Platelet Responses after Stimulation with Lipopolysaccharide from Porphyromonas gingivalis. Emiko ISOGAI,1 Hiroshi IsoGAI,2 Kimiharu HIROSE,1 Hiroko ...
Bioscience Microflora Vol. 16 (2), 79-82, 1997
Note
Platelet from
Responses
after
Porphyromonas
Stimulation
with
Lipopolysaccharide
gingivalis
EmikoISOGAI,1 HiroshiIsoGAI,2 KimiharuHIROSE,1 HirokoMIMURA,1 KoichiKIMURA,3 ShunjiHAYASHI,3 MasanobuHAYASHI,4 NobuhiroFUJII3 and YoshimiBENNO5 Department ofPreventive Dentistry, School ofDentistry, Health 1 Sciences University ofHokkaido, lshikari-Tobetsu 1757, Hokkaido 061-02, Departments of 2Animal Experimentation and3Microbiology, Sapporo Medical University, Sapporo 060,4Department of Radiation Biology, Faculty ofVeterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069, 5Japan Collection ofMicroorganisms,RIKEN, Wako, Saitama 351-01, Japan Received April 12,1997; Accepted forpublication, June10,1997 Avarietyofbacterial components hasbeenknowntoinduceplatelet aggregation. Here,wereportthatPorphyromonas gingivalis lipopolysaccharide (Pg-LPS) caused directaggregation ofrabbit,butnothumanplatelets. Pg-LPS alsoappeared toinducechemiluminescence response (CLR)inhumanpolymorphonuclear neutrophils (PMNs) inwholebloodor in serum.Humanplateletaggregation wasobserved onlyinthePMN-platelet mixturesystemafterstimulation withPgLPS. Keywords: Porphyromonas gingivalis; lipopolysaccharide; platelets; polymorphonuclear neutrophils
Adult
periodontitis
thought
to be
is
initiated
by
of periodontopathic gingivalis LPS) 10,
response
P.
17, in
20).
and
including
of
immune
proliferation
Porphyromonas
biological has
a similar
cells
(Pg-
activities
(9,
been
mainly
obtained
that
Pg-LPS
was
and purified by a method described previously (9). Escherichia coli 0111:B4 (E-LPS, Difco Laboratories, Detroit, MI, USA) was used as an additional control. The blood from each rabbit or human was collected and mixed in 1/10 volume of 3.8% (w/v) sodium citrate; which served as a source of platelets. The citrate blood was gently mixed, then centrifuged at 150 xg for 10 min at room temperature. Platelet-rich plasma
disease
lipopolysaccharide
Pg-LPS
host has
infectious
infection
gingivalis
a variety
information tal
5).
exhibits 16,
an
bacteria,
(4,
12,
a chronic
or
12).
(3, even
7, 9,
greater
However, using
this
experimen-
animals. We
(PRP)
reported
human
PMNs
radicals
(9).
PMNs
in
isolated
it
from
PMNs
as
a potent
greater
from
tems in
(2,
6, 18,
response
19). to
specific
stimuli
oxygen
radicals
potentiate
lated
with
P.
and tion.
hr.
may
by
a wide
In via
cell
variety
been
this
(15).
of
with study,
contact
was
that PAF
to PMNs
was
hemin
cells
was
grown
(GAM,
with
three
in
extracted
Nissui and
were
times
anaerobically
of
to
used
the
In
stimu-
chronolume
with
Co.,
Tokyo,
menadione
collected
by
the
(pH
Japan)
at 37•Ž
actual
hot-phenol
saline
donors
measure
platelet
mixture
solu-
1000
method
LPS
79
of
Sigma)
was
used
to
experiments,
placed rpm solution
in and
and
represent
was
the
of
added.
within
4 hr.
Tokyo,
Japan)
used
To
50 ƒÊl
40
to
of
mg/ml
in
buffer
transmission.
450 ƒÊl
PRP
a tube,
at 37•Ž. test
adjust 100%
PRP,
whether
In and
50
which
with
to warm
release to
HEPES-Hanks
lumiaggregometer
allowed was
added
ex-
to represent
0% of
108/
each
for
reagent,
a mixture
2.0 •~
and
was
450 ƒÊl
50 ƒÊl
sepaprevi-
to
aggregation
transmission
were
Ficoll
diluted
at
plastic
described
Co.,
(lusiferase-luciferin
7.4)
as
PPP
PRP
PMNs
completed
experiment,
A
the
adjusted
and
platelet-
in capped
double
Percoll), was
100%
of
use.
by
were
solution then
stored
solution
each
water,
addition
(AHS/Japan
of
for
centrifugation
pyrogen-free by
Gifu
centrifuging
concentration
instrument
aggregation.
by was
and
concentration
the
until
same
original
to
ATP.
plate-
in
The the
platelet
by
temperature the
Experiments
distilled 381
PRP
A lumiaggregometer
cell-
human
obtained
(Ficoll-Conray
as
the
109/ml
10 min.
room
periment.
types
suggested
(PPP)
(8).
ml sys-
and
to 3.1 •~
from
ously
described of
at
ration
oxy-
removed,
for
obtained
hy-
of
many
xg
tubes
or
a number
synergistically
initiated
plasma
1800
progressive
been
on
It has
(1).
poor
that
Pg-LPS.
Bacterial
Pg-LPS
act
injury
Medium
washed
by 14).
oxygen
controls has
is produced
13,
were
supplemented 24
PAF
(6,
gingivalis
Anaerobic
healthy
effects
stimulation
tissue
let responses
with
adjusted
quantities
(PAF)
mediator
was
exaggerated
producing
than
rapidly
was
inducing
demonstrated
with
Platelet-activating-factor
of
produce
been
a constitutive
response,
radicals
to
has
patients
exhibit
peractive
blood
Recently,
periodontitis
gen
whole
capable
was
stirring
Then the
50 ƒÊl platelet
at of
80
E. ISOGAI,et al.
Table
1.
Comparison
of platelet
response
after stimulation
of rabbit
a. ATP release ratio expressed as a value obtained by LPS stimulation/value b. ND: not detectable, NT: not tested.
and human platelets
Germany). was
proceed,
trations:
(9,
stimulation 100 ƒÊg/ml)
(8).
The
was
system
after
platelets, 1 and
dent 1.
Dose-dependent
lation
of
lets.
Pg-LPS. •œ-•œ:
Each
from
point
represents *
Pg-LPS
stimulated
concentration
of rabbit
rabbit
5 replicates.
tween LPS
response
:
=
the
mean
and
standard
and
(p
