Microbiol. Immunol. Vol. 36 (5), 507-516, 1992
Induction of Cytokines in Human Peripheral Mononuclear Cells by Mycoplasmas
Masakazu
KITA,*,1
Yasukazu
OHMOTO,2
Yoshikatsu
Blood
HIRAI,2
Nozomi YAMAGUCHI,1and Jiro IMANISHI1 Department of Microbiology,Kyoto Prefectural Universityof Medicine, Kamikyo-ku,Kyoto 602, Japan, and 2Cellular TechnologyInstitute, Otsuka Pharmaceutical Co., Ltd., Tokushima 771-01, Japan
1
(Accepted for publication, January
28, 1992)
Abstract Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasmapneumoniae, M. hyorhinis, M. arginini, M. salivarium,
M.
interleukin-1ƒÀ
orale,
M.
gallisepticum
(IL-1ƒÀ),
IL-2,
or IL-4,
A.
laidlawii
IL-6,
for
tumor
48
hr,
necrosis
and
the
factor-ƒ¿
activities
of
(TNF-ƒ¿)
and
interferon (IFN) in the supernatants were determined by ELISA or bioassay. mycoplasma
only duced
species
by M. pneumoniae. was
antigenically
plasma-stimulated
activities. kinds
immunological
IL-1ƒÀ,
IFN
was
confirmed cultures
did
Furthermore,
cells in human many
induced
PBMC
of cytokines
IL-6
induced to
not
and
be
IFN-ƒ¿.
experiments
by mycoplasma
IL-2
7 species, On
detectable
were induced
as well as by mycoplasma induced
although
by 5 of the mainly
contain
the cytokines
TNF-ƒ¿,
the
amounts
and
alone.
These
the
other of
by mycoplasmal
contamination
was
IFN
hand,
IFN-ƒÀ
All
induced
promyco-
and
IL-4
contaminating
results
suggest
in cell culture
that affect
in vitro.
Some mycoplasma species are found among the normal flora of the genital, urinary, and respiratory tracts. The major human pathogen in this group, Mycoplasma pneumoniae, is the causative agent of atypical pneumonia. In addition to their pathogenic properties, mycoplasmas are common contaminants of cell cultures. It has been reported that as much as 60 to 90% of all established cell lines may be contaminated with mycoplasmas (19). Mycoplasma has drastic effects on the physiology and the structural integrity of the infected host cells. Furthermore, a very disparate group of metabolic changes involving DNA, RNA and protein accompany mycoplasma infection (24). On the other hand, mycoplasma has a variety of effects on the immune system, including macrophage activation (11), enhancement of NK activity (6, 9, 10), an increase in T cell cytotoxicity (1, 8, 18) or the suppression of cytotoxic response (26), and the enhancement of B cell proliferation and maturation etc. (5, 12, 27). The proliferative response induced by M. pneumoniaein human lymphoid cell cultures reflects a mitogenic or an antigen-specific T cell response (3, 4). Mitogenic or 507
508
M.
antigenic which
stimulation regulate
Since caused blood
of lymphocytes
the
these
by
immune
effects
cytokine
of
cells
factor-ƒ¿
ET AL
in vitro
triggers
the
production
of
cytokines
response. mycoplasma
production,
mononuclear
necrosis
KITA
produce
(TNF-ƒ¿)
infection
we
immune or
1 ƒÀ (IL-ƒÀ,
interferon
(IFN)
MATERIALS
the
whether
interleukin-
and
on
examined
in
AND
system
not
IL-2,
response
be
peripheral
IL-4,
to
may
human
IL-6,
tumor
mycoplasmas
.
METHODS
Mycoplasmastrains and cultureprocedures. M. pneumoniaeMac, M. hyorhinisBST-7, Acholeplasmalaidlawii PG-8, M. arginini G-230, M. salivariumPG-20 , M. orale CH19299, and M. gallisepticumwere cultured for 2-4 days at 37 C in PPLO broth (Difco Laboratories, Detroit, Mich., U.S.A.) supplemented with a final concentration of 20% heat-inactivated horse serum, 10% fresh yeast extract (Flow Laboratories Inc .), 0.5% glucose or 0.5% arginine and 1,000 U/ml penicilln G. Mycoplasmas were harvested
by
buffered
centrifugation saline
at
(PBS),
and
7,000 •~
g for
stored
30
at -80
min,
washed
C until
use.
three All
times
with
phosphate-
mycoplasma
species
were
provided by Dr. Yamamoto of the National Institute of Animal Health. To determine colony-forming units (CFU) of mycoplasmas, the number of colonies of mycoplasmas in serial diluted samples was counted on PPLO agar plate after 7-day incubation at 37 C or the number of mycoplasmas was counted in a bacteriacounting
chamber
plasmas
was
under
dark-field
equivalent
to
microscopy.
