Pleurisy was induced in ether-anesthetized animals by the intrathoracic. (it.) injection of LPS. (250 ng/cavity) in a final volume of 100 il. Experiments included.
Requirement
for lymphocytes
LPS-induced
pleural
and
eosinophil
resident
macrophages
accumulation
Patricia T. Bozza, Hugo C. Castro-Faria-Neto, Carmen Penido, Maria das Gra#{231}as,M.O. Henriques, Patricia M.R. Silva, Marco Ricardo Ribeiro dos Santos and Renato S.B. Cordeiro Departamento
de Fisiologia
Cruz/FIOCRUZ,
Rio
e Farmacodindmica
de Janeiro,
and
*Departamento
. eosinophils
. icrophages
of
e Biologia
have demonstrated neutrophils and
lasting
significantly inhibited the eosinophilia triggered by LPS. The i.t. injection of liposomes containing dichboromethylene diphosphonate significantly reduced (65%) the number of resident macrophages after 5 days. Under this condition, the eosinophil infiltration induced by LPS was completely inhibited. Accordingly, the i.t. injection of supernatant from macrophage monolayers, obtained from the pleural cavities of LPS-injected rats, into naive recipient animals led to a twofold increase in the number of pleural eosinophils. In conclusion, our data suggest an important role for resident macrophages and T bymphocytes in the eosinophil accumulation induced by LPS. J. Lcukoc. Biol. 56: 151-158; 1994. (LPS)
de Bioquimica
We tion
trathoracic (i.t.) injection of LPS (250 ng/cavity) into rats induced a significant eosinophil accumulation that developed within 24 h, was maximal at 48 h, and returned to control values within 120 h. This eosinophil influx was preceded by a huge neutrophil influx within 4 h and accompanied by a mononuclear cell accumulation between 24 and 48 h. Pretreatment with an antineutrophil monocbonal antibody (RP-3, 2 ml per animal) selectively reduced the number of circulating neutrophils within 8 h but failed to alter the LPS-induced eosinophilia. Similarly, platelet depletion with an anti-rat platelet antiserum did not alter the LPS-induced eosinophil accumulation. Cycbosporine (.50 mg/kg, 12 and 2 h before) partially inhibited (51%) the LPS-induced pleural eosinophilia, whereas the eosinophilia was not changed by prior degranulation of pleural mast cells with polymyxin B (10 tg/cavity, 24 h before). Moreover, selective depletion ofT lymphocytes using an anti-Thy 1.0 monocbonal antibody
endotoxin
Andrea P. Larangeira, A. Martins, Molecular,
. T
rat
and
dose-dependent
pleural
in
cavity.
vitro,
that LPS, mononuclear
indicating
INTRODUCTION
LPS
that
the
characterized [ 2-4], later
by
an
infiltration
induced
by
within
acute
neutrophil
influx
followed by mononuclear phase of the reaction
cell
accumulation
[5].
LPS
during
is
2-3
h the
to the migrainduces a long’ in
to attract
the
eosinophils
observed
in
generated endogenously accumulation was
vivo
is
[6]. Inprevented
by glucocorticosteroids and a protein synthesis inhibitor but not by platelet-activating factor (PAF) antagonists or inhibi’ tors of arachidonic acid metabolites [7]. Moreover, it was demonstrated that the cell-free pleural washings from LPSinjected rats contained a heat-stable protein that induces selective eosinophil accumulation in naive recipient animals [7]. The aim of this study was to investigate the cell source of LPS-induced eosinophil chemotactic factor. By means of selective cellular depletion, we provide evidence for the involvement
of
sinophil
T
lymphocytes
accumulation
MATERIALS
and
induced
macrophages
by
in
the
eo-
LPS.
AND METHODS
Animals Male ing
Wistar 18-25
rats
weighing
150-180
Cruz
Foundation
g (Oswaldo
g and
Swiss
breeding
mice unit)
weigh-
were
used.
Pleurisy Pleurisy was induced in ether-anesthetized intrathoracic (it.) injection of LPS (250 volume of 100 il. Experiments included of control line. The
animals animals
animals ng/cavity) equivalent
receiving the same were killed with an
volume overdose
by the in a final numbers
of sterile of ether
saat
different times (1-24 h) after the it. injection of LPS or saline, and the thoracic cavity was washed with 3 ml of heparinized saline (10 Ui/mb) and the fluid volume evaluated with a graduated syringe. Total leukocyte counts were per-
on
cytocentrifuged
Gr#{252}nwald-Giemsa
Leukocyte
Oswaldo
accumulation
failed
eosinophilia
accounted for by a factor(s) deed, LPS-induced eosinophil
performed
Lipopolysaccharides (LPS) of the cell wall of gram-negative bacteria elicit a wide range of effects on biological systems and mimic several of the pathophysiobogical events observed during infections with these organisms (for a review see ref. 1). One of the hallmarks of the inflammatory response induced by LPS is its attraction of leukocytes to the focus of
in addition cells,
eosinophil
However,
formed in a Coulter counter after ing with isotonic liquid. Differential
inflammation.
