
elastase
may
be
one
such
factor.
Since
PMNs
are
ubiquitous
at
sites
of
inflammation,
it
is
possible
that
Lc
1
degradation
is
a
permissive
mechanism,
which
ensures
that
sufficient
inflammation
occurs
to
destroy
the
provocative
stimulus.
However,
it
is
equally
possible
that,
in
some
circumstances,
the
mechanism
may
be
pathological
and
that
the
inactivation
of
Lc
1
leads
to
chronic,
uncontrolled
inflammation.
Introduction
Although
glucocorticoids
have
been
used
empirically
in
the
treatment
of
inflammatory
diseases
for
40
years,
their
mechanisms
of
action
are
only
now
being
eluci-
dated.
Studies
in
animals
show
that
the
anti-inflam-
matory
actions
of
steroids
can
be
mediated,
at
least
in
part,
by
a
family
of
structurally
related
proteins
named
lipocortins
1
through
6.
These
anti-inflammatory
pro-
teins,
which
are
synthesized
in
response
to
both
naturally
occurring
and
synthetic
glucocorticoids
(1,2),
inhibit
phospholipase
A2
(PLA2).
This
prevents
the
release
from
phospholipid
of
arachidonic
acid,
the
common
precur-
sor
of
both
prostaglandin
and
leukotriene
inflammatory
*
Department
of
Medicine,
Charing
Cross
and
Westminster
Medical
School,
Fulham
Palace
Road,
London,
W6
8RF,
UK.
t
School
of
Pharmacy
and
Pharmacology,
University
of
Bath,
Claverton
Down,
Bath,
BA2
7AY,
UK.
Address
reprint
requests
to
S.
F.
Smith,
Department
of
Medicine,
Charing
Cross
and
Westminster
Medical
School,
Fulham
Palace
Road,
London,
W6
8RF,
UK.
mediators.
Although
lipocortins
are
produced
by
human
cells
(3,4)
and
human
lipocortin
genes
have
been
cloned
(5),
the
exact
role
of
these
proteins
in
human
health
and
disease
has
yet
to
be
understood.
As
yet,
there
are
no
published
data
on
lipocortin
in
the
human
lung,
whether
healthy
or
abnormal,
although
lipocortins
are
present
in
rat
(6)
and
bovine
(7)
lungs.
However,
there
is
evidence
that
human
lung
cells
can
produce
steroid-inducible
inhibitors
of
arachidonic
acid
release
(8),
one
of
which
may
be
a
lipocortin.
A
previously
published
study
has
shown
that
lipocor-
tin
1
(Lc
1;
37
kDa),
the
most
widely
distributed
and
best
characterized
member
of
the
lipocortin
family,
can
be
proteolyzed
in
vitro
by
an
elastase
(probably
porcine
pancreatic
elastase)
(9)
to
a
number
of
products,
with
molecular
weights
ranging
from
18
to
33
kDa.
The
func-
tional
significance
of
one
such
fragment
(33
kDa)
is
of
particular
interest.
Unpublished
studies
(Flower
et
al.)
show
that
in
vivo,
the
33-kPa
form
loses
the
ability
to
suppress
inflammation;
furthermore,
it
fails
to
reduce
prostaglandin
production
and
release
by
peritoneal
SMITH
ET
AL.
macrophages
maintained
in
vitro,
although
it
appears
to
retain
90%
of
its
PLA2
inhibitory
capacity
in
one
cell-
free
in
vitro
assay
system
(9).
In
addition,
when
the
37-kDa
protein
is
proteolyzed, as
well
as
the
33-kDa
form,
a
3-kDa
fragment
is
liberated
from
the
N-terminal
portion
of
the
parent
molecule.
There
is
some
evidence
that
peptides
with
sequence
homology
to
this
3-kDa
frag-
ment
may
have
some
inherent
anti-inflammatory
activi-
ty.
For
example,
similar
peptides
are
able
to
block
formyl-
methionyl-leucyl-phenylalanine
(fmlp)-induced
chemo-
taxis
in
polymorphonuclear
neutrophils
(PMN)
(10),
although
these
effects
occur
at
high
concentrations
that
are
probably
nonphysiological.
It
therefore
seems
likely
that
proteolysis
of
Lc
1
could
have
significant
patho-
logical
effects.
Elastase
activities
in
the
human
lung
are
greatly
in-
creased
by
tobacco
smoking
(11);
in
addition,
elevated
elastase
activities
have
been
implicated
in
the
etiology
of
many
pulmonary
diseases
including
smoking-related
disorders
and
some
environmentally
induced
diseases
(12),
as
well
as
certain
congenital
diseases
(13).
The
first
aim
of
the
present
study
was
to
establish
whether
or
not
Lc
1
is
present
in
the
human
lung.
To
do
this,
lung
lavage
fluids
from
healthy
human
subjects
and
from
patients
with
various
lung
diseases
were
analyzed
for
Lc
1
and
for
its
breakdown
products.
The
second
aim
of
the
current
investigation
was
to
determine
whether
or
not
Lc
1
was
degraded
(inactivated?)
by
purified
human
neutrophil
elastase
(NE)
and,
if
so,
to
find
out
if
the
actions
of
NE
on
lipocortin
could
be
reproduced
by
exposure
of
recombinant
Lc
1
to
media
in
which
elastase-secreting
cells
(PMN,
and
alveolar
macrophages,
AM)
had
been
cultured.
Methods
Materials
Double-lumened,
balloon-tipped
catheters
(5Fand
6F)
were
obtained
from
Kimal
Scientific
Products
Ltd.
(Ux-
bridge,
Middx.,
UK).
Centricon
units
were
purchased
from
Amicon
Ltd.
(Stonehouse,
Glos.,
UK).
The
PhastSystem
electrophoresis
apparatus
and
SDS-
polyacrylamide
gradient
Phastgels
to
use
on
it
were
obtained
from
Pharmacia
Ltd.
(Milton
Keynes,
Bucks,
UK),
while
the
dot-blotting
apparatus
and
nitrocellulose
paper
were
purchased
from
Bio-Rad
Laboratories
Ltd.
(Hemel
Hempstead,
Herts.,
UK).
The
Chromoscan
3
gel
scanner
was
from
Joyce-Loebl
(Gateshead,
Tyne
and
Wear,
UK).
All
tissue
culture
media
were
obtained
from
Gibco
Ltd.
(Paisley,
Renfrewshire,
UK).
Rainbow
molecu-
lar
weight
markers
were
purchased
from
Amersham
In-
ternational
plc
(Amersham,
Bucks,
UK).
Goat
anti-rabbit
IgG
conjugated
to
horseradish
peroxidase
was
obtained
from
Sigma
Chemical
Co.
Ltd.
(Poole,
Dorset,
UK).
Neutrophil
elastase
and
its
substrate,
insoluble
elastin,
were
ordered
from
Elastin
Products
Ltd.