One ƒÊg/ml
approximately
2 •~
107
(wet
weight)
of
myco-
CFU/ml.
Mycoplasma-infected K-562 cells. In order to prepare the cell lines contaminated with mycoplasmas, mycoplasma-free K-562 cells were infected with each species of mycoplasma
at
doses
of
10 ƒÊg/ml.
After
a
few
passages,
these
cells
were
used
as
mycoplasma-infected cells. Mycoplasma infection of K-562 cells was determined by staining with bisbenzimide trihydrochloride (Hoechst 33258, Sigma Chemical Co., St. Louis, Mo., U.S.A.). Mycoplasma infecting K-562 cells were eliminated by treatment with MC210, a mycoplasmacidal agent (Dainippon Pharmaceutical Co.) for 1 week in vitro. Production of cytokines. Heparinized venous blood was obtained from healthy volunteers tested and proven to be serologically negative for IgM or IgG antibodies to M. pneumoniaeby determining with indirect immunofluorescence method. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-paque density gradient centrifugation. Following three PBS washed, PBMC were suspended at a
density
fetal
of
mycin. of 5% at
1 •~ 106
bovine
serum, Viable
at
15,000 •~
remove
for
10
min,
IL-1 ƒÀ
assay.
The
for
stored
IL-1ƒÀ
activities
U/ml
to 48
these
supplemented penicillin
PBMC
hr
in
a
at humidified
were
collected filter
100 ƒÊg/ml final
fully
membrane
with
and
cultures a
cultures
through
at -80
medium
100
added
from
filtrated
and
1640 and
incubated
Supernatants
mycoplasma,
RPMI
were
and
37 C. g
in
glutamine
mycoplasmas
0.01-100 ƒÊg/ml, CO2
cells/ml 1 mm
atmosphere
of
centrifugation
0.1 ƒÊm
porosity
to
C. in
the
supernatants
were
of
concentrations
by of
10% strepto-
measured
using
INDUCTION
a
human
IL-1ƒÀ
titration
ELISA
plates
kit
were
of PBS, pH 7.
OF
CYTOKINES
(Otsuka
coated
Pharmaceutical
with
Following
BY MYCOPLASMA
anti-IL-1ƒÀ
overnight
Co.,
Ltd.).
monoclonal
incubation
509
Briefly,
antibody
in
at 4 C, the wells were
micro-
100 ƒÊl/well
blocked
with
1% skim milk in PBS for at least 1 hr at room temperature and washed three times with
PBS
(100
pi)
4 C for
containing in
24
0.05%
0.1%
BSA-PBS
hr.
antibodies
The
were
the
plates
were
IgG
and
incubated
added
using
of
(Tween-PBS).
added
were
to
then
to each
washed,
incubated
100 ƒÊl
were
plates
23
well
C for
the
washed
and
supplemented
at
wells
and
three
standard
plate
2 hr
of
or
100 ƒÊl
for
100 ƒÊl
Finally,
the
times,
incubated with
2 hr.
Samples
was of
of
incubated
rabbit
at 23 C.
goat
enzyme
at
anti-IL-1ƒÀ
Subsequently,
POD-labeled
100 ƒÊl
IL-1ƒÀ
anti-rabbit
substrate
(1
mg/ml
in 0.1 Msodium citrate buffer, pH 5) was added to each well
O-phenylenediamine and
Tween-20
at
room
temperature
2 N H2SO4
a Titertek
to
for
each
Multiscan.
well This
5 min.
and kit
the
The
reaction
absorbance
at
is specific
for
IL-1ƒÀ,
was 492
and
stopped nm
does
by
was not
adding
determined
measure
other
cytokines. IL-2
assay.
IL-2
activity
was
also
measured
by
the
same
ELISA
as
IL-1ƒÀ,
using
anti-IL-2 monoclonal antibody (ANOC 101, Otsuka Pharmaceutical Co., Ltd.) and rabbit anti-IL-2 antibodies (OCT 113, Otsuka Pharmaceutical Co., Ltd.) instead of
anti-IL-1ƒÀ
assay
monoclonal
was
specific
antibody
for
IL-2,
and
and
did
rabbit
not
anti-IL-1ƒÀ
measure
antibodies.
other
This
ELISA
cytokines.