Instituto
Brazil
Abstract: In this study we investigated the involvement of inflammatory cells in the pleural accumulation of eosinophils induced by lipopolysaccharide (LPS). In-
Key Words: lymphocytes
in
Abbreviations: lene
APAS,
PAF, blood
red
Reprint
anti-rat
mAb, TNF-a,
requests:
tumor
PT
Fundac#{227}o Oswaldo
Bozza,
Cruz,
Cl2MD1
antibody;
necrosis
NRS,
factor
dichloromethyLPS,
normal
rabbit saline;
Journal
December
of Leukocyte
2,
1993;
Biology
accepted
seRBC,
a.
de Fisiologia e Farmacodin#{226}mica, Rio de Janeiro, Rj 21045 900,
Brazil.
Received
May-
performed
interleukin’8;
phosphate-buffered
Departamento Av. Brasil 4365,
washwas
the
were
IL-8,
PBS,
by
counts
antiserum;
interferon’y; factor;
pleural analysis
stained
cell
platelet
monoclonal
platelet-activating cell;
Mast
IFN-’y,
lipopolysaccharide; rum;
smears
method.
diphosphonate;
diluting the leukocyte
March
Volume
21,
56,
1994.
August
1994
in a Neubauer
chamber
ing
fluid
the
Allergic
pleural
under
light
in toluidine
blue
microscopy
after
Pleural
dilut-
dye.
The animals were sensitized subcutaneously with a mixture ovalbumin (50 tg) and aluminum hydroxide (5 mg) in a final volume of 200 gil. Fourteen days later ovalbumin (12 pg/cavity) was administered it. and after 24 h the were killed Nonsensitized it. were used
Blood
cell counts
and
pleurisy animals as negative
transfer
was evaluated as in which ovabbumin controls.
.
.
.
.
Six hours after the i.t. injection of LPS line into donor rats, the pleural cavity jl of sterile saline, and the pleural (400g) for 10 mm at 4#{176}C.The pleural
pleurisy
animals above. jected
washing
.
15
>-
described was in-
(250 ng/cavity) or sawas washed with 500 fluid was centrifuged wash free of cells was
I-. *
C.)
12
A
*
‘0 9.
Blood samples were obtained from the tail vein 3 or 8 h after administration of antiplatelet antiserum or antineutrophil monocbonal antibody (mAb), respectively. The samples were diluted in Turk fluid (2% acetic acid) for total leukocyte counts or in buffered EDTA-formalin solution for platelet and red blood cell (RBC) counts. Buffered ELffA-formalin solution was prepared by adding 3 ml of 0.077 M EDTA, ml of 10 x concentrated phosphate-buffered saline (PBS), 150 l of 40% formalin, and distilled water to a final volume of 20 ml. The counts were performed in Neubauer chambers under light microscopy. Differential analysis was performed in blood smears stained by the May-Gr#{252}nwald-Giemsa method.
Co
< Ui 1
0 2
z 0 z
.
0
0’
20
*
>-
I-
Treatments Specific neutrophil depletion was achieved 8 h after the intraperitoneal injection of anti-rat neutrophil mAb (RP-3, 2 ml per animal) [8]. The ascites of a nonhybridized parent cell line, P3-X63.Ag8.653, was used as a control. Platelets
C.)
15
B
#{149}0
*
were depleted by an intravenous (iv.) injection of a rabbit anti-rat platelet antiserum (APAS) 3 h before the LPS injection. Control animals received an i.v. injection of a normal rabbit serum (NRS). T lymphocyte depletion was achieved
*
by the treatment ofmice with a rat anti-murine Thy 1.0 mAb (G7, American Type Culture Collection, Rockvible, MD). The supernatant of the G7 hybridoma was semipurified and 20 x concentrated by ultrafiltration (Amicon, Beverly, MA) with a 100-kDa cutoff membrane. The retentate, after being further washed with five volumes of PBS to help deplete substances smaller than 100 kDa, was injected intraperitoneally (100 l) daily during 6 days. The mice were used 24 h after the last injection ofthe antibody. The success ofT cell depletion was ascertained by immunoperoxidase staining of spleen cells, and the depletion was greater than 90% after the end of the treatment. Polymyxin B (10 tg/cavity) was used as a mast cell degranulating agent [9], and was injected intrathoracically 24 h before LPS. Cycbosporine (25-50 mg/kg) injection.
was given orally Cycbosporine
12 and 2 h before the LPS or antigen was solubilized with corn oil, and the
vehicle was administered biposomes were prepared 75 mg of phosphatidylcholine dissolved in chloroform.
to control groups. Encapsulated as described earlier [10]. In brief, and 11 mg of cholesterol were The thin film that formed after ro-
4
3
2
tary evaporation was dispersed by 10 ml of PBS. For dichboromethylene diphosphonate (C12MDP)-containing liposomes, 1.9 g of CI2MDP was dissolved in the 10 ml of PBS in which the lipid film had been dispersed. After being washed, the liposomes were resuspended in 4 ml of PBS, and 0.1 ml (containing about 1 mg of liposome-entrapped C12MDP) was injected i.t. in the rat. Macrophage ebimination in situ was obtained 5 days after an it. injection of C12MDP-containing liposomes. Control animals received PBS-containing liposomes.