(Pacific,
MO).
Recombinant
human
Lc
1
and
monospecific
antibodies
to
the
whole
molecule
were
the
gifts
of
Biogen
Inc.
(Cambridge,
MA).
All
other
chemicals
were
of
AnalaR
grade
and
were
purchased
from
BDH
Ltd.
(Poole,
Dorset,
UK).
Subjects
Peripheral
lung
lavage
fluid
(PLF;14)
was
collected
from
6
volunteers
(1
male;
6
smokers;
median
age
26
years,
range
22-31
years)
who
underwent
bronchoscopy
solely
for
research
purposes.
Informed
consent
was
ob-
tained
from
all
subjects
by
a
clinician
who
was
not
in-
volved
in
the
study,
and
approval
was
obtained
from
the
Local
Ethical
Committee
of
Charing
Cross
Hospital.
In
addition,
PLF
was
obtained
during
routine
fibre-
optic
bronchoscopy
of
11
patients
(5
male;
4
nonsmokers,
4
exsmokers,
and
3
current
smokers;
median
age
68
yrs,
range
56-78
years).
The
final
diagnoses
on
these
pa-
tients
were:
fibrosing
alveolitis,
n
=
3;
pleural
effusion,
n
=
2;
mesothelioma,
bronchial
carcinoma,
and
hemop-
tysis
of
unknown
origin,
each
n
=
1;
no
final
diagnosis,
n
=
3.
Central
lung
lavage
fluid
(CLF)
(14)
was
collected
from
4
male
patients
(1
nonsmoker,
3
current
smokers;
median
age
61
years,
range
57-69
years);
and
the
final
diagnoses:
bronchial
carcinoma,
resolving
pneumonia,
hemoptysis
of
unknown
origin,
and
no
final
diagnosis,
each
n
=
1.
An
additional
22
patients
(14
male;
8
nonsmokers,
4
exsmokers,
and
10
current
smokers;
median
age
56
years,
range
25-75
years)
underwent
bronchoscopy
with
bron-
choalveolar
lavage
(BAL)
as
a
routine
part
of
their
diag-
nostic
work-up.
The
final
diagnoses
on
these
patients
were:
normal
lung,
n
=
5;
bronchial
carcinoma,
n
=
5;
fibrosing
alveolitis,
n
=
3;
sarcoidosis,
n
=
3;
pulmonary
effusion,
n
=
2;
asperillosis,
mesothelioma,
bronchiec-
tasis,
infection,
no
final
diagnosis,
each
n
=
1.
One
sub-
ject
had
mesothelioma
with
fibrosing
alveolitis,
and
therefore
there
are
n
=
23
diagnoses.
The
subjects
found
to
have
normal
lungs
were
investigated
for
unexplained
cough,
hemoptysis,
or
cancerphobia.
No
subject
included
in
this
study
was
undergoing
treat-
ment
with
glucocorticoids
at
the
time
of
bronchoscopy.
Coliection
and
Processing
of
Lavage
Fluids
and
Preparation
for
Western
Blotting
PLF
was
collected
as
previously
described
(14).
A
6F
double-lumened,
balloon-tipped
catheter
was
passed
through
the
biopsy
channel
of
an
unwedged
BF1T
Olym-
pus
bronchoscope
and
wedged
by
inflation
of
the
balloon
at
the
level
of
the
seventh
or
eighth
generation.
A
20-mL
portion
of
sterile,
warmed
0.15
mole/L
saline
was
instilled
through
the
second
lumen
and
into
the
lung,
below
the
level
of
the
wedged
balloon.
It
was
aspirated
immediately
into
a
sterile
trap,
and
the
process
was
repeated
until
100
mL
saline
had
been
instilled
into
the
lung
and
aspir-
ated
into
the
same
trap.
CLF
was
collected
as
previously
described
(14).
Briefly,
a
5F
double-lumened,
balloon-tipped
catheter
was
pass-
ed
through
the
bronchoscope
as
previously
described
and
saline
was
instilled
into
the
airways-this
time
above
136
LIPOCORTIN
IN
HUMAN
LUNG
LAVAGE:
ROLE
OF
INFLAMMATORY
CELLS
the
level
of
the
balloon,
at
the
fifth
or
sixth
generation.
Sterile,
warmed
0.15
mole/L
saline
was
instilled
in
4
mL
portions
and
aspirated
as
previously
described,
until
a
total
of
20
mL
had
been
instilled
into
the
airway
and
col-
lected
into
a
sterile
trap.
BAL
was
carried
out
as
previously
described
(15).
Warmed,
sterile
0.15
mole/L
saline
(50
mL)
was
instilled
through
the
biopsy
channel
of
the
bronchoscope
that
was
wedged
at
the
third
or
fourth
generation.
The
BAL
fluid
(BALF)
was
withdrawn
immediately
into
a
sterile
trap
and
the
process
repeated
a
further
three
times.
All
PLF,
CLF,
and
12
of
the
BALF
were
processed
in
the
same
way.
A
sample
of
1
to
2
mL
was
removed
for
a
total
cell
count
using
a
Neubauer
hemocytometer
and
for
a
differential
cell
count
following
Wright-Geimsa
staining.
The
remaining
fluid
was
filtered
through
wire
mesh
and
centrifuged
at
300g
at
40C
for
15
min
to
pellet
the
cells.
(Cells
from
two
BALF
samples
were
used
for
the
culture
studies
described
in
the
following
section).
Lavage
supernatants
were
stored
in
fractions
at
-
20°C
or
lower
until
analysis.
Tbtal
protein
was
measured
by
the
method
of
Lowry
et
al.
(16)
using
bovine
serum
albumin
as
a
standard.
Samples
(2
mL)
of
BALF
and
PLF
were
concentrated
ap-
proximately
15-fold
and
0.2
mL
samples
of
CLFwere
con-
centrated
approximately
10-fold
by
centrifugation
in
Centricon
units,
with
molecular
weight
cut-off
10
kDa,
for
3
hr
at
3800g.
The
final
protein
concentration
was
adjusted
to
2.5
mg/mL
with
0.15
mole/L
saline.
Samples
were
boiled
for
5
min
with
equal
volumes
of
sample
buf-
fer,
pH
8,
containing
Tris-HCl
(10
mmole/L),
EDTA
(1
mmole/L),
5%
(w/v)
SDS,
10%
(v/v)
mercaptoethanol
and
0.02%
(w/v)
bromophenol
blue.
The
reduced
samples
were
stored
at
200C
prior
to
Western
blotting.
Culture
of
BALF
Cells
and
Incubation
of
Cell
Secretions
with
Recombinant
Human
Lipocortin
The
cells
from
12
BALF
samples
were
cultured
as
follows:
BALF
cells
and
supernatants
were
separated
as
previously
described.
The
cells
were
washed
in
Hanks'
buffered
salt
solution
(without
Ca2+
or
Mg2+),
then
pelleted
by
repeat
centrifugation
at
300g
for
15
min
at
40C.