IL-4 assay. IL-4 activity was measured using Intertest-4 (Human interleukin-4 ELISA test kit; Genzyme). IL-6
assay.
IL-6
activity
was
also
measured
by
the
same
ELISA
as
IL-1ƒÀ,
using anti-IL-6 monoclonal antibody (ANOC 602, Otsuka Pharmaceutical Co., Ltd.) and rabbit anti-IL-6 antibodies (OCT 601, Otsuka Pharmaceutical Co., Ltd.) in
place
This
of
assay
TNF-ƒ¿
by
anti-IL-1ƒÀ
ELISA
using
assay.
two
specific
IFN
for
TNF-a
FL
was
and
and also
did
rabbit
not
measured
antibodies
anti-IL-1ƒÀ
measure by
the
(ANOC
and
human as
did
not
activity effect
ammion
in
other
culture
inhibition cells
previously
measure
same
705
was
performed
as indicator
described
cells
(13).
cytokines. ELISA
and
as
IL-1ƒÀ,
706.
Otsuka
This ELISA assay
cytokines.
supernatants
assay
antibodies.
other
Co., Ltd.) to recognize the different epitope. Antiviral
virus
IL-6,
monoclonal
cytopathic
or WISH
challenge
for
activity
TNF-ƒ¿,
assay.
conventional
antibody
specific
anti-TNF-a
Pharmaceutical was
monoclonal
was
and
Briefly,
determined
in microtiter vesicular
5 •~ 104
by
plates
stomatitis
WISH
cells
virus in
a
with
0.05
as ml
were incubated with 0.05 ml of serial diluted samples or standard preparation in microtiter plates at 37 C in a 0.5% CO2 humidified atomosphere. After 24 hr incubation, the cells were challenged by VSV, and incubated for 18 to 24 hr. The viable cells were stained with 1% neutral red, then the dye extraction solution (0.1 sodium phosphate : ethanol =1 : 1) was added. The absorbance at 540 nm was determined 69/19) which
using and
IFN-ƒÁ
is expressed Sheep
antiserum
a Titertek (Gg as
Multiscan.
203-901-530) international against
The were units
Sendai
international
used per
ml
virus-induced
to
calibrate
references units
IFN-ƒ¿ of
IFN
(MRC activity,
(IU/ml). HuIFN-ƒ¿
(provided
from
Japan
510
M.
Chemical from
Research
Toray
Co.,
Industries,
Ltd.), Inc.)
KITA
ET AL
monoclonal and
antibodies
monoclonal
against
antibodies
HuIFN-ƒÀ
against
(provided
HuIFN-ƒÁ
(provided
from Midori Juji Co., Ltd.) were used to characterize the antigenic properties of antiviral activity in culture supernatants. They contained more than 3,000 neutralizing units/ml. Equal volumes of antibodies and samples were mixed and incubated for 1 hr at 37 C. The samples were then assayed for residual IFN activity as described above. RESULTS
IFN Production in PBMC Cultures by M. pneumoniae PBMC from 2 healthy volunteers with no previous at
exposure
doses
Fig.
of
1.
to M. pneumoniae,
10 ƒÊg/ml.
Kinetics
The
of IFN
were
culture
serologic
cultured
in the presence
supernatants
production
in human
indication
collected
PBMC
at
culture
of recent
or
of M. pneumoniae various
by M. pneumoniae.
time
were
Human
PBMC were cultured in the presence of M. pneumoniaeat a dose of 10 itgiml. The culture supernatants
collected
Table
1.
at various
Induction
times
of IFN cells
were
assayed
in human
culture
for
peripheral
by mycoplasmas
IFN
activity.
blood
mononuclear
INDUCTION
OF
assayed for IFN activity. peaked at 48 hr (Fig. 1). To
determine
cultures
at
the
doses
of
to
of
IFN,
and
the
mycoplasma
100 ƒÊg/ml,
in culture were measured. ml
BY MYCOPLASMA
511
IFN activity was first detected after 6 hr in culture and
optimal
0.01
CYTOKINES
dose,
and
IFN
In this experiment,
optimal
dose
was
7 species
activities
were
in
added
to PBMC
supernatants
after
M. pneumoniae induced
48
hr
22 to 150 IU/
10 ƒÊg/ml.
