233 lnterleukin-1-dependent mitogenic responses induced by protoscoleces of Echinococcus multilocularis in murine lymphocytes. Takako. Kizaki,*. Masamichi.
lnterleukin-1-dependent
mitogenic
of Echinococcus
protoscoleces
responses
induced
multilocularis
by
in murine
lymphocytes Takako
Kizaki,*
Robert
A. Good,
*Hokkaido kaido,
Abstract:
Masamichi Ishige,* Wang and Kazunori Ono#{233}t
Institute
Japan,
of Public
and
Mitogeme
*All
and
Health
Children’s
Bingyan,t
rinstitute
Hospital,
of Immunological
University
of protoscoleces (PSCs) of on murine lymphocytes were studied. Spleen cells from normal BALB/c mice showed significant proliferative responses when eocultured with PSCs. Proliferative responses were observed in both the T and B cell populations. The PSCs also stimulated cells of the macrophage/monocyte lineage to secrete interleukin-1 (IL-i). Depletion of plasticand Sephadex G-l0adherent cells from the spleen cell population significantly reduced the proliferative responses to PSCs and the low responsiveness was restored by addition of plastic-adherent cells to these cultures. Furthermore, addition of anti-IL.! serum to the spleen cell cultures stimulated with PSCs completely suppressed the proliferative responses. These findings demonstrate that the mitogenic effect of PSC on lymphocytes depends on IL-i secreted by cells of macrophage/monocyte lineage. J. Leukoc. Biol. 53: 233239; 1993. Echinococcus
Mitsuru
effects
multilocularis
Science,
of South
injection coelom
Florida,
of of
fection),
Key
Words:
#{149} interleukin-1
protoscoleces
multilocularz.s
of E.
#{149}mitogenic
ef-
INTRODUCTION Echinococcus tion of its life adult, strobilar an intermediate
requires cycle:
two mammalian a definitive (final)
stage develops in host in which
usually develops in Alveolar hydatid
multilocularis, vascularized mass
which in the
number
of rodent
species
infiltrating
structures
consisting
of
dense cyst.
As
Although, whelmingly germinal trusions or
blood
characteristic
natural
the cells give
rise
to
the
of E. multilocularis
tumor-
humans
and barrier,
egg
metastatic
Hok-
PSCs species
hydatidosis
into (i.e., can
the perivisceral experimental
be
established
in[5].
In
our
previous
study
of E. multilocularis murine lymphocytes cells from BALB/c depressed
proliferation
tion in pearance variety
A unusual
parasitoses
but
we
and
concanavalin of these of
tuitous
[12],
demonstrated
that
induced proliferative in vitro. On the other mice infected with
PSCs
responses in hand, spleen PSCs showed
interleukin-2
(IL-2)
produc-
(Con A) responses. The apimmunologic states with a
suggests
that
it
rather may have evolved that both host or parasite
may
not
as need
an to
be
for-
essential survive
a
far
the direct contribution of the mitogenic influences of parasites to immune regulation has not been well analyzed. To investigate the mechanism underlying this seemingly complex phenomenon, the present study focused on an analysis of cellular components involved in the proliferative responses induced by PSCs of E. multilocularis in vitro. Elucidation of the cellular interactions that occur may confrontation
between
better
them.
understanding
However,
of
the
thus
immunoregulatory
processes that are seen in the host-parasite relationship. We show herein that PSCs stimulate T and B lymphocytes and that both of these cellular responses are entirely dependent on IL-i secreted by cells of macrophage/monocyte lineage that are stimulated with PSCs.
be
single sus-
over-
detachment cellular
[3, 4]. Furthermore,
the
Female BALB/c from Japan SLC
disis
foci
Animals mice and (Hamamatsu,
C3H/HeJ Japan).
mice
were
obtained
or
hydatid
via
AND METHODS
a
may
ingestion
route, the infiltrating
Sa/4.ioro,
In both case, the survival of a proportion of infected parasites (PSCs) implies the existence of a mechanism(s) that enables some E. multilocularis PSCs to avoid the host cellular attack. Indeed, several immunologic abnormalities have been identified in mice infected with E. multilocularis [6-8] or Echinococcus granulosus [9-11].
MATERIALS
in
debilitated
alveolar
and
University,
multivesicular,
there
are
a
K. Day,l
Hokkaido
multilocularis rodent
E.
Noorbibi
Petersburg
a secondary
permit
of
as a solid
within a and partially
distribution distant
and
embedded and
humans,
subsequent
the
intestine metacestode
host-tissue
(PSCs) hosts
most common or PSCs from
their
of are
tissues,
PSCs
comple-
in which
metacestode
vesicles
infections,
via
occurs
and
limiting
hypersusceptible including in
rarely
no
protoscoleces
hosts,
by
Cysts
small
connective
a result,
ceptible killed. ease
of
thousand
small cystic
develops organs 2].
with
numerous
stroma
several
the the
[1,
for
host
the viscera. disease is caused
Echinococcus
like
hosts
St.
certain
modification fect
Kumagai,*
of prolymph that
are
Abbreviations:
C,
complement;
Con
A,
concanavalin
A;
ELISA,
en-
zyme-linked immunosorbent charide; mAb, monoclonal PSCs, protoscoleces; TdR, Reprint requests (and
Hygiene, National 359, Japan. Received
after
Journal
of
September
Leukocyte
assay; IL-l, interleukin-1; LPS, lipopolysacantibody; PBS, phosphate-buffered saline; thymidine. present address): T. Kizaki, Department of Defense Medical College, 3-2 Namiki, Tokorozawa,
28, 1992;
Biology
accepted
Volume
October
25, 1992.
53, March
1993
233
Parasites The E. multilocularis was maintained by serial intraperitoneal inoculation of E. multilocularis PSCs into cotton rats (Sigmodon hispidus). Hydatid cysts were minced under aseptic conditions, and PSCs were separated from connective tissues by extensive decantation in phosphatebuffered saline (PBS) and stored in PBS supplemented with 100 U/mI penicillin and 100 p.g/ml streptomycin (Gibco Laboratories, Grand Island, NY). Viabilities of PSCs
were
clusion
assessed
test
cell
the
blue
trypan
and lymphocyte
suspensions described
previously
duced
using
dye
ex-
spleen
cell
were For
[12].
proliferation
assay
prepared by determination
proliferation,
(4 or
8)
the
x 10
method of PSC-in-
cells
in
tis-
sue culture medium consisting of RPM! 1640 (Gibco) supplemented with 10% heat-inactivated FCS, 100 U/mI penicillin, 100 p.g/ml streptomycin, and 2 mM Lglutamine (Gibco) were cultured in flat-bottomed 96-well microtiter plates (200 j.tl/well) (Costar, Cambridge, MA) at 37#{176}Cin a 5% CO2-humidified atmosphere for 2 to 4 days with periments, 110;
Genzyme,
added rabbits sence in
appropriate serially
PSC
concentrations. rabbit anti-IL-la
diluted
Boston,
MA)
or
normal
In
some serum
ex(IP-
serum
was
rabbit
to cultures. IP-1 10 was generated by immunizing with purified recombinant mouse IL-la in the abof other exogenous cytokines. Cells were cultured quadruplicate
thymidine
wells,
MA) was added for (counts per minute, scintillation
the
counter
expressed
as
Peritoneal
to
final cpm)
8 h of
5 days
was
j.tCi/well
tritiated
Nuclear, culture.
Boston, Radioactivity in a liquid
determined
Tokyo,
Japan).
incorporation
of IL-i
Results
are
later,
cells
by
105/ml, PSCs
1 mI/well in
the (LPS,
Detroit,
MI)
(Wako BALB/c
harvested
or
coli
and
Pure mice.
purified
activity
was
al. [14] Thymocytes
fractionated glutinin,
on
the basis nonagglutinated
Difco) p.Ci/well) were a liquid
234
cells adjusted (200 (16 then
after to 2.5 p.1/well)
ng/ml) was added harvested
scintillation
Journal
055:B5,
Mizel from
of
to
the
for
method
final
radioactivity
slight were
by peanut were used with
Cells
in 96-well (PHA,
h. of
was
[3H]TdR culture. counted
(0.5 Cells using
Biology
them
to
followed
were
Nonadherent
adhere
by vigorous
prepared
cells
Handwerger Briefly,
on a lected
wool column. incubation
nylon after
preparation less than
cells
according
were
37#{176}C.Thereafter, incubated with cells.
T-enriched
were
cells
collected
the PSCs
to
prepared
after
1
nonadherent in the presence
according
to
the
and Schwartz [17] with slight spleen cells (2 x 108) were applied Nonadherent 1 h at
for
cells 37#{176}C.The
contained more than B220 cells as determined
2%
tibody staining. B-enriched
were
prepared
80%
by
were colresultant
CD3 cells by fluorescent
treating
and an-
spleen
cells
with a 1:500 dilution of anti-Thy 1.2 monoclonal antibody (mAb) and a 1:10 dilution of guinea pig complement (C, Cedarlane Laboratories, Ontario, Canada). The resultant preparation contained less than 5% CD3 cells and more than 80% B220 cells as determined by fluoresantibody some
staining. experiments,
cells
T or
depleted
cells
of
prepared
B lymphocytes
plastic-
and
as described
were
isolated
Sephadex
G-10-ad-
earlier.
Proliferative responses in murine by E. multilocularis PSCs The
mitogenic
cells
was
for
Volume
53,
March
1993
effect
analyzed
in
BALB/c
mice
of
observed 2 or
of E. an
were
PSCs. in
3 days
system.
cocultured
Significant
spleen
cells 1 and
(Figs.
amounts
Viable
a peak at 100 PSCs/culture. (200 PSCs/well) the paralleling a reduction have
PSCs
on
Spleen with
stimulated 2).
negligible
may
induced
of
[‘H]TdR.
resulted
spleen
cells
from
various
proliferative
reached centration decreased,
cytotoxicity
lymphocytes
multilocularis
in vitro
porated
con-
responses with
PSCs
living alone
The
PSCs incor-
response
At a higher conproliferative response in cell viability (Fig. 1). from
a direct
effect
by
PSCs themselves or may be attributable to their products, as reported for E. granulosus cyst fluid [18]. The magnitude of the response and time course (Fig. 2) suggested that the exposure to the parasite component activated lymphocytes polyclonally. By contrast, killed PSCs evoked only
a weak
responses
1). Furthermore, medium in which several
counter.
of Leukocyte
Sweden).
method of modifications.
The
agas
PBS.
incubated 72 8 h
allowing
plates,
cells
h of incubation at responder cells were or absence of adherent
were -20#{176}C
with mice
phytohemagglutinin
samples for the
and
psala,
centrations
h. Cul-
at
[15]
times
and
48
stored
et al. C3H/HeJ
three
106/ml with
and
for
agglutinability thymocytes
washing x
Nonadherent
normal
Laboratories,
air and
with
lipopolysac-
Difco
according
and
EDTA at cells (2 x
stimulated
5 p.g/ml
C02/95%
determined
et
and
of
by
of 96-well
RESULTS in
dishes were
0.2% These
were
were filtrated of IL-I activity.
of Lachman modifications.
were plates
absence
Escherichia
measurement
responder
plates)
at 37#{176}C in 5%
supernatants
IL-i
24-well
presence
charide ture before
in
washing.
isolated
wells
intraperitoneal
liquid paraffin Japan) in
PBS containing three times.
were
to the
herent induced
were
by exposure to I h and washed
cells
directly
In
tissue culture medium by adherence to polystyrene for 24 h. Adherent cells (macrophages/monocytes) collected 37#{176}Cfor
Adherent
from
were
1 ml of Osaka,
Cell fractionation
cent
by cells.
release
cells
injection of Industries,
(i.p.)
England
(Aloka,
exudate
Chemical
0.5
New
[3H]TdR
Measurement
Four
and
([3H]TdR,
zyme).
the method of Ly and Mishell [16]. Briefly, macrophages were depleted from spleen cell suspensions by incubation on plastic petri dishes (2 h at 37#{176}C in complete medium), followed by application onto a Sephadex C-b culumn (10 x 100 mm, Pharmacia LKB Biotechnology AB, Up-
[13].
Cell preparation Spleen
by
In some experiments, IL-la activities in the supernatants from spleen cell cultures or cultures of macrophage/monocyte lineage from peritoneal exudate cells were determined using an Intertest-laX ELISA kit (Gen-
days
before
at
neither PSCs
the
highest
concentration
(Fig.
homogenized PSCs nor culture had been maintained at 4#{176}C for assay was effective as a stimulus for
When IL-la was directly evaluated using anti-IL-la antibodies, significant amounts were detected in supernatants from both spleen cell cultures and cultures of macrophage/monocyte lineage stimulated with PSCs for 2 or 3 days (Table 2).
3
Influence of adherent cells on PSC-induced proliferative responses in nonadherent spleen
To examine whether the IL-i release from cells of macrophage/monocyte lineage stimulated with PSCs was requisite for proliferative responses of spleen cells to PSCs, we then analyzed the proliferative responses to PSCs in spleen cells depleted of adherent cells. Figure 3 shows that the nonadherent cell fraction mounted markedly
2 C 0
lower
responses
siveness, of adherent
0
a I-.
0 0
stantial
1
C
than
however, cells.
unfractionated
cells.
The
was completely Adherent cells
recovered alone did
by
proliferation
in
the
presence
We
then
1 a on the spleen serially
0
rabbit
0
25
100
50 PSC/
200
examined spleen
cells
the influence cell responses
were
stimulated
diluted
rabbit
serum.
As
anti-IL-ia proliferative
can
serum but responses
on PSC-induced cells of antibodies against to PSCs. Unfractionated
with
PSCs
anti-IL-ia be
seen
not
responaddition show sub-
of PSCs.
Influence of anti-IL-i a antiserum proliferative responses in spleen
I-
Fig.
cells
in
the
antiserum in
Figure
not normal of spleen cells
presence or
4,
serum to PSCs
ILof
normal
addition
of
suppressed in a dose-
well
1. Dose-response
curve of [3H]TdR incorporation in spleen cells BALB/c mice stimulated with E. multiloculari.s PSCs. PSCs alone (0) or spleen cells (2 x 106/ml, 200 Ill/well) that various concentrations of living PSCs (#{149}) or PSCs killed by
from
normal
Living received freezing
at -70#{176}C(A)
were
cultured
poration in the final 8 h was determined are expressed as mean incorporation sentative
of three
separate
at 37#{176}C for 72 h. [ H]TdR for triplicate cultures. ± SD.
This
set
of data
2
incorResults
0
is a repre-
experiments.
E proliferation
(data
demonstrate
PSCs
that
are
required
not
shown).
appropriate
These
concentrations
to stimulate
of
lymphocyte
a 0
results living
proliferation.
C
0
IL-i release from cells of macrophage/monocyte lineage or spleen cells stimulated with E. multiocularis PSCs
I-
0
a 0
Macrophages from mice been reported to regulate immune responses [19-21]. PSCs
on
macrophage
functions,
macrophage/monocyte quantified.
PSCs for concentrations mined by stimulated separate sentative
exudate
significant
from peritoneal Furthermore, the
manner.
cells
from
with
were
cells
PSCs
stimulated
0
I-
of
was with
I LJ
h in the presence or absence of LPS. IL-i in the culture supernatants were deterevaluating the [3H]TdR incorporation by PHAthymocytes from C3H/HeJ mice. Two experiments were carried out and a repreresult is shown in Table 1. As shown in this
natants alone. ent
release
cultured
C
48
table,
enhanced
IL-i
lineage
Peritoneal
0
infected with parasites have mitogenor antigen-induced To examine the effects of
IL-i
IL-i
release
activities
were
cell cultures PSCs did not induced
by
LPS
detected
in
containing suppress but in a dose-depend-
super-
PSCs rather
0 24
72
48
96
hours Fig. 2. Time course study of PSC resjonses in spleen cells from normal BALB/c mice. Spleen cells (2 x 10 /ml, 200 I.tl/well) were stimulated with () or without (0) PSCs (100 PSCs/well) at 37#{176}C for 24 to 96 h. [‘H]TdR incorporation in the final 8 h was determined for quadruplicate cultures. Results are expressed as mean incorporation (cpm) ± SD. This set of data is a representative of three separate experiments.
Kizaki
et al.
Mitogenic
effects
of E.
Multilocularis
235
TABLE
1.
of E. muttilocularis
Effect
PSCs
on
IL-l
Release
from
in either depleted
Macrophages/Monocyte? lL-l
PSCs/ml 0
production
stimulated
PSCs
alone
PSCs
2,691
± 435
56,282
2000
aMacrophes/monocytes cells
were
LPS
+
± 4,961
prepared
from
by i.p. injection of BALE/c mice with I ml of as described in Materials and Methods. These cells were stimulated with PSCs in the presence or absence
paraffin 105/ml)
either
cell-enriched and Sephadex
passed
extensively of cells siveness
exudate
pentoneal
induced
or B plastic-
over
fraction. Spleen G-l0-adherent
a nylon
wool
column
cells cells
or
treated
with anti-Thy 1.2 + C and stimulated with PSCs in the presence or absence of plastic-adherent cells. As summarized in Table 4, proliferative responses to PSCs were not observed in the T-enriched population that had been
82,041 ± 7,163 105,687 ± 5,447 139,103±8,162
N.D.b 6,755 ± 2,508 11,037±2,458
200 1000
were
with
T of
liquid (2 x of 5
tig/mi LPS for 48 h in 24-well plate. IL-i activity in the supernatant was determined as [5H]TdR incorporation by thymocytes from C3H/HeJ mice. All values of [5H]TdR incorporation are expressed as mean cpm ± SD. This set of data is typically representative of two separate
depleted
of adherent
adherent to PSCs.
depleted
of
responses
to plastic Similarly,
adherent
than
proliferation
cells.
partially a
cells
was
still
considerably
cells,
observed
was almost cells.
addition the responpopulation
B-enriched
mounted
unfractionated
responsiveness tion of adherent
However,
restored
in
lower
although this
significant
population.
completely
restored
The
by
addi-
experiments. bNot
determined.
dependent
manner.
Thus,
data
presented
in
Figures
3
and 4 and Table I may cell responses to PSCs from adherent cells.
permit us to conclude that spleen are dependent on IL-la released
Proliferative responses subpopulations
to PSCs in lymphocyte
To analyze prepared
the nylon
cell
cells)
Thy
1.2-
and
1#{176})
b Sc
population wool-nonadherent cells
responding
(B-enriched
cells
to PSCs, (T-enriched
cells)
from
we
E a. 0
spleen
cells. These cell fractions were stimulated with optimal doses of Con A, LPS, or PSCs. Three separate experiments were performed and representative results are summarized in Table 3. As shown in this table, PSCs in-
C 0
duced
0
lymphocyte
transformation
unfractionated
riched marked
10
1
spleen
in
cells,
cells, although as that to other
cultures
T-enriched
containing cells,
or
the responsiveness was mitogens (Con A and LPS).
B-ennot as
a.
0 0 C
Influence of adherent cells on the PSC-induced proliferative responses in T- or B-enriched cells We
then
examined
phage/monocyte
the
lineage
TABLE 2. Cultures
influence on
the
of
cells
IL-la
macro-
responses
of Spleen
Concentrations in Supernatants or Cultures of Macrophage/Monocyte Stimulated with pSCSa IL-la
of
proliferative
Cell
Lineage
concen
tration
0
(pg/mi)
adherent
-
cell
I
Culture
period
Spleen
PSC
cells
monocytesb 5.55 ± 1.14 17.14±3.08 3.23 ± 0.29 70.44 ± 2.97
-
+ 72
-
+
aspleen were
cells
cultured
(2 x 106/ml) for
48
or
or macrophages/monocytes 72
h
in
the
presence
responder
1.47 ± 0.33 181.31±5.85 0.37 ± 0.07 306.77 ± 17.33
(2 x 105/ml) or
absence
of
PSCs
(500/mi) in a 24-well plate. Culture supernatants were filtered and IL-la concentrations were determined using an Intertest-laX ELISA kit. Results are expressed as mean concentration ± SD. bMacrophages/monocytes were prepared from peritoneal exudate cells induced Materials and
236
by i.p. injection Methods.
Journal
of
Leukocyte
with
1 ml of liquid
Biology
paraffin
Volume
as described
53, March
in
1993
cell
Fig. 3. Effect of Adherent cells tion on plastic onto a Sephadex 1 h of incubation (whole) or the were stimulated
adherent
I
I
non-adherent on PSC-induced
proliferative
responses.
were depleted from spleen cell suspensions by incubaPen-i dishes at 37#{176}Cfor 2 h, followed by application C-b column. Nonadherent cells were collected after at 37#{176}C.Thereafter the untreated spleen cells nonadherent responder cells (2 x l06/ml, 200 Ill/well) with PSCs in the presence or absence of adherent cells
at 37#{176}C for 72 h. Adherent here directly to the wells ing. [5H]TdR incorporation quadruplicate (cpm) ± SD.
whole cells
+
______
I
Macrophages/
(h) 48
-
+
cultures.
cells were isolated by allowing them of 96-well plates, followed by vigorous in the final 8 h was determined Results
are
expressed
as
mean
incorporation
to adwash-
for
to be Thus, peared PSCs.
,.
effective in unfractionated the B cell fraction depleted to release substantial IL-la
spleen cells (Fig. 4). of adherent cells apupon stimulation with
2 DISCUSSION
5