The
cells
were
then
suspended
in
Dulbecco's
modi-
fied
Eagle's
medium
or
low-protein
hybridoma
medium
and
plated
out
at
a
concentration
of
106/mL.
After
a
1-hr
adherence
period,
the
conditioned
media
and
floating
cells
were
removed
and
separated
by
centrifugation
at
300g
at
40C
for
15
min.
The
media
(CM
1)
were
stored
at
-
200C.
The
adherent
cells,
mainly
AM,
were
cultured
in
fresh
medium
for
a
further
3
hr.
Following
that,
the
conditioned
media
(CM
3)
were
removed,
separated
from
any
residual
nonadherent
cells
as
previously
described,
and
stored
as
previously
described.
Fractions
of
CM
1
and
CM
3
were
concentrated
by
lyophilization
or
by
centrifugation
in
Centricon
units,
(cut-off,
10
kDa),
at
3800g
for
3
hr
at
4°C.
Pure
recombi-
nant
Lc
1
(80
Ag/mL)
was
incubated
at
37°C
with
con-
centrated
fractions
of
CM
1
and
CM
3
from
the
equivalent
of
2
to
20
x
106
elastase-secreting
cells
(PMN
and
AM)/mL
for
periods
of
up
to
48
hr.
Fractions
of
the
in-
cubation
mixtures
were
removed
at
various
time
points,
and
the
reaction
was
terminated
by
boiling
for
5
min
with
an
equal
volume
of
sample
buffer
prepared
as
describ-
ed
earlier.
Because
the
amounts
of
CM
varied
between
patients,
samples
were
not
taken
from
every
subject
at
every
time
point.
Collections
were
staggered
so
that
suf-
ficient
samples
were
available
to
carry
out
statistical
analysis
on
each
time
point.
In
order
to
determine
whether
the
BALF
cells
had
released
any
Lc
1
into
the
medium,
samples
of
CM,
to
which
no
exogenous
recom-
binant
Lc
1
had
been
added,
were
prepared
for
Western
blotting
as
previously
described.
Prepared
samples
were
stored
at
-
200C
until
analysis.
Western
Blotting
for
Lipocortin
1
SDS-polyacrylamide
gel
electrophoresis
was
perform-
ed
on
8
to
25%
gradient
Phastgels
using
a
PhastSystem
electrophoresis
apparatus
according
to
the
manufactur-
er's
instructions.
A
1-uL
fraction
of
lavage
fluid,
prepared
as
previously
described,
was
applied
to
each
gel
and
electrophoresed
for
approximately
30
min
at
10
mAmps
per
gel,
maximum
voltage,
100
V.
Pure
recombinant
human
Lc
1
(80
Ig/mL)
was
prepared
as
described
above
and
used
as
a
control
on
every
gel.
Molecular
weight
markers
(14.3-200
kDa)
of
variously
dyed
proteins
were
used
on
each
gel.
Samples
of
recombinant
Lc
1
(80
jAg/mL)
which
had
been
incubated
with
CM,
as
previously
described,
were
electrophoresed
in
the
same
way.
Protein
bands
were
transferred
from
gels
to
nitro-
cellulose
paper
by
diffusion
under
pressure
for
18
hr
at
room
temperature
using
Tris-glycine-methanol
buffer,
pH
8
(Tris
6.25
mmole/L
+
glycine
250
mmole/L:
metha-
nol,
4:1),
as
a
carrier.
Completion
of
protein
transfer
was
monitored
by
observing
the
colored
bands
of
rainbow
markers.
The
nitrocellulose
sheets
were
agitated
in
Tris-buffered
saline
(TBS;
0.15
mole/L
NaCl,
10
mmole/L
Tris-HCl,
pH
7),
containing
3%
(w/v)
bovine
serum
albumin
for
1
hr
at
room
temperature
in
order
to
block
any
remaining
pro-
tein
binding
sites.
The
sheets
were
then
shaken
gently
for
24
hr
at
40C
(lavage
supernatants),
or
2
hr
at
room
temperature
(CM/Lc
1
mixtures)
with
a
specific
rabbit
anti-recombinant
human
Lc
1
antibody
diluted
1
in
1000
with
washing
buffer
[WB;
10
mmole/L
Tris-HCl,
pH
7,
0.3
mole/L
NaCl,
0.5%
(v/v)
Nonidet
P-40]
containing
3%
(w/v)
bovine
serum
albumin.
The
remaining
steps
were
performed
at
room
temperature.
Nonspecifically
bound
antibody
was
removed
by
exhaustive
washing
in
WB
(without
serum
albumin).
Following
this,
the
blot
was
exposed
for
1
to
2
hr
to
goat
anti-rabbit
IgG,
conjugated
to
horseradish
peroxidase
diluted
1
in
1000
in
WB,
con-
taining
3%
(w/v)
bovine
serum
albumin.
After
further
washing
in
WB
followed
by
TBS,
the
Lc
1
bands
were
visualized
by
incubation
of
the
blots
in
TBS
containing
137
SMITH
ET
AL.
diaminobenzidine,
125
,ig/mL
and
H202,
0.04
1L/mL.
After
a
30-
to
60-min
incubation,
the
reaction
was
ter-
minated
by
washing
the
blot
in
TBS.
Any
lavage
supernatants
that
were
negative
for
Lc
1
on
Western
blotting
were
immunoblotted
using
larger
amounts
of
sample.
Briefly,
50-IAL
portions
of
concen-
trated,
reduced
lavage
fluid
were
applied
directly
to
nitrocellulose
paper
using
a
dot-blotting
apparatus
ac-
cording
to
the
manufacturer's
instructions.
Any
Lc
1
in
the
sample
was
then
visualized
using
the
antisera
and
detection
system
described
previously.
Identification
of
the
Partially
Degraded
Lc
1
Band
Duplicate
samples
of
pure
recombinant
Lc
1
and
Lc
1
partially
degraded
by
incubation
with
a
sample
of
CM
1
were
electrophoresed
as
previously
described
on
a
single
gel.
The
gel
was
then
bisected
and
one
set
of
samples
Western
blotted
as
described
above.
The
second
set
of
samples
was
blotted
using
a
first
antibody
raised
to
a
peptide
with
an
identical
amino-acid
sequence
to
residues
13
to
26
of
human
Lc
1.
This
region
of
the
N-
terminal
portion
of
Lc
1
contains
the
tyrosine
phos-
phorylation
site
situated
at
position
21
(5).
The
antibody
was
diluted
1
in
100
with
WB
contain-
ing
3%
(w/v)
bovine
serum
albumin
and
was
left
bathing
the
nitrocellulose
sheet
for
24
hr.
The
rest
of
the
blot-
ting
procedure
was
as
previously
described,
except
that
the
horseradish
peroxidase-conjugated
second
anti-
body
was
diluted
1
in
500,
not
1
in
1000,
and
was
left
in
contact
with
the
blot
for
3
hr.
