Lung Repair and Granuloma
Formation*
Tubercie Bacilli Stimulated
Neutrophils
Release Chemotactic
Factors for
Monocytes
Veena
Ruth
T
B. Antony,
M.D.;
Steven
N. Haroda,
M.D.;
and John
he
common
and other
granulomata.
A. Sahn,
underlying
E. Repine,
host
which
,originate
cytes (MN).’
granulomatous
Since neutrophils
inflammation,2
F.C.C.P;
M.D.
response
granulomatous
repair
These
granulomata
phagocytes
M.D.,
from
in tuberculosis
processes
include
is formation
mononuclear
peripheral
blood
precede
monocytes
we hypothesized
trophils
initiate
these
inflammatory
chemotaxins
which attract
monocytes.
BCG
colonies
granuloma
intrapleurally,
numbers
recoverable
formation
from
on pleural
pleural
fluid,
by tuberboth in
treated
was associincreased
and
Injection
caused
±
responses
by releasing
Our results
supported
neutropenia
of macrophages,
RESULTS
mono-
at areas of
that neu-
this premise
(Fig 1). First,
neutrophils
stimulated
cle bacilli released
chemotactic
factors for monocytes,
vivo
and
in vitro.
Second,
in nitrogen
mustard
rabbits
given
BCG
ated
with
decreased
of
nitrogen
mustard
given in a single bolus dose (1.75 mg/kg) 60 hours
prior to initiation
of the experiment.
In some experiments,
immediately following
BCG instillation,
the pleural
spaces of nitrogen
mustard-treated
rabbits were reconstituted
with neutrophils
isolated from peripheral
blood.4 Exudative
pleural effusions developed
and were sampled
sequentially
via thoracocentesis
over the next 120
hours. Pleural
fluid was analyzed
for cytology
(Papanicolaou
technique) and pleural fluid supernatants
evaluated
for their chemotactic
activity for monocytes
using modified
Boyden chambers.5
Supernatants from serum-free
mixtures
of BCG and neutrophils
incubated
at
37”C for 4 hours also were evaluated
for their chemotactic
activity for
monocytes
in vitro.
nant
cell
eFrom the Pleural
mental
Medicine,
Pulmonary
Colorado
Supported
Space Laboratory
Webb-Waring
fluids
and the Laboratory
of ExperiLung Institute
and Division
of
of Medicine,
University
of
Sciences,
Department
School
of Medicine,
Denver.
in part by grants
from the
National Institutes
of Health
and T32 HL 67685), the American
Heart Association,
the Council Ibr Tobacco Research
USA Inc., the Kroc, Swan, Hill
and Kleberg Foundations.
Dr. Repine is an Established
Investigator of the American
Heart Association.
Reprint requests: Dr Antony, University
of Colorado Health Science
Center, Box C-272, 4200 East Ninth, Denver, 80262
(HL
24248
the
SEMI
±
recovered
in nitrogen
neutrophils
pleural
spaces
influxes
cells/p.l
from
of normal
of cells
rabbits
(peak
by 4 hours).
pleural
mustard-treated
in pleural
For
of 11,800
the
numbers
and
pleural
of BCG
decreased
surfaces
experiment,
nitrogen
at autopsy.
first
24
(>60%).
In contrast,
was an absence
of
count
<200
cells)
by the
chemotaxins
for
recoverable
from
pleural
on the visceral
and
parietal
Importantly,
in the reconstitution
usual
influx
of macrophages,
and granuloma
that neutrophils
monocytes.
This
chemotactic
activity
for monocytes.
chemotactic
activity
for monocytes
instillation
trophils
in
and
pleural
neutropenic
fluid.
rabbits
did
In
not
decreased
was
Furthermore,
with
contrast,
show
sup-
fluids obtained
from
a marked
degree
of
was seen
correlated
of
fbrmation.
The
might be produc-
suggestion
when
supernatants
from pleural
rabbits
treated
with BCG showed
BCG
of neu(<10%),
of neutrophils
into pleural
spaces
rabbits
given BCG
intrapleurally
numbers
of BCG colonies
aforementioned
suggested
ported
normal
colonies
granulomata
instillation
mustard-treated
was followed
ing
spaces
rabbits
there
spaces
(absolute
injection
of BCG.
Furthermore,
absence
was followed
by a lack of macrophages
increased
New Zealand
white rabbits
were immunized
with intradermal
bacillus Calmette-Gu#{233}rin (BCG) and sensitization
was confirmed
by
protein purified
derivative
(PPD)
challenge.
Rabbits then received
intrapleural
BCC via a percutaneous
technique.3
For some experiments,
rabbits were made neutropenic
(<200 neutrophils/d)
with
into
reproducible
[mean
3,000
trophils
METHODS
and
hours
after
BCG
instillation,
this cellular
influx
consisted
primarily
of neutrophils
(>75%,
Table 1). However,
96 hours
following
injection
of BCG,
macrophages
were the predomi-
following
decreased
surfaces.
of BCG
large
the
number
pleural
evidence
maximal
at 24 hours
after
of neufluids
from
of chemotactic
HYPOTHESIS:
Neutrophils
Initiate
Macrophage
Responses
to I
Neutrophils are attracted
AFB (BCG).
a
by
AFB stimulated
neutrophils
release
chemotaxins
for
monocytes.
Macrophages
localize
AFB
(granutomas).
FIGURE
1.
Hypothetical
neutrophil-initiated
sponses to tuberculosis.
CHESTI83I5IMay,l9e3lSupplement
Downloaded From: http://publications.chestnet.org/pdfaccess.ashx?url=/data/journals/chest/21359/ on 06/17/2017
mechanism
macrophage
of
re-
95S
Table 1-Pleural
Fluid
Nitrogen
Mustard-Treated
BCG
Characteristics
Neutropenic
Intrapleuraily
in Normal
and
Rabbits
Given
to infection
3 Antony
tory
Chemotactic
Total
CellNeutrophils
Count
(X103/ml)
(% total
Cells)
(% total
Cells)
VB, Repine
monocytes
(MN/10 HPF)
human
5 Snyderman
eds.
8±1
4±1
R, Pike
chemotaxis
32±2f
67±4t
8±1
4±4
8±1
Fibrotic
Lung
Factor
9±1
A Growth
96hr
2.0±0
5±2
6±2
5±3
Human Lung
tValue
significantly
neutropenic
rabbits
activity
bits
ftr
monocytes
reconstituted
while
with
activity
fbr monocytes
I3CG and neutrophils
monocytes
seen
in
determinations)
(p<O.OS)
increased
injected
with BCC.
to value
nitrogen
HPF)
for
mustard-treated
showed
which
rabmixtures
activity
of
BCG
of
for
than
(15
±
that
2)
or
spaces
of rabbits
given
BCG.
Second,
in
on pleural
of the usual
macrophage
on the basis of the direct
on monocytes,
trophils,
the
usual
trophils
the only
were
nitrogen
tion
since
following
macrophage
variable
mustard-treated
was
followed
appeared
that
of monocytes.
chemotactic
fluids
by
the
neutrophils
This
activity
of normal
into
and
since
appearance
were
was
with
Since
pleural
neuneu-
spaces
neutrophil
in the
substantiated
was found
neutrophil-reconstituted
of
injecit
recruitment
when
in pleural
rabbits,
trophils
formation
in in vitro
observations
mixtures
support
participate
in monocyte
and suggest
that this
in granulomatous
inflammation
of neutrophils
the possibility
recruitment
mechanism
and
and
that
the
JI,
macro-
Quie
PC,
S. Rennard,
that
important
modulators
tin to augment
human
To accomplish
cultured
in
medium
present
fibrotic
1%
but
BCG.
neu-
and granuloma
may be involved
repair.
REFERENCES
WC. The granulomatous
inflammatory
exudate.
Int Rev
Rap Pathol 1969; 8:1-55
2 Montgomery
LG, Simon WS. The cellular reaction of the pleura
alone
seen
in
contrast,
alveolar
gelatin
extracellular
matrix
serum
the
growth
2ag/ml
lOOnM,
the
derived
patients
p
of
fibronectin
growth
with
in the fibrosis
the numbers
factor for lung
macrophages
in cell
Cells
number
p
cultured
growth
above
>0.2.
In
that
marked
both
fibronectin
factor
showed
up to 185%
macrophage
and
a dra-
together
with
above
control
derived
both
comparisons
increased
amounts
factor
within
fibrotic
lung disease
characteristic
of fibroblasts
(0
in cell number
ranging
from
0.2 g/ml
and alveolar
macro-
alveolar
<0.001,
fol-
cultures
macrophage
of most
patients
with
growth
factor
addition,
mM)
factor
+
presence
component
a growth
activated
with
growth
increase
(fibronectin
human
chromatography
(control),
fibroblasts
cultured
macrophage-derived
but
from
to the fibroblast
with
alveolar
increase
medium
5 p.g/ml
a viable,
purified
structures
Following
(HFL-1)
modified
+
in
aftinity
To
are
of fibronecwas evalu-
cultured
72 hr, then counted.
or alveolar
macrophage-derived
matic dose-dependent
88% above control
factor
Thus,
elements
ablumin
fibroblasts
Fibronectin,
little
mediates
matrix.
the ability
replication
factor (0 to lOOnM),
is released
by the
showed
phage-derived
that
extracellular
sepharose
defined
(fibronectin
B.S.;
a glycoprotein
to the
bovine
in the alveolar
lung
disease.
factor
S. Adelberg,
human
lung fibroblasts
medium
(Dulbecco’s
maintain
state.
by
M.D.;
of cell number,
lung fibroblast
this,
defined
+
to
plasma
in
Disease*
for
concept
fibroblasts
were
with
fibronectin
not in neutropenic
rabbits
given
intrapleural
BCG.
This
phenomenon
also was validated
by finding
similar
monocyte
chemotaxins
The present
and
Callin
1978
of fibronectin,
derived
growth
fibroblasts
that
The
of macrophages,
involved
impression
for monocytes
and
surfaces.
reconstitution
influx occurred.
injected
Press,
lowed by gel filtration,
was added
to 2 pg/ml)
alone
or together
influx
could
not be exeffect of nitrogen
mustard
rabbits
for monocyte
chemotaxis.
of fibroblasts
nonreplicating
a lack
absence
plained
Methodology
M.D.
amount
Eagle’s
neutropenia
was associinflux,
increases
in the
from pleural
fluids
and
granulomata
cells and granulocytes
from
21(Suppl 97): 77-89
1968;
to an accumulation
of collagen,
the alveolar
of patients
with
fibrotic
lung
disease
are
by increased
numbers
of fibroblasts
and in-
attachment
transferrin)
nitrogen
mustard-treated
rabbits,
ated with an absence
of macrophage
number
of BCG colonies
recoverable
of well-defined
the
ated.
were
Considerable
evidence
was obtained
to support
the premise that neutrophils
play an important
role in granulomatous
inflammation.
First,
neutrophils
preceded
influxes
of macropleural
creased
evaluate
(10 ± 4) alone.
into
M.D.;
n addition
structures
characterized
DISCUSSION
phages
P Bitterman,
I
chemotactic
was greater
mixtures
SA. InflammaActa Cytol
pleurisy.
Fibroblasts
and R. G. Crystal,
(Table 1). Supernates
from
also contained
chemotactic
(80 ± 6 MN/10
supernates
from
neutrophils
compared
neutrophils
tuberculous
Role of Fibronectin
-
6±4
(of 18 or more
Surg 1933;
-
78±2t
30±2t
20±lt
5±0
SEM
MC.
New York: Raven
2.5±0
±
RN, Good JT, Sahn
in leukocyte
48hr
tMean
JE, Harada
in experimental
A. Isolation
of mononuclear
blood. Scand J Clin Invest
phage
Normal rabbits given BCG
Ohr
1.7±0.4
2±0.3
l2hr
10.6±3,t
76±3t
48hr
3.6±1
20±4t
96hr
2.5±0
4±1
Neutropenic
rabbits given 8CC
Ohr
1.7±0.4
2±0.3
l2hr
3.0±1
5±2
J Thor
tuberculosis.
(in press)
4 Boyum
Activity
mycobacterium
responses
for
Macrophages
with
2:429-39
growth
with control).
of the matrix
alveolar
macrophage-
the alveolar
structures
may play a significant
of
role
of these disorders,
by amplifying
in the alveolar
structures
of these
patients.
1 Spector
5From
the National
Institutes
of Health,
Heart,
Lung
Bethesda.
96s
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and
Blood
Lung
Institute,
Defense.
National
Injury and Repair