Feb 7, 1984 - Nettesheim. P. Respiratory carcinogenesis studies with the Syrian ... penitoneal macrophage population following intrapenito- neal injection.
Journal
of Leukocyte
Biology
37:349-358
(1985)
Enhancement of Hamster Alveolar Macrophage Phagocytic Activity by Lymphoki nes Raymond
H. Widen
and Lois J. Paradise
Department of Medical Microbiology College of Medicine, Tampa, Florida
and Immunology,
University
of South
Florida
Modulation of phagocytic activity of resident hamster pulmonary alveolar macrophages was accomplished by incubation of the cells in lymphokines prepared by stimulation of hamster splenocytes with concanavalin A or alloantigens in mixed lymphocyte cultures. Alveolar macrophages preincubated in either of these lymphokine preparations possessed significantly greater ability to ingest lgG or 1gM plus complement-coated sheep erythrocytes, via their Fc or complement receptors, respectively, than macrophages exposed to control preparations. Ingestion of yeast particles also was enhanced with macrophages incubated in supernatants from cultures of stimulated splenocytes. Supernatant fluids from either mitogenor alloantigen-stimulated splenocytes possessed migration inhibitory activity with characteristics similar to MIF from other animals; the phagocytosis-enhancing activity shared some of these characteristics. Key words:
alveolar
macrophages,
phagocytosis,
lymphokines
INTRODUCTION Alveolar macrophages are the predominant phagocytes in the airways of the healthy lung and their role in its defense is well established [6]. This defense is attributable in part to the ability of these cells to ingest and kill invading microorganisms [7]. Either microbial infection of the lung or exposure to microbial products induces alterations in Fc and complement receptor-mediated (immune) binding and phagocytosis by alveolar macrophages and leads to increased ingestion of unopsonized particles
(nonimmune
alterations Received Reprint University
phagocytosis)
observed 7, 1984;
February requests:
Raymond
of South
© 1985 Alan
are
Florida
R. Liss,
due
accepted H.
Widen,
College
Inc.
[10,14,27,29]. to
changes
September Department of Medicine,
However,
in 18.
the
activity
it is not of
known
if the
the
resident
alveolar
and
Immunology.
Box
1984. of Medical
12901
North
Microbiology
30th Street. Tampa.
FL 33612.
10,
350
macrophages or if they are present in the lavage fluids.
Widen
and
Paradise
associated Furthermore,
with
activities of the inflammatory whether the altered binding
cells also ingestion
and
are due to direct effects of the stimulating agent or to responses to factors released stimulated lymphocytes was not determined. Syrian hamsters are desirable hosts for studying respiratory infections immunity because of the low incidence of natural, chronic, respiratory disease, similarity respiratory
of the anatomy of and, hence, particle deposition tracts, and the similar disposition of lymphoid
accessory
tissues
species
study,
we
A) and
mixed
lymphocyte
(Con the
in both
In this immune
and
nonimmune
same
supernatant
AND
METHODS
MATERIALS
effects
in the hamster and human elements in the lung and
of
lymphokines
from
(MLR)-stimulated
phagocytic
activity
of the
the reaction
of the factors and compared
and the
[6,12,17].
measured
Characteristics were studied
by
activity
responsible with those
concanavalin
hamster
of hamster
for altered of a migration
splenic
alveolar
activities inhibitory
A cells
on
macrophages.
of the macrophages factor (MIF)-like
fluids.
Hamsters Inbred Charles
MHA
River
and
Ind.)
were
8-16
Alveolar
were old
.
from
Essential (MEM-A;
hamsters
for
were
Mass.
cells
or tissues
purchased
All hamsters
They were anesthetized sodium, Eli Lilly and
before
used
macrophages Trachea and the
lungs
Medium GIBCO, were
were
from received
by intraperitoneal Company, Indianacollected.
Hamsters
all studies.
at 400
of carbon “resident
suspensions in RPM! per
ml
and
Kan.). Concanavalin was added to one-half cells
were
Ten
minutes
instilling
2-ml
samples
of
containing 100 U penicillin Island, N.Y.) intratracheally g for
10 mm
and
ice
of the procedure hamsters. Cells cold
Eagle’s
particles, alveolar
Minimum
and 100 tg streptomycin/mi 5 times. The pooled lavage
the cells
and diffuse macrophages.”
for were
were
resuspended
staining
for
in MEMcells possessed adherence to
nonspecific
esterase,
of Lymphokines
Single-cell prepared
streptomycin
by
a modification en bloc from
90% of the cells were viable. Since about 95% of these of alveolar macrophages as measured by morphology,
glass, phagocytosis they were designated Preparation
were obtained by lungs were removed
(MEM) Grand
centrifuged
A. More than characteristics
were
libitum. (Brevital
exsanguinated when
male
Inc. , Wilmington,
Macrophages
washed
fluids
and
Alveolar [ 16]
rabbits
chow ad methohexital
weeks
3-5-week-old
Laboratories,
water and commercial injection of sodium polis,
CB strains,
Breeding
incubated before
(1 x 106 viable cells/ml) 1640 (GIBCO) containing 10%
fetal
bovine
A (Miles-Yeda, Rehovot, of the suspension; the
serum
of spleens from MHA hamsters 100 U penicillin and 100 tg (FBS;
Israel) remainder
at 37#{176}Cfor 24 hr in a humidified the
incubation
milliliter of control cell suspension. 1,000 g for 30 mm, the supernatant
period
was
terminated,
After the cells were fluids were stored
KC
Biological,
at a concentration served as the atmosphere 3 tg
Lenexa,
of 3 jzg/ml control. The
of 5% Con
A was
CO2
added
in air.