Blot
Scanning:
Use
and
Validation
The
Lc
bands
detected
on
each
individual
track of
every
blot
were
scanned
using
a
Chromoscan
3
gel
scan-
ner.
Each
scan
was
0.3
mm
wide.
A
scan
length
of
5
mm
was
long
enough
to
include
the
bands
of
native
and
degraded
Lc
1.
The
scanner
was
set
in
reflectance
mode
to
measure
optical
density
at
530
nm.
The
machine
defm-
ed
the
limits
of
each
band
according
to
preprogrammed
parameters,
and
it
calculated
the
total
and
relative
den-
sities
of
all
the
bands
within
a
5
mm
scan.
It
could
there-
fore
calculate
the
percentage
of
the
total
Lc
1
detected
in
the
parent
(37
kDa)
and
proteolyzed
(34
kDa)
forms.
The
between-blot
reproducibilities
of
the
absolute
and
relative
densities
of
the
37
and
34
kDa
Lc
1
bands
were
determined
by
pooling
the
control
data
from
every
Western
blot
and
calculating
the
coefficient
of
variance
of
these
parameters.
Western
blots
with
identical
samples
of
pure
Lc
1
on
every
track
or
with
a
different
amount
of
pure
Lc
1
(0-500
ng)
on
every
track
were
analyzed
in
the
same
way
to
determine
within-blot
reproducibili-
ty
and
to
determine
whether
optical
density
was
linear
with
respect
to
the
amount
of
Lc
1
applied
to
the
gel.
Effect
of
Human
Neutrophil
Elastase
on
Recombinant
Human
Lc
1
Human
neutrophil
elastase
(NE)
was
active
site
titrated
using
constants
taken
from
Nakajima
et
al.
(1?).
Recom-
binant
human
Lc
1
was
incubated
with
active
(NE)
at
37°C
in
low
protein
hybridoma
medium
in
molar
ratios
of
0.01
to
1
(NE)
to
1
(Lc
1).
Fractions
of
incubation
mix-
tures
were
removed
at
intervals
up
to
48
hr.
The
reac-
tion
was
terminated
and
the
samples
analyzed
by
Western
blotting
as
previously
described.
Determination
of
Elastase
Activity
(EA)
Elastase
activities
(EA)
of
samples
of
CM
and
BALF,
and
PLF
supernatants
were
measured
against
insoluble
elastin
(from
bovine
ligamentum
nuchae)
tritiated
as
previously
described
(18).
EA
was
assayed
by
the
method
of
Banda
and
Werb
(18),
modified
as
previously
describ-
ed
(15).
Human
NE
active
site,
titrated
as
earlier
describ-
ed,
was
used
as
a
standard.
Statistics
The
Mann-Whitney
U
test
was
used
to
compare
lavage
data,
while
the
Wilcoxon
signed
rank
test
for
paired
data
was
used
to
analyze
the
effect
of
CM
on
Lc
1.
The
Spear-
man
rank
correlation
coefficient,
corrected
for
tied
values,
was
calculated
to
assess
the
relationship
between
variables.
In
all
analyses,
p
<
0.05
in
a
two-tailed
test
was
taken
as
statistically
significant.
Results
Validation
of
Scanning
Technique
There
are
two
ways
of
analyzing
the
degradation
of
native
Lc
1
(i.e.,
from
37
to
34
kDa)
from
a
Western
blot
scan.
The
first
involves
measuring
the
reduction
in
op-
tical
density
of
the
37
kDa
band.
This
method
requires
a
linear
relationship
between
optical
density
and
the
amount
of
protein
in
the
band. However,
the
relation-
ship
was
nonlinear,
particularly
at
high
concentrations
of
Lc
1.
Furthermore,
when
equal
amounts
of
exogenous
Lc
1
were
analyzed
on
each
track,
the
reproducibility
of
total
optical
density
measurements
was
very
poor
(lible
1).
The
alternative
method
of
estimating
degrada-
tion
is
to
measure
the
total
density
of
the
37
kDa
(parent)
band
plus
that
of
the
34
kDa
(degraded)
band
and
calcu-
late
the
proportion
of
the
total
density
contributed
by
each
band.
Using
this
method,
it
was
found
that
the
con-
tribution
of
the
37
kDa
band
to
the
total
optical
density
was
very
consistent
within
and
between
blots
(Thble
1)
and
irrespective
of
the
total
amount
of
Lc
1
applied
to
the
gel.
Therefore,
this
measurement
has
been
used
throughout
the
study.
Comparative
Analysis
of
BALF,
PLF,
and
CLF
Lipocortin.
LC
1
was
detected
by
Western
blotting
in
PLF
from
all
healthy
volunteers
and
in
8
of
the
11
PLF
samples
from
patients.
In
addition,
Lc
1
was
detected
in
11
of
the
12
BALF
samples
tested,
but
it
was
not
detected
in
CLF
from
any
subject.
However,
when
samples
of
negatively
staining
lavage
fluids
were
dot-
138
LIPOCORTIN
IN
HUMAN
LUNG
LAVAGE:
ROLE
OF
INFLAMMATORY
CELLS
lkble
1.
Validation
of
scanning
technique.
Coefficient
of
Parameter
Condition
n
x
+
SDa
variance
Total
densityb
Within
blot
7
3144
+
1006
32%
Total
density
Between
blot
45
2240
+
1822
81%
37
kDa,
Within
blot
14
91
+
3
2%
%
of
total
37
kDa,
Between
blot
22
93
±
5
5%
%
of
total
aSD,
standard
deviation.
bTotal
density
is
measured
in
arbitrary
units.
blotted,
Lc
1
was
then
detected
in
all
CLF
and
PLF
and
in
all
but
one
of
the
BALF
(data
not
shown).
The
proportions
of
native
(37
kDa)
to
degraded
(34
kDa)
Lc
1
varied
considerably
between
subjects.
However,
PLF
from
volunteers
contained
at
least
70%
Lc
1
in
the
native
form
(Fig.
1),
which
was
significantly
higher
than
in
PLF
from
patients
(lible
2).
In
contrast,
in
BALF,
the
proportion
of
37
to
34
kDa
Lc
1
was
more
variable
and
there
was
no
statistically
significant
dif-
ference
in
the
percentage
of
Lc
1
in
the
native
form
bet-
ween
BALF
from
patients
with
normal
and
abnormal
lungs
(Thble
2).
0
C)
a)
-o
Ca)
a)
>1
-rp
0
C-
a)
.,,i
4-)
(I
z
--
C)
4-p
(a
z1
90
-
80
-
70
-
60
-
50
-
40
-
30
-
20
-
10
-
0-
PLF-
.
-1-~
~ ~
0
0
9
.