We determined if other mycoplasma species were capable of inducing IFN. The results showed that M. pneumoniae,M. salivarium, M. orale, M. gallisepticumand A. laidlawii induced IFN production, although M. arginini and M. hyorhinisdid not (Table 1). Characterizationof IFN Inducedby Mycoplasmas The antiviral activity induced by mycoplasmas was characterized by a neutralizing
test
using
The M.
by
IFN-ƒ¿
and
were
These
not
to
results
not
by
was neutralized
reduced
demonstrated
mycoplasma
supernatants
neutralized
On the other
was
antibodies.
culture
Accordingly,
M. salivarium.
mycoplasmas
to
mostly
IFN-ƒÁ.
-r
in PBMC
M. salivarium
and
sponse
-ƒÀ and
activities
gallisepticum
induced
of
anti-IFN-ƒ¿,
IFN
infection
by the
IFN-ƒÁ
hand,
was
exposure
that
the
was
partially
IFN
mainly
by
induced
anti-IFN-ƒÀ
produced antigenically
M.
that
amounts
M.
of
pneumoniae
by all the species
antibodies
in
orale and
whereas
to equal
induced
activity to
by
antibody,
antibodies
the IFN
after
induced
anti-IFN-ƒ¿
PBMC identical
(Table
cultures to
2).
in
IFN-ƒ¿,
rebut
IFN-ƒÀ.
Inductionof TNF-cc in PBMC Culture by Mycoplasmas In
order
to
study
mycoplasmas, TNF-a species of mycoplasmas As more
shown
than
in
whether
or
not
PBMC
produce
TNF
activities in PMBC culture supernatants were determined by ELI SA.
Table
3,
TNF-a
was
induced
by
of IFNs
induced
all
mycoplasma
0.1 ƒÊg/ml.
Table
2.
Characteristics
by mycoplasmas
in
the
presence
stimulated species
of
by at
doses
7
512
M.
Table
Table
4.
Table
3.
Induction
5.
induction
of
Induction
KITA
ET AL
of TNF-cr by various mycoplasma in human PBMC culture
TNF-a,
IL-1ƒÀ,
of cytokines
K-562 cells infected
IL-2,
IL-4
and
in co-culture
with various
IL-6
by
of human
mycoplasmasa)
species
mycoplasmasa)
PBMC
and
INDUCTION
OF
CYTOKINES
BY MYCOPLASMA
513
Inductionof Interleukinsin PBMC Culture by Mycoplasma As mycoplasmas possess the ability to induce human PBMC to produce IFN and TNF, it is possible that mycoplasmas induce the production of other cytokines. IL-1ƒÀ,
IL-2,
IL-4
mycoplasma The
and
were results
IL-6
activities
therefore
in
determined
demonstrated
that
PBMC
culture
supernatants
stimulated
by
by ELISA. IL-1 ƒÀ
and
IL-6
were
induced
by
all
myco-
plasma species, whereas IL-2 was induced by only one, M. pneumoniae. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IL-4 activity (Table 4). Inductionof Cytokinesin Co-Cultureof Human PBMC and Mycoplasma-InfectedK-562 Cells In order to confirm the induction of cytokines by mycoplasma-contaminated cells, mycoplasma-infected K-562 cells were tested for their ability to induce the production of cytokines. The results demonstrated that cytokines were induced by K-562 cells infected with various mycoplasmas in co-culture with human PBMC in the same manner as that by mycoplasma alone (Table 5). DISCUSSION
The present study demonstrated that in vitro cultures of human PBMC are capable of producing many cytokines in response to infection with mycoplasmas. Our results showed that human PBMC produced significant levels of IFN in response to M. pneumoniae, M. salivarium, M. orale, M. gallisepticum and A. laidlawii, and that the IFN
induced
by these mycoplasmas
was mainly
the a-type.
Cole et al (7) have reported that IFN was induced by M. pneumoniaeand M. synoviein human lymphocyte cultures, but not by M. salivarium, M. orale, M. gallisepticumand A. laidlawii. They observed a marked variation in the levels of IFN induced by mycoplasmas in lymphocytes from the same donor on different occasions and from different donors in the same experiment. However, we did not observe a marked variation in IFN production in PBMC from the same donor. On the other hand,
it was
natants
of
reported M.
that
arthritidis
IFN-ƒÁ (14,
was
20).
induced
However,
by we
M.
arginini
could
not
and detect
by
culture
IFN-ƒÁ
superactivity
supernatants of cells culture stimulated by M. arginini. This discrepancy due to the differences in species of mycoplasma and animals used. Human used
in
the
PBMC present
produced study.