per
removed by centrifugation at at -70#{176}C. For some experi-
Modulation ments,
Con
A was
of Hamster
removed
(Pharmacia, Uppsala, remove greater than
from
hamsters (5 x 106 viable were mixed with an equal
cells pretreated with mitomycin Louis, Mo.) for 30 mm. Control syngeneic
spleen
cells.
by centrifugation With both
After
was (2.5
Boston,
[18].
Mass.)
fluids procedure sample
by adsorption
G-75 to we
any blastogenic inducing cells also were stimulated of spleens from MHA
RPMI 1640 containing of the same concentration
antibiotics and 10% of CB strain spleen
C (50 zg/ml medium; Sigma Chemical Company, St. cultures of MHA cells received mitomycin C-treated
incubation
monitored jzCi/ml;
to Sephadex
was shown by these authors of Con A from medium and
effectively eliminated A had been added. Spleen [4]. Single-cell suspensions cells/ml volume
351
of the MLR
for 48 hr, the cells
by blastogenic specific activity
were
at -70#{176}C. of lymphokines,
removed stimulation
assays measuring the incorporation of 2 Ci/mmole; New England Nuclear,
Inhibition
For phages
indirect
agarose
in Dulbecco’s
biose;
Polysciences,
added
to wells
Oxnard, 200
Macrophages
and the supernatant fluids were stored Con A and MLR methods of preparation
of splenic cells [3H]thymidine
Migration
supernatant
Sweden) [22]. This 99 % of a radiolabeled
found that use of the procedure activity of medium to which Con to release lymphokines in MLR strain FBS)
Alveolar
inhibition
Eagle’s
of a 96-well, Dilutions after
the
flat-bottomed of splenic
agarose
droplet were measured, incubation at 37#{176}Cfor
PA,
had
using 24 hr.
I 13],
assays
medium
Inc. , Warrington,
Calif.).
l/well,
migration
modified
(GIBCO) 2 tl
cell
supernatant
2
plate fluids
Two
considered The determined and then For control Danvers, 100, 50, 0.22-j.m
to be significant. ability of L-fucose by adding 0.1 adding to splenic molecular
weight
cultures Mass.)
inhibition
estimation,
daltons, respectively. stored at -70#{176}C until
of Erythrocyte-Antibody
(EA)
-
fluids
and
The used.
to the wells, of each after from
each test performed in migration in test wells! by more than 20% was
of macrophage
supernatant
were
II, Falcon,
immediately and was determined
migration
in medium to macrophages fluids and incubating as usual.
were passed through the ultrafiltration XM100, XM5O, and PM3O with
and 30 thousand membrane and
Preparation
to block
added
(Indu-
cells,
diameters
an ocular micrometer, both Percent inhibition of migration
M L- or D-fucose cell supernatant
l0
(Microtest
were
macro-
agarose
x
perpendicular
the average distance of migration from the droplets, with triplicate or quadruplicate: % inhibition of migration = (1 migration in control wells) x 100. Inhibition of migration
hamster
0.4%
containing
microtitration
hardened.
MHA
and
from
Con
[21] in the
was wells
A-stimulated
or
membranes (Amicon Corp., molecular weight separations at fractions
were
filtered
through
Erythrocyte-Antibody-Complement
(EAmC)
the
A 5% (vol!vol) IgG fraction of
Downington, were washed cytosis similarly.
Pa.) and
assays. After
suspension of sheep erythrocytes, washed 3 times, mixed with rabbit antisheep erythrocyte antiserum (Cappell Laboratories,
and incubated for 30 mm at 37#{176}C,provided suspended (0.5% by volume) in MEM-A
EAm the
were second
prepared
with
washing,
the
1gM
hemolysin
resuspended
EAm
EA. without
These FBS
(Difco, complexes
complexes for phago-
Detroit, were
Mich.) mixed
a
352
Widen
with were
and
Paradise
a nonhemolytic concentration of hamster incubated at 37#{176}Cfor 10 mm, yielding
washing, without
the FBS.