0
0
0
+
0
N
NORMAL
ABNORMAL
NORMAL
ABNORMAL
FIGURE
1:
Percentage
of
37
kDa
Lc
1
present
in
PLF
and
BALF
from
subjects
with
normal
and
abnormal
lungs.
See
text
and
'lible
2
for
statistically
significant
differences
between
groups
and
for
correla-
tions
with
other
lavage
parameters.
Table
2.
Recovery
of
fluid
and
cells,
protein
levels,
elastase
activities
and
proportions
of
native
Lc
1
in
different
types
of
lavage
fluid
from
subjects
with
normal
and
abnormal
lungs.
Percent
of
fluid
Cell
profile,
Tbtal
protein,
37
kDa
Lc
1,
Group
n
recovered
Cells,
x
106
%
total
mg
EA,
ng/mg
protein
%
totala
Normal/volunteer
PLF
6
33(21-40)
16.3(12.2-21.3)
AM
94(81-100)
7.33(5.25-17.55)
27(3-103)
95(73-98)
PMN
4(0-14)
LYM
4(0-7)
Abnormal
PLF
11
31(10-68)
4.7(0.1-9.6)b
AM
87(58-99)
1.88(0.39-14.15)b
36(0-559)
53(0-100)"
PMN
2(1-25)
LYM
8(0-24)
Normal
patient
BALF
5
67(31-75)c
27.5(5.4-108.8)
AM
91(80-96)
15.88(5.59-47.85)
175(72-395)C
63(0-97)b
PMM
1(0-5)
LYM
4(3-20)
Abnormal
BALF
7
39(18-58)
15.3(4.1-58
0)d
AM
55(45-79)e
18.50(7.56-41.70)d
255(38-2860)
32(8-73)
PMN
35(7-56)f
LYM
11(1-15)
Abnormal
CLF
4
33(15-45)
0(0-0)g
AM
ND
0.39(0.02-0.73)g
NA
ND
PMN
ND
LYM
ND
a
Only
samples
positive
for
Lc
1
on
Western
blotting
are
included;
see
Figure
1.
b
<
Normal
PLF.
c
>
Normal
PLF.
d
>
Abnormal
PLF.
e
<
Abnormal
PLF
and
normal
BALF.
f
>
Abnormal
PLF
and
normal
BALF.
g
<
Abnormal
PLF
and
abnormal
BALF.
139
SMITHET
AL.
When
PLF
and
BALF
were
compared
to
each
other,
it
was
found
that
PLF
from
volunteers
had
a
significantly
greater
percentage
of
Lc
1
in
its
native
form
than
BALF
from
patients
with
normal
lungs
(Table
2).
Other
Variables.
Although
there
was
no
statis-
tically
significant
difference
in
the
percentage
of
in-
stilled
fluid
recovered
in
PLF
from
volunteers
compared
to
that
from
patients,
more
protein
was
recovered
from
volunteer's
PLF
(lible
2).
More
cells
were
recovered
from
volunteer's
PLF,
but
the
cell
profiles
were
not
significant-
ly
different
to
those
from
patients.
EA/mg
protein
did
not
differ
between
the
groups
and
neither
did
EA/lavage
(data
not
shown),
despite
the
significant
difference
in
protein
recoveries.
There
were
no
significant
differences
in
fluid
recover-
ies,
levels
of
total
protein,
or
numbers
of
BALF
cells
in
BALF
from
patients
with
normal
lungs
compared
to
those
with
abnormal
lungs
(Thble
2).
However,
a
signifi-
cantly
greater
proportion
of
the
recovered
cells
in
ab-
normal
BALF
were
PMN
(lible
2).
EA/mg
protein
did
not
differ
between
the
two
sets
of
BALF.
When
BALF
and
PLF
from
subjects
with
healthy
lungs
were
compared,
a
greater
percentage
of
the
instilled
fluid
was
recovered
after
BALF,
but
the
total
protein,
number,
and
type
of
cells
recovered
was
not
significant-
ly
different
between
the
lavage
types.
However,
the
EA/mg
protein
was
significantly
greater
in
BALF
than
in
PLF
(Thble
2).
Comparison
of
BALF
and
PLF
from
subjects
with
ab-
normal
lungs
showed
no
difference
in
the
percentage
of
instilled
fluid
recovered.
However,
protein
levels
were
higher
in
BALF
than
in
PLF.
Significantly
more
cells
were
recovered
in
BALF
from
patients
with
abnormal
lungs,
and
the
proportion
of
PMN
was
greater
than
in
abnor-
mal
PLF
(Thble
2).
EA/mg
protein
tended
to
be
greater
in
abnormal
BALF
than
in
PLF
from
patients
with
ab-
normal
lungs,
but
this
difference
was
not
statistically
significant.
The
percentage
of
instilled
fluid
recovered
after
CLF
was
not
significantly
different
to
that
after
other
types
of
lavage.
Cells
were
not
detected
in
any
of
the
CLF,
and
protein
levels
were
significantly
lower
than
in
other
lavage
types
(Table
2).
EA
was
not
assayed
in
these
samples,
because
of
the
small
volumes
of
material
available.
Correlations
ofLipocortin
with
Other
Variables.
No
statistically
significant
correlations
were
found
between
the
proportion
of
Lc
1
in
the
37-kDa
form
and
any
variable
in
PLF
from
patients
or
volunteers.
However,
in
BALF
from
patients
with
abnormal
lungs,
there
was
an
inverse
correlation
between
the
proportion
of
LC
1
remaining
in
the
native
(37
kDa)
form
and
the
number
of
PMN
in
the
lavage
(n
=
7
pairs,
rs
=
-
0.8214,
p
<
0.05).
In
addition,
there
was
a
tendency
towards
an
inverse
correlation
between
the
percentage
of
native
(37
kDa)
Lc
1
and
EA/mg
protein
(n
=
7
pairs,
rs
=
-
0.7500,
p
<
0.10)
in
BALF
from
patients
with
abnormal
lungs.
No
such
relationships
were
observed
in
BALF
from
pa-
tients
with
apparently
normal
lungs.
Analysis
of
BALF
Cell
Secretions
in
CM
1
and
CM
3
and
Their
Effect
on
Recombinant
Lc
1
Under
the
conditions
employed
in
this
study,
Lc
1
could
not
be
detected
in
any
sample
of
CM
before
the
addition
of
exogenous
recombinant
protein.
The
cellular
equivalents
of
the
CM
1
and
CM
3
in-
cubated
with
recombinant
Lc
1
are
shown
in
TAble
3.
PMN
were
present
in
CM
1,
but
not
CM
3.
EA/105
elas-
tase
secreting
cells
(ESC)
was
higher
in
CM
1
than
in
CM
3
(CM
1,
3.3
[0.5-10.0
ng/105
cells];
CM
3,
0.5
[0-9.7
ng/105
cells]
median
[range]
CM
3
<
CM
1,
p
<
0.05).