152
Journal
of Leukocyte
Biology
Volume
56,
August
1994
0 0
24
12
48
72
TIME Fig. 1. Time (C) accumulation
( 250 for
course in
at
least
eight by
the
(#{149}) or
ng/cavity)
indicated
ofmononuclear
an
animals. asterisk.
cell
pleural saline
cavity (0).
Statistically
Results
(A),
96
(H)
neutrophil
induced are significant
120
by
(B), the
it.
expressed differences
and
eosinophil
injection as
of
(P
LPS
± SEM
means
.
>
diphosphonate (Germany).
C) ‘0 SC
Statistical
analysis
Data are statistically bowed by significance
C,)
reported as means by means of analysis the Newman-Keuls-Student set at P < .05.
± SEM and were analyzed of variance (ANOVA) foltest with the level of
RESULTS
x 0. 0
z (I,
0 Lii
stimulus
SAt.
LPS
LPS
LPS
SAL
LPS
triatmmtl
-
-
XP3
RP-3
-
-
Fig.
2. Effect
eosinophil
Time course The
it.
neutrophil numbers mononuclear
of cell accumulation
injection
of LPS
accumulation of neutrophils cell
(Fig.
(250
induced
ng/cavity)
induced
within declined
4 h (Fig. 1B). and significant
1A)
eosinophib
and
The
by LPS
(Fig.
At
24
increases 1C)
antineutrophil
(XP3)
a marked h,
the in
counts
of the
from
mAb
the
traperitoneally platelet
(P
‘
by
mast cell or enhance
degranulation the eosinophil
with polymyxin accumulation
B in-
LPS.
+
3
0
Effect of cyclosporine treatment and lymphocyte depletion on the LPS-induced eosinophil accumulation
“5.
0 >
I>
ofcyclosporine
± SEM
(P
(B), br
Cells
+ Cells
‘
-
Fig.
it.
and
[10]. resident
through
accumulation
demonstrated LPS-injected
mice
treatment without
liposomes. the
sinophil
in
this
monocytes of pleural
cells
indicate the LPSway to iden-
in
work is under for this effect. intravenous injection induced pronounced
that
eosinophil
LPS-induced
results
role
macrophages,
of circulating the involvement
depletion
our
important
depletion
to point
against
number verified
depleted in vivo of 1’ lymphocytes by means of an anti-Thy 1.0 mAb. Eosinophil accumulation induced by LPS in mice shows the same pattern of response as that observed in rats and also seems to depend on a newly generated heat-stable protein [11]. In agreement with the data obtained with cycbosporine in rats, T lymphocyte depletion with anti-Thy 1.0 mAb in mice was very potent in inhibiting eosinophil migra-
=
an
macrophage
C12MDP-containing
< 0
as a whole,
play
is interesting
only
least
differences
liposome-treated
by
lymphocytes
long-basting
given
liposomes
SEM
groups.
and
those
significant
saline-challenged
(250 fluid
respectively. for
C12MDP
tsseans
statistically
LPS pleural
T
induced eosinophilia, and further tify the T cell subset responsible It has been reported that C12MDP-containing liposomes
eosinophilia
the
h
eosinophilia expressed
plus
C12M an
24 columns
(striped
Results
the before
LPS-challenged fluid
liposomes
column).
solid
and
pleural
on
5 days
it.
performed
and
saline-
PBS-containing
eight
given
were open
rats,
( crosshatched
CI2MDP
were
The
LPS-challenged
control
containing
Liposomes
ng/cavity)
For
induced
that
induced
(P
iCY’
x
SAL-m#{216}
of
with
asterisk,
LPS-challcngecl
l)leral
wash
fluids.
Biology
Volume
56,
August
several
main
liposonse-treated
stimulation
1994
target
other cell
[1, 30,
reports for
31].
showing cytokine
that production
macrophages following
are
the LPS
Both
lymphocyte
and
macrophage
depletion
IL-5 accounts for the mouse triggered by antigen but not
completely
abolished the LPS-induced eosinophil migration, suggesting that cooperation between these cells might take a place in this reaction. Indeed, T cell bymphokines can regulate macrophage activation and lead to the production and secretion of cytokines. For example, a T cell supernatant and IL-2 are
12.
able to necrosis
13.
induce factor
macrophage (TNF)
production of [32-34]. Moreover,
IL-i
or IL-2
tumor and
interferon-y (IFN-’y) enhance the production of TNF-a, TNF-f3, IL-i, and IFN-’y by monocytes and macrophages in the presence of LPS or cytokines [35-37]. Nevertheless, the precise mechanisms of cooperation between lymphocytes and macrophages for the production of LPS-induced eosinophibotactic factor are still not clear and studies are under way to further clarify this point. In conclusion, our results indicate that cumulation induced by LPS is independent neutrophils, and platelets. Furthermore, resident macrophages seem to contribute of the eosinophil chemoattractant protein
eosinophil acof mast cells, lymphocytes and to the production induced by LPS.
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Silberstein,
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