Assay
EAmC
for Fc and
With measured. cells!ml 35-mm washing
were
C3b
a slight A l00-tl
resuspended
serum as a source EAmC complexes.
to 0.5 % concentration
Receptor-Mediated
of complement and After additional
by volume
in MEM-A
Phagocytosis
modification of methods volume of a suspension
[3], phagocytosis of EAmC and EA was of alveolar macrophages ( 1 X 106 viable
MEM-A) was placed on each of four glass coverslips (12 mm diameter) plastic tissue culture dishes. After 1 hr, nonadherent cells were removed with warm (37#{176}C)Hanks balanced salt solution (HBSS) 3 times. Dilution
supernatant fluids from stimulated ing 5 % FBS were added to the
or control spleen cells dishes in 2-ml volumes.
cultures After
in MEM-A incubation
for
in by of
contain48 hr,
the cultures were washed with warm HBSS in preparation for Fc and complement receptor binding and phagocytosis assays. For Fc receptor assays, 0.5 % EA suspensions were added to the dishes and incubated for 1 hr at 37#{176}C.One coverslip from each plate was examined for rosetting, to ensure coverslips noningested
that EA did bind to the surfaces of the macrophages. The remaining three were dipped for 15 s into HBSS diluted with 4 parts of water to lyse EA. Cells were stained with Wright’s stain. Percentages of macrophages
rosetting
(three
or more
EA adhering
ing (one or more EA 200 to 300 macrophages
visible per
in an identical
except
Phagocytosis cells
fashion of Yeast
For
measurement
were
exposed
that
phosphate
buffered to 2 x
yeast After
l0
of a macrophage)
a macrophage) Complement
EAmC
complexes
were
in HBSS,
of phagocytosis to
supernatant
saline
(0.85%
cells!ml
RPMI
of yeasts, fluids
coverslip
from
per
[19]. The cells 1640 containing cerevisiae, NaCl;
pH
1640
containing
and
prepared
for Wright’s
or
of adherent
control
splenic
cell
were then washed 3 times in HBSS 10% FBS. An equal volume of a which had been boiled in 0. 15 M
7.0),
washed 20%
twice FBS,
staining.
was
(Difco, were The
were
in HBSS
and
added.
Killing
Detroit, removed,
percentages determined
susof
Mich.). washed of macroby counting
Analysis
Results are reported as the mean ± value. Percentage data were submitted
compared
counts of performed
preparations
stimulated
phages ingesting 1 or 2, 3 or 4, or 5 or more yeast cells yeast cells in at least 200 macrophages per coverslip. Statistical
phagocytiz-
from were
used.
was verified by plating on Sabouraud dextrose agar varying intervals of incubation at 37#{176}C,coverslips
vigorously
and
were calculated receptor assays
Cells
cultures for various time intervals and covered with 0.5m1 RPMI suspension of killed Saccharomyces pended
to the surface
within coverslip.
by Student’s
t-test
SE for at least three separate to the arcsin transformation
experiments analysis and
[28].
RESULTS Hamster
alveolar
Con A-stimulated spleen plexes than macrophages
macrophages
preincubated
cells possessed greater incubated in control
for 48 hr in supernatant
fluids
from
ability to phagocytize EAmC comsupernatant fluids (Table 1). Results
Modulation
of Hamster
Alveolar
Macrophages
353
TABLE 1. Phagocytosis of EAmC by Alveolar Macrophages Incubated Supernatants From Con A-Stimulated or Control Spleen Cell Cultures Supennatant Supernatant
source’
% Cells
dilution
None
32±2
-
Con
A
1/5
40
±
5
Control
+
Con
A
1/5
39
±
3
1/5
53
± 4
1/20
35 38
± 4 ± I
55
±
A stimulated
Control Control Con
-
Con
A
+
Con
A
1/20
A stimulated
aAlveolan
1/20
macnophages
supennatant
fluids
were
from
Con
incubated
for
A-stimulated
48
hr
on control
using
Student’s
supernatant medium
did
not
control
fluids alone
differ
t-test.
with
in their
effect
approximately
90%
Supernatant
or
of the
Con
fluids
macrophage
significant
A.
and
neither
EAmC
lymphokine
that
enhancement
cells
numbers versus
Enhanced supernatants of
EAmC from macrophages
(with.
or
+ , and
Con
+
difference was
(None)
A
supernatant
between
significantly
control
different
from
were
in control
fluids
macrophages
preincubated
with
bound
in medium
addition
EAmC
of Con
(data
not
A.
alone,
Con
A-stimulated
splenocyte
and
control
of EA
(EA)
shown),
preparations
phagocytosis
cultures
phagocytosis
indicating
by was
adsorption
not due
phagocytosis following incubation Con A-stimulated splenocytes was ingesting
41 ± 3 % in lymphokine-stimulated
two
or
more
cells,
2).
hamster
effect
(3 1 ± There
was
of G-75
of Con
for enhancement 80#{176}C(Table 2). (Table 2). of alveolar associated
or
Removal
to Sephadex
to a direct
complexes P < 0.05).
Binding
enhanced
(Table
in
In all cases,
component of complement (C3b) were present. or of untreated erythrocytes did not occur.
from
in
without,
in medium.
control
on the macrophages (Table 2). The lymphokine responsible phagocytosis was stable to heating 30 mm at 56#{176}Cbut not A and MLR fluids retained activity when diluted in medium with
alone
phagocytosis.
Fc receptor-mediated
A from
showed
on