Therefore,
because
for
any
given
subject,
Lc
1
was
in-
cubated
with
CM
1
and
CM
3
derived
from
equivalent
numbers
of
elastase
secreting
cells,
the
EA/Lc
1
ratio
was
significantly
higher
when
Lc
1
was
incubated
with
CM
1
than
CM
3
(lible
3).
There
were
no
significant
correla-
tions
between
EA
and
PMN
or
AM
number
in
CM
1
or
CM
3.
All
but
two
of
the
CM
1
tested
were
able
to
degrade
recombinant
Lc
1
(Thble
4),
although
some
samples
were
much
more
active
in
this
respect
than
others.
CM
3
were
generally
less
active
than
CM
1
(Fig.
2),
although
the
groups
were
not
statistically
significantly
different
to
each
other,
because
of
the
variability
between
different
CM
1
samples
in
the
capacity
to
degrade
Lc
1.
In
CM
1
the
disappearance
of
parent
(37
kDa)
Lc
1
was
positively
correlated
to
the
PMN/Lc
1
ratio
in
the
samples
after
1
and
6
hr
incubations,
and
it
tended
to
be
correlated
after
24
hr
(Tible
4;
1
hr,
n
=
10
pairs,
rs
=
-0.7200,p<
0.05;
6hr,
n
=
6pairs,
rs
=
-0.9241,
p
<
0.05;
24
hr,
n
=
10
pairs,
rs
=
-0.6398,
p
<
0.10).
In
contrast,
the
number
of
AM
was
inversely
correlated
with
the
disappearance
of
37-kDa
fonn
of
Lc
1
at
the
6-hr
time
point
(RTble
4;
n
=
6
pairs,
rs
=
-0.6891,
p
<
0.05).
The
disappearance
of
parent
Lc
1
was
not
significantly
correlated
to
EA/Lc
1
ratio
in
CM
1
at
any
time
point.
There
were
no
statistically
significantly
correlations
be-
tween
the
percentage
of
native
Lc
1
remaining
and
any
of
the
parameters
measured
in
CM
3.
Identification
of
the
Partially
Degraded
Form
of
Lc
1
Although
the
antibody
raised
to
an
amino-acid
se-
quence
in
the
N-terminal
portion
of
native
Lc
1
cross-
reacted
with
the
37-kDa
protein,
it
failed
to
cross-react
with
the
34
kDa
form
produced
following
incubation
for
3
hr
with
CM
1
from
a
mixed
population
of
cells
(Fig.
3).
Effect
of
Pure
Human
NE
on
Recombinant
Lc
1
Incubation
of
pure
NE
and
Lc
1
at
a
molar
ratio
of
0.1:1
or
0.5:1
resulted
in
complete
degradation
of
the
37-kDa
protein
to
the
34-kDa
form
within
0.5
hr,
while
at
a
ratio
of
1:1,
no
lipocortin-derived
protein
of
any
molecular
weight
could
be
detected
on
a
Western
blot
following
a
140
LIPOCORTIN
IN
HUMAN
LUNG
LAVAGE:
ROLE
OF
INFLAMMATORY
CELLS
Ibble
3.
Cell
profile
from
which
CM
1
and
CM
3
were
derived
and
the
effect
of
these
cells
on
pure
recombinant
Lc
1.
Ratio/Lc
1
Ratio/Lc
1
Control
CM
1
CM
3
medium
Parameter
n
>
10
n
>
9
n
=
2
ESC,
x
105a
1.0(0.4-2.0)
1.0(0.4-1.0)
0
AM,
x
105
0.7(0.3-10O)b
1.0(0.4-1.0)c
0
PMN,
x
105
O.
1(0-0.5)d
O(0_0)e
0
EA,
NEE
ngf
3.0(0.5-9.1)
0.3(0-9.7)e
0
Table
4.
Time
course
of
Lc
1
degradation
by
CM
1
and
CM
3.
37
kDa,
37
kDa,
%
remaining
%
remaining
Control
Time
of
CM
1
CM
3
medium
incubation,
hr
n
>
6
n
=
6
n
=
3
0
100
100
100
0.5
94(29-108)g
-
102
1.0
91(0-l0l)g
101(55-1
10)
100
3.0
81(0-102)8
100(0-114)
99
6.0
42(0-97)g
101(0-105)
102
18.0
4(0-105)g
101(0-114)
-
24.0
32(0-105)g 97(0-99)g
101
48.0
0(0-105)g
86(0-101)
100
aElastase
secreting
cells.
b
+
ve
correlation
with
37
kDa
remaining
in
CM
1,
6
hr.
C>
CM
1.
d
-ve
correlation
with
percentage
of
percentage
of
37
kDa
remaining
in
CM
1,
1
hr
and
6
hr.
e<
CM
1.
fneutrophil
elastase
equivalents.
9<
time
0
hr.
See
text
for
rs
values.
p
<
0.05
for
all
data.
0
0
x
-J
a)
(n
0
a)
4-)
0
.rl
z
C-
U1)
4-)
C,,
4z
U1)
-4-
0.5
1
3
6
18
24
48
Time
(hr)
FIGURE
2:
Median
percentage
of
37
kDa
human
recombinant Lc
1
re-
maining
after
different
times
of
incubation
with
samples
of
con-
ditioned
media
(CM
1
and
CM
3).
CM
1:
Percentage
of
37
kDa
re-
maining
<
time
0
hr
at
all
incubations
times
of
1
hr
or
more,
p
<
0.05.
CM
3:
Percentage
of
37
kDa
remaining
<
time
0
hr
only
after
24
hr
incubation,
p
<
0.05.
FIGURE
3:
Comparison
of
Western
blots
of
pure
recombinant
human
Lc
1
usingantibodies
raised
to
the
whole
Lc
1
molecule
(tracks
A-D)
or
to
a
peptide
consisting
of
residues
13
to
26
from
the
N-terminal
portion
of
the
molecule
(tracks
E-G).
Track
D
contains
molecular
weight
markers.
The
upper
horizontal
line
corresponds
to
a
molecular
weight
of
37
kDa,
the
lower
to
a
weight
of
34
kDa.
Tracks
C
and
G
contain
untreated
pure
recombinant
human
Lc
1.
A
small
proportion
is
in
the
34-kDa
form,
but
only
the
parent
protein
is
detected
by
antibodies
to
the
N-terminal
sequence. Tracks
A,
B,
E,
and
F
contain
recombinant
human
Lc
1
pretreated
for
3
hr
with
a
sample
of
CM
1.
Tracks
A
and
B
show
that
the
majority
of
the
Lc
1
has
a
molecular
weight
of
34
kDa.
No
bands
are
visible
on
tracks
E
and
F,
demonstrating
that
the
34
kDa
portion
of
the
molecule
lacks
the
N-terminal
sequence.
0.5
hr
incubation
(Fig.
4).