TNF-a Murine
in response spleen
cells
to all the released
species TNF-ƒ¿
in
may
be
of mycoplasma when
they
were
incubated with mycoplasmas or with mycoplasma-infected L929 cells (11, 16). Moreover,
it was reported
that
TNF-a
was induced
by a various
species
plasmas by a protein kinase C-independent pathway (2, 22, 23, 25). were coincident with these.
of myco-
Our results
Several B cell stimulatory factors inducing B cell growth and differentiation have been characterized and molecularly cloned, including IL-1, IL-2, IL-4 and IL-6. Among them, IL-1 and IL-6 possess a number of biological important activities in immune function and the inflammation response. All species of mycoplasmas
514
M.
KITA
ET AL
shown here induced the production of IL-1 and IL-6 in culture of human PBMC. Quentmeier et al (21) have reported that M. fermentans-derived high-molecularweight material induced IL-6 release in cultures of murine macrophages and human monocytes. However, there have been no reports indicating that IL-6 is produced by various mycoplasma species. Preliminary results showed that heat-killed mycoplasmas also induced the production of cytokines. Therefore, some substances other than mycoplasma-derived factors in culture media may exist. We did not identify a substance from mycoplasmas which induces the production of cytokines. However, the study to identify a substance which induces the cytokines is in progress, and the results will be reported in separate papers. Makhoul et al (17) have reported that M. pneumoniaemembranes lack the ability to induce IL-2 production in human PBMC. However, low levels of IL-2 were induced only by M. pneumoniae. A few factors were considered as the cause of the conflicting results from these experiments as follows : 1), the different strains of mycoplasma used (M. pneumoniaeEaton FH and M. pneumoniaeMac) ; 2), a difference in the IL-2 assay sensitivity (a bioassay using the IL-2-dependent T cell line and ELISA) and 3), different culture conditions. Since a low level of IL-2 was detected in this study and since rat lymph node cells produced IL-2 in response to M.pulmonis membranes (15), the largest cause of the discrepancy seems to be a difference in sensitivity. Yoshida et al have reported that the rate of mycoplasmal contamination in cell lines maintained in Japan might be higher than 26% and the mycoplasmas detected by an immunoblot were M. hyorhinis, M. fermentans, M. orale, M. hominis and A. laidlawii (28, 29). In most cases the number of viable mycoplasmas in contaminated cell lines ranged from 105 to 108 CFU/ml. This number of mycoplasmas was equivalent
to
approximately
0.01-10 ƒÊg/ml
in
this
study.
Furthermore,
we
examined
whether or not human PBMC produce cytokines in co-culture with mycoplasmainfected cells. As cytokine activities were detected in the co-culture supernatants in the same manner as the previous results, human PBMC produced the same cytokines when they were incubated with mycoplasma or with mycoplasma-contaminated cells. These contamination
results affect
suggest the
that results
many
kinds
of
cytokines
of immunological
induced
experiments
by
mycoplasma
in vitro.
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BY MYCOPLASMA
515
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Immunol.
86-92.
Sher, T., Yamin, A., Rottem, S., and Gallily, R. 1990. In vitro induction of tumor necrosis factor a, tumor cytolysis, and blast transformation by Spiroplasma membranes. J. Natl. Cancer Inst. 82: 1142-1145. Stanbridge, E. J. 1981. Mycoplasma detection: an obligation to scientific accuracy. Isr. J. Med. Sci. 17: 563-568. Sugama, K., Kuwano, K., Furukawa, M., Himeno, Y., Satoh, T., and Arai, S. 1990. Mycoplasmas induce transcription and production of tumor necrosis factor in a monocytic cell line, THP-1, by a protein kinase C-independent pathway. Infect. Immun. 58: 3564-3567.
516
26)
27)
28) 29)
M.
KITA
ET AL
Teh, H., Ho, M., and Williams, L.D. 1988. Suppression of cytotoxic responses by a supernatant factor derived from Mycoplasma hyorhinis-infected mammalian cell lines. Infect . Immun. 56: 197-203. Wayner, E.A., and Brooks, C.G., 1984. Induction of NKCF-like activity in mixed lymphocytetumor cell culture: direct involvement of mycoplasma infection of tumor cells . J. Immunol. 132: 2135-2142. Yoshida, T., and Takeuchi, M. 1987. Test for mycoplasmal contamination in cell lines collected in IFO. IFO Res. Comm. 13: 52-58. Yoshida, T., Yanai, N., Kawase, M., Mizusawa, H., Yamamoto, K., and Takeuchi , M. 1989. Identification of mycoplasmas contaminating animal cell lines. IFO Res . Comm. 14: 13-19. (Received for publication, September 24, 1991; in revised form, December 5, 1991)
Induction of Cytokines in Human Peripheral Mononuclear Cