eosinophil chemotaxis during allergic airway inflammation ... of eosinophils to the site ofallergic reactions. ..... in absolute methanol, and stained with eosin and.
c-c
chemokine-induced airway inflammation
eosinophil
Nicholas W. Lukacs, Theodore J. Standiford7 Robert M. Strieter and Steven L. Kunkel Department
of Pathology
and
*Depar.tmeru
of Internal
chemotaxis Stephen
Abstract:
The
chemotactic may be mulation, studies
production
of
have
identified
play crucial site ofallergic
chemokines
roles in recruitment reactions. In this
an established -mediated specffic
specific
model ofschitosome allergic responses
C-C
chemokines
protein-la
of eosinophils study we have egg to examine
RANTES,
and
aUractant protein-i (MCP-1)J in ment. We have previously identified in eosinophil
accumulation
ing
airway
studies using lish that both phil way
chemotactic responses.
lungs beginning ing at 48-96 in
vitro
eosinophil
eosinophil
durthose
allergic demonstrated
air-
within
the
When
lung
allergen-challenged
incubated in la, RANTES,
vitro
with antibodies specific for MCP-1, a signfficant reduction
phil way
chemotactic
factors
J.
responses.
Leukoc.
produced
during
Biol.
60:
a significant
were
preMIPin
immune
of the
sponsible and are ciated The to the Specific
reaction,
bronchial
characterized
mucosa
with
macrophages, been reported
for the induction thought to induce
573-578;
air1996.
such
by the
as
16J. These of the first
baso-
and lymphocytes to be the primary of
the
infil-
neutrophils,
bronchial bronchial
[2]. re-
cell
mucosal obstruction
with the asthmatic response [5-14J. elicitation of leukocyte subsets has been production of several different chemotactic neutrophil, mononuclear, and eosinophil
differences. chemotactic
injury assoattributed factors. chemo-
been identified. These into two distinct superC-C (beta) chemokines designated by the posiand reflect functional
The C-X-C family of chemokines is primarily for neutrophils and is typified by interleukin-8
(IL-8). The C-C family of chemokines is primarily chemotactic for mononuclear phagocytes, lymphocytes, and/or eosinophils and contain closely related proteins that indude monocyte chemoattractant protein-1,2,3 (MCP-1,2,3), macrophage
MIP-la studies eosino-
allergic
proportion
diseases
asthma [1J. The long-term pathological effects of asthma have been attributed, in part, to infiltrating leukocytes that surround the bronchus and infiltrate into the airway [2-41. The immune response associated with asthma has been described as having histopathological features of a chronic,
demon-
from
eosinophil chemotaxis was observed with only and RANTES neutralization. Altogether, these indicate that RANTES and MIP-la are major
Care,
include
divisions have been two cysteine residues
homogenates
or
affect and
[15, tion
These of pen-
mice
diseases
plateauUtiliz-
chemotacand peak ac-
in situ.
Critical
(chemokines) have have been divided C-X-C (alpha) and
24-h lung homogenates. morphometric analysis
accumulation
and
tactic cytokines chemoattractants gene families,
assays
in eosinophil homogenates
G. Kunkel,
Medicine,
worldwide
phils, eosinophils, Eosinophils have
allergen-challenged
chemotactic
increases 8-h lung
with samples from correlated with the
bronchial
from
pulmonary
recruitMIP-la
to estabeosino-
of eosinophils
Lab
population
tration
airway
eosinophil chemotaxis RANTES are potent
Allergic of the
chemo-
extend
a and
homogenates
strated significant tic activity with tivity data
and
at 8 h, peaking at 24 h, and h after allergen (SEA) challenge.
whole-lung
mice,
lung
for
of Pulmonary and
cell-mediated
inflammatory
We
accumulation
(SEA) role of
monocyte
factors in lungs during Morphometric analysis
a peribronchial
ing
in the
may
to the utilized
antigen the
eosinophil a role
inflammation.
in vitro MIP-1
that
(macrophage
(MIP-la),
allergic
airway responses eosinophil accudamage. Recent
in
Divz-.sion
of Pathology
Robin
allergic
I NTRODUCTION
eosinophil-specffic
factors during allergic a pivotal event resulting activation, and airway
W. Chensue,t
Medicine
University of Michigan Medical School; and t Department The Veterans Affairs Medical Center, Ann Arbor, Michigan
during
inflammatory
Abbreviations:
SEA,
inflammatory IL-8,
interleukin-8;
fields;
PBS,
HBSS,
adjuvant;
Hanks’
IFA, of
balanced
antigen;
monocyte salt
Freund’s Nicholas
Michigan
egg
MCP-i,
(MIP-la),
Medical
W. School,
a, macrophage
MIP-1 saline;
solution;
TNF,
Lukacs,
Ph.D.,
protein-i. HPF,
CFA,
adjuvant; 1301
eotaxin,
chemoattractant
phosphate-buffered
incomplete
Correspondence: Univ.
schistosome
a;
protein-i
protein-la
high-power
complete
tumor
Freund’s
necrosis
Dept.
of
Catherine,
factor.
Pathology,
Ann
Arbor,
MI
48109-0602.
Key Word.: protein-la
schrstosome .
monocyte
egg antigen chemoattractant
.
macmphage protein-i
Received
inflammatory .
RANTES
8,
April
10.
1996;
revised
August
7. 1996;
accepted
August
1996.
Journal
of
Leukocyte
Biology
Volume
60,
November
1996
573
and
RANTES
lar
[15].
interest
The
in the
C-C family
allergic
members
response
Morphometric
of particu-
are
because
there
appears
Mice
to be a strong correlation with the continued expression of C-C chemokines and chronic diseases that feature mononuclear and eosinophilic leukocyte recruitment [16]. In particular, eotaxin, MIP-iga, and RANTES may be important in allergic teins have
responses for several reasons. shown to be chemotactic for
airway
been
differentially and more
chemotactic importantly,
23].
the
With
model
use
we have
for lymphocyte chemotactic for of
the
previously
an important role in eosinophil airway challenge [24J. In this
study
the
direct
sinophil accumulation was examined. The
subsets eosinophils
[17-19J, [20-
allergic
airway
SEA-induced demonstrated
These promonocytes,
that
MIP-la
accumulation
effect
of C-C
in vivo
airway that
be
nearly
samples from anti-RANTES gesting
that
abrogated
allergic antibody, MIP-la
mediating
by
and
eosinophil
RANTES
have
accumulation
eosinophils
were
anti-MIP-la in vitro,
important
whole-lung
tissue
Products,
Racine,
was
quent
filtration
Arbor,
MI).
1:1
ratio
or sug-
roles
in
model.
Isolation
ME)
were
maintained
under
standard
and soluble preparation
Harbor,
Eosinophils
protein Soluble
egg
mice
as previously
antigens
of infected the
mice
soluble
an
were
after
from
ultracentrifuge
The
in the
characterized
schistosomiasis
ofcirculating
the
and
ground
g for
2 h and
mansoni-infected
were
as inducing
a Th2-type
ice
was
the
primarily
in l0-
a Th2-type
in
CBA/J
normal
were
nasal
to release
then
spun
supernatant
in
collected.
glycoproteins,
which
granulomatous
cell
0 and
7 of the
to localize
lenge
with
the
antigen
mice
upon
later
by intratracheal
PBS
or with
after
the
in
previously
Journal
Mice
The
10%
control the
described
of
and
Leukocyte
the
were
This
initial into
mice
given
eggs an intra-
intranasal the in 25
killed and
The mice
neutrophil
72
displayed and
of the of sterile
at various h) and was
a
the
times
were
be
the
easily by
and
at a
and
with
to
staining
calf
be
adherence
techniques
and
lymphoand
the
approximately
in
the
chemotaxis
lymphocytic.
on
serum
in tissue and
stained,
used
h
streptomycin.
yields
morphologically
granular
L-glutamine,
(eosinophils
primarily
were
fetal
and
procedure
cells
48
Cells
5%
differentially
populations
a pool
After
mM
by plastic
This
of the
20
cells and
counted. pure
collected.
of penicillin
cytospinned,
peritoneum
induces
peritoneum.
cells
pyruvate,
the
protocol
the
the
removed
differentiated
nuclei
into
nonadherent
remainder
into
This
recruited
respectively,
1 h. The
eosinophil
The
cells
stained
chemotaxis
described
above.
assays
60,
tL
of
chemokine,
or HBSS
cate
wells
bottom
suspension
taxis
chamber
(GIBCO,
for
and
(Sigma
and
x
For
and
vehicle
stained
with
counted
in
10
anti-serum
30
37
fixed
was
eosinChemo-
(400
through or
as
were #{176}C,then
number
toluidine bottom
of cells from
preincubated assayed
2% to the
media or
absolute
x magnification).
1000 the
95%
in
with
migrated
anti-MCP-l, at
chamber.
removed,
conditioned
mice
mm
top
size
Corp.)
murine
counterstained
HPFs
anti-RANTES, for
in the
were
expressed
was
in duplipore
at 37#{176}Cin humidified
had
experiments,
airway-challenged control,
placed
and
that
placed A 3-tim
of peritoneal-eicited
filters
eosin
were
chamber.
incubated
Cells
neutralization
of
NY)
recombinant
Nucleopore
was
The
[28J.
described
specimen,
Island,
p1
were
mm.
75
were
previously
chemotaxis 250
of eosinophils
HPF.
Grand
106/mL)
Chemical).
filter
as
supernatant
(polyvinylpyrrolidone-free,
(1
CO2
tactic
(1:1)
assemblies
Chemotaxis
ing
performed
assembly
ophil
lution
were
diluted
of a blind-well
filter
in the
with
both a 1:200
anti-MIP-la for
per SEAdi-
neutraliz-
chemo-
cellular
activity.
Production of antibodies murine C-C chemokines Rabbit
examined
multiple-site
significant
eosinophil
November
specific
for
MCP-1
antibodies
were
White
rabbits
lungs
infil-
anti-munne
binant vant
murine (CFA).
chemokine
Volume
assay
150
of infiltra-
[271.
Biology
(HBSS).
Ann
assays
6 days
tL
magnitude mice
chal-
lungs
rechallenged
were
mice
saline
then
48,
Sciences,
solution
injection
sodium
mg/mL,
for
with
of the
mansoni
of SEA
SEA-challenged
included
Briefly,
S.
infiltrate
formalin.
SEA-challenged which
mice
were
(1, 8, 24,
buffered
established
of phosphate-buffered
of 10 tg
The resulting g) and subse-
(Gelman in chemotactic
supplemented
2 mM
collected,
methanol,
was
isolated
airway.
cellular
(vehicle).
vehicle
response, as
to the little
challenge in
Only
inflammatory
574
alone
fixed the
14 the
10 p1
examination.
intratracheal and
day
in
administration
PBS
histologically.
tration
response
L241.
described 5000
a tissue-tearer
using
(10,000
filter
mice.
lavaged
of eosinophils
placed
response
procedure
with On
of SEA
induced
histological
in both
following
previously
protocol.
of 10 tg
(PBS)
tion
the
The
associated
of PBS.
used
thioglycollate
to be
populations
polycarbonate
have
response
of the airway
intraperitoneally
challenge
inflated
as
mice
immunized
at days
response
by
antigen-sensitized
100
were
blue To induce
syringe saline
RPMI-1640
dishes
Briefly,
livers
26J.
and induction
ice
1 mL spin
then
balanced
2-ME,
U and
air/5%
Sensitization
s in
a high-speed
peritoneally
Chemotactic
from
on
preparation
are
completely
on
30
was
eosinophils
mice
can
isolated
were
The
supernatant
f25,
S.
eggs
egg.
airway.
(SEA)
acutely
Briefly,
incubation
the
at 100,000
antigens
been
a 3-day
antigens
from
1251.
for
a 1.2-urn
elicited
egg
Chemotaxis
prepared
described
homogenized
supernatant
were
assays,
conditions.
egg antigen
of only
an
alveolitis.
WI) after
Hanks’
of S. man.soni
filters
Egg isolation
was
through
suspended
(Bar
pathogen-free
counted
of eosinophils
70-80% Laboratories
no
To count
enumeration to
nearly
was
mouse
per
eosinophils
the
adjacent
model
or
isolated
The
with
culture
Jackson
little
(Biospec
percentage from
immediately
for
individual
of lung.
The
assured
multiple
The
(HPF)
sections
followed. This
post-
lung
Animals purchased
with
Isolated supernatant
METHODS
mice
was
and .
fields
step
pee-
points
Wright-Giemsa
l000x
high-power
criteria
in this
with at
were
time
Lung homogenates
cytes)
CBAIJ
100 multiple
vehicle
paraffin
in
viewed
and
or
saline
at various
stained
using
within
eosinophils
or
embedded
to an airway.
airway
Adherent
Female
a strict
observed
the
were
from
point
eosinophils
SEA
differentially
counted
in juxtaposition
100
AND
lungs
were
time
eosinophils
with
with
paraformaldehyde
of eosinophils
at each
(FCS),
MATERIALS
fixed
sections
inflammation
after
of
in this
challenged
of 4%
identffication
those
inflammation in vitro eosin-
a specific anti-MCP-1,
step
were
in eo-
preincubation
mice with but not
The
50-tm
the
and 1 mL
challenge.
lung
ophil chemotactic activity in biological lung samples reflected the peribronchial eosinophil accumulation in vivo. Furthermore, the eosinophil chemotactic activity could
with
of peribronchial
plays
chemokines
during allergic data demonstrated
immunized
served
the
analysis
1996
MIP-la
immunization MIP-la Rabbits in incomplete
and of New
or MCP-l were
boosted Freund’s
Zealand (Genzyme) with adjuvant
in complete the
appropriate (IFA).
Polyclonal
prepared with
by recom-
Freund’s
adju-
recombinant antibodies
were
titered
by
specifically mIL-la,
1,
direct
munne
mMIP-13,
h-eotaxin,
necrosis
hIL-8,
antibodies
by
failure
mMCP-1,
end
of
by
were
(ELISA)
and
murine
mMIP-la,
hMIP-la,
was the
mIL-3,
IL-6,
hTNF,
using
mMCP-
m-eotaxin,
Dorherty
hMCP-l,
mIL-la,
hIL-8,
hRANTES,
Native
and
Rahway,
and
Poly-
mMIP-la, is
m-
not
.c 0. 0
available.
Cl) 0
a)
Statistical
significance
was
determined
by
analysis
of variance
P