Decreasing
the
NE:Lc
1
ratio
to
0.01:1
reduced
the
rate
of
loss
of
lipocortin-derived
protein.
However,
even
at
this
low
ratio,
all
the
protein
was
in
the
34-kDa
form
by
0.5
hr.
After
a
24-hr
incuba-
tion
with
NE
at
a
ratio
of
0.01:1
(NE:Lc
1),
no
lipocortin-
derived
protein
was
detectable
on
a
Western
blot
(Fig.
4).
Discussion
Lipocortin
1
was
detected
in
all
but
one
of
the
lavage
samples
analyzed,
irrespective
of
the
clinical
status
or
smoking
history
of
the
subjects.
We
believe
this
to
be
the
first
report
of
the
presence
of
any
member
of
the
lipocor-
tin
family
in
human
lung
lavage
fluid.
In
addition,
it
has
been
shown
that,
although
in
healthy
volunteers
the
Lc
1
was
predominantly
in
its
native
form,
in
many
of
the
lavage
samples
from
patients,
a
high
proportion
of
the
protein
was
partially
degraded.
Our
in
vitro
studies
sug-
gest
that
this
degradation
could
be
caused
by
one
or
more
141
SMITH
ET
AL.
FIGURE
4.
Western
blot
of
recombinant
human
Lc
1
following
prein-
cubation
with
NE
at
different
molar
ratios.
(A)
Track
G
contains
molecular
weight
markers.
Tracks
A-D
contain
Lc
1
pretreated
with
NE
at
a
ratio
of
0.5:1
(NE:Lc
1)
for
1,
10,
30,
and
60
min
respective-
ly.
After
a
1-min
exposure,
the
majority
of
the
Lc
1
is
partially
degraded.
By
10
min
it
is
all
partially
degraded,
and
by
60
min
it
is
completely
proteolyzed.
Tracks
E
and
F
contain
Lc
1
pretreated
with
NE
at
a
ratio
of
1:1
for
10
and
30
min,
respectively.
Following
a
10-min
exposure,
all
the
Lc
1
is
partially
degraded,
while
by
30
min
it
is
completely
proteolyzed.
(C).
Track
H
contains
molecular
weight
markers.
Tracks
A-G
contain
recombinant
human
Lc
1
pretreated
with
NE
at
a
molar
ratio
of
0.01:1
(NE:Lc
1)
for
0,
0.5,
1,
3, 6,
24,
and
48
hr,
respectively.
Within
30
min
all
the
Lc
1
has
been
proteolyzed
to
the
34
kDa.
products
of
the
PMN,
while
studies
with
a
specific
anti-
body
indicate
that
this
partially
degraded
Lc
1
lacks
the
N-terminal
portion
that
we
believe
to
be
essential
for
full
anti-inflammatory
function
in
vivo
and
in
cellular
systems
in
vitro
(Flower
et
al.,
unpublished
observation).
In
previous
studies,
lipocortins
have
been
detected
in
lung
tissue
from
the
rat
(6),
from
cattle
(7)
and
in
feline
tracheal
rings
(19).
The
most
likely
source
of
the
Lc
1
detected
in
our
lung
lavage
fluids
is
the
AM,
since
studies
in
the
rat
(5,20)
and
on
cell
lines
from
various
species
(6)
show
that
monocytes
and
macrophages
are
frequent-
ly
rich
sources
of
lipocortins.
Lipocortins
require
Ca2
+
to
remain
attached
to
the
plasma
membrane,
and
thus,
lavage
of
the lung
with
a
calcium-free
solution
(NaCI)
may
result
in
some
of
the
Lc
1
becoming
detached
from
the
AM
surface
and
solubilized
in
the
supernatant
frac-
tion
of
the
lavage
fluid.
However,
the
possibility
that
Lc
1
exists
in
a
free
form
at
the
epithelial
surface
can-
not
be
excluded.
In
the
current
study,
amounts
of
Lc
1
could
not
be
quantified.
This
is
probably
because
factors
such
as
am-
bient
temperature
and
the
activity
of
conjugated
horseradish
peroxidase
are
difficult
to
control
but
may
modify
the
density
of
staining.
In
contrast,
the
relative
proportion
of
37
kDa
to
proteolyzed
(34
kDa)
Lc
1
was
very
reproducible
(lible
1)
and
has,
therefore,
been
determined.
Taking
into
account
the
degree
of
resolution
obtained
with
SDS-polyacrylamide
electrophoresis,
it
is
probable
that
the
34-kDa
band
observed
throughout
the
current
study
is
the
same
or
very
similar
to
the
33-kDa
species
generated
from
human
recombinant
Lc
1
by
Huang
et
al.
(9).
These
authors
showed
their
fragment
to
be
clip-
ped
30
residues
from
the
N-terminal
of
the
precursor
(37
kDa)
molecule.
In
addition,
an
earlier
study
by
the
same
group
(6)
demonstrated
that
some
of
the
endogenous
Lc
1
isolated
from
rat
peritoneal
extracts
was
clipped
in
the
same
region.
Thus,
it
seems
that
both
in
vivo
and
in
vitro,
this
region
of
Lc
1
is
the
most
vulnerable
to
pro-
teolysis.
In
the
current
study,
it
has
been
shown
that
the
partially
degraded
Lc
1
occurs
in
the
human
lung
('lble
1)
and
that
NE
and
possibly
other
products
of
the
PMN
are
capable
of
causing
such
degradation
(Thble
3).
In
ad-
dition,
this
study
has
shown
that
the
34-kDa
form
lacks
the
N-terminal
end
of
the
parent
protein
since
it
does
not
cross-react
with
an
antibody
to
this
epitope
of
the
molecule
(Fig.
3),
increasing
the
probability
that
we
have
studied
the
same
molecular
species
as
Huang
et
al.
(9).
Although
the
results
of
cell-free
PLA2
assays
(9)
suggest
that
the
clipped
form
inhibits
PLA2,
other
authors
con-
sider
cell-free
systems
to
be
flawed
(21).
Therefore,
because
we
believe
bioassay
to
be
more
relevant
to
the
situation
in
man,
we
are
confident
that
measurement
of
the
ratio
of
37:34-kDa
forms
is
actually
a
measure
of
the
ratio
of
functional:nonfunctional
Lc
1.
The
results
of
the
in
vitro
studies
(Thble 3)
show
that
the
recombinant
Lc
1
was
much
more
actively
degrad-
ed
by
conditioned
media
from
mixed
populations
of
cells
containing
AM
and
PMN
than
that
from
adherent
AM
142
LIPOCORTIN
IN
HUMAN
LUNG
LAVAGE:
ROLE
OF
INFLAMMATORY
CELLS
143
alone,
suggesting
that
the
degraded
Lc
1
observed
in
lung
lavage
samples
may
be
the
result
of
PMN
activity.
This
was
supported
by
the
observation
that
degradation
of
37
kDa
Lc
1
was
related
to
the
number
of
PMN
in
CM
1
(Thble
3).
It is
not
clear
from
these
data
what
factor
(or
factors)
released
into
CM1
by
the
PMN
is
responsible
for
the
breakdown
of
Lc
1.
Even
though
pure
NE
is
able
to
proteolyze
Lc
1
(Fig.
3),
no
relationship
was
observed
between
EA
and
proteolysis
of
Lc
1
in
our
in
vitro
ex-
periment.
There
are
a
number
of
possible
reasons
for
this;
EA
is
a
measure
of
activity
from
all
elastolytic
en-
zymes,
some
of
which
may
not
degrade
Lc
1,
thus
in
the
in
vitro
study,
metalloelastase
from
the
AM
(22)
may
have
made
a
significant
contribution
to
the
EA
measured.
Furthermore,
it
is
possible
that
two
or
more
enzymes
act
in
sequence
to
degrade
Lc
1-for
example,
studies
with
the
pure
protein
show
that
NE
first
degrades
Lc
1
to
the
34-kDa
form
(Fig.
4).
This
may
cause
a
three-
dimensional
change
to
the
lipocortin
structure
that
makes
it
more
susceptible
to
attack
by
other
PMN
pro-
teases,
for
example,
cathepsins.
Equally,
an
unidentified
protease
could
make
the
initial
clip
in
the
parent
Lc
1
and
NE
could
perform
subsequent
degradation.
In
view
of
the
animal
studies
showing
macrophages
to
be
rich
in
lipocortins
(5,20),
it
is
perhaps
surprising
that
endogenous
Lc
1
was
not
detected
in
media
where
AM
had
been
cultured
(CM).
However,
the
CM
samples
were
taken
from
very
small
numbers
of
cells
and
had
media
from
a
larger
cell
population
been
investigated
or
the
sensitivity
of
the
detection
system
been
increased,
Lc
1
might
have
been
detected.
Alternatively,
because
the
tissue
culture
media
contain
Ca2
+,
any
Lc
1
pro-
duced
by
the
AM
may
have
remained
attached
to
the
plasma
membrane
and,
thus,
would
not
be
detected
in
CM.
Alternatively,
Lc
1
released
into
the
CM
may
have
already
been
proteolyzed
to
small
peptides
that
would
not
be
detected
on
a
Western
blot.
A
final
possibility
is
that
human
AM
synthesize
a
member
of
the
lipocortin
family
other
than
Lc
1,
which
would
not
cross-react
with
our
antibody.
Further
studies
will
be
necessary
to
dif-
ferentiate
between
these
possibilities.
The
results
of
the
immunoblotting
experiments
in-
dicate
that
some
samples
of
lavage
fluid
contain
more
Lc
1
than
others.
While
the
reasons
for
differences
be-
tween
subjects
undergoing
the
same
type
of
lavage
are
unclear,
the
low
LC
1
levels
in
CLF,
compared
to
PLF
and
BALF,
may
reflect
the
paucity
of
AM
in
the
large
airways
washed
during
the
central
lavage
procedure.
However,
it
is
also
possible
that
in
some
lavages,
particularly
in
CLF,
which
contain
relatively
high
protease
activities
(14),
the
Lc
1
was
degraded
to
small
peptides
that
would
not
be
detected
on
a
Western
blot.
The
lavage
data
(Tble
2)
suggest
that
the
proportion
of
native
to
partially
degraded
Lc
1
reflects
both
the
health
of
the
subject
and
the
region
of
the
lung
sam-
pled.
PLF
contains
proportionally
less
34
kDa
Lcl
than
BALF.
Unlike
BALF,
PLF
contains
no
material
from
above
the
seventh
generation,
so
it
is
likely
that
much
of
the
34
kDa
Lc
1
in
BALF
is
from
the
airways.
Analysis
of
more
concentrated
samples
of
CLF
or
immunoblotting
with
the
antibody
to
the
N-terminal
epitope
would
clarify
this
point.
It
is
possible
that
the
mucociliary
escalator
is
a
route
for
clearance,
either
of
nonfunctional
lipocortin
or
of
cells
(probably
AM)
carrying
nonfunctional
lipocor-
tin.
Alternatively,
more
degradation
of
lipocortin
may
occur
in
the
upper
respiratory
tract
than
in
the
periphery,
a
possibility
supported
by
earlier
studies
from
this
laboratory
showing
that
protease
activity
per
unit
albumin
and
the
number
of
PMN
(expressed
as
a
percent-
age
of
the
total
cell
population)
are
both
higher
in
CLF
than
in
PLF
(14).
Also,
it
is
known
that
phosphorylation
of
the
tyrosine
residue
at
position
21
of
Lc
1
down-
regulates
its
PLA2-inhibitory
capacity
(23)
and
that
once
phosphorylated,
it
is
more
susceptible
to
degradation.
Thus,
it
is
possible
that
in
the
upper
respiratory
tract,
cells
are
stimulated
(for
example,
to
phagocytize
and
degrade
large
inhaled
particles
deposited
in
the
upper
airways),
and
that
the
subsequent
downregulation
of
lipocortin
by
phosphorylation
results
in
an
increase
in
proteolysis
of
Lc
1.
Summary
In
summary,
we
have
demonstrated
that
Lc
1
is
present
at
the
epithelial
surface
of
the
human
lung,
usually
in
a
variable
mixture
of
native
and
partially
degraded
forms.
This
study
suggests
a
role
for
the
PMN
in
inactiva-
tion
of
this
protein
that
may
be
important
in
inflam-
matory
lung
diseases.
Further
studies
to
bioassay
and
quantify
the
Lc
1
will
be
necessary
before
the
physio-
logical
significance
of
these
observations
can
be
fully
evaluated.
An
influx
of
PMN
during
inflammation
may
be
a
mechanism
of
downregulating
lipocortins
and
would
per-
mit
the
inflammatory
response
necessary
to
destroy
a
provocative
stimulus.
Removal
of
the
stimulus
with
reduced
PMN
numbers
would
halt
lipocortin
degrada-
tion.
Equally,
it
is
possible
that
in
chronic
inflammation,
the
inactivation
of
lipocortin
is
pathological,
perhaps
when
the
stimulus
cannot
be
destroyed,
for
example,
in
asbestosis
or
pneumoconiosis.
In
such
situations
a
massive
increase
in
lipocortin
synthesis,
induced
by
high
doses
of
exogenous
glucocorticoids,
may
then
be
the
only
way
of
halting
the
inflammatory
process.
The
authors
thank
the
Medical
Research
Council
for
financial
support
and
Biogen
Inc.
Cambridge,
MA,
for
their
generous
gift
of
recombinant
human
Lc
1
and
for
specific
antibodies
to
the
whole
molecule.
We
also
thank
R.
Thylor
for
technical
assistance
in
prepar-
ing
the
antiserum
to
the
tyrosine
phosphorylation
sequence
of
Lc
1.
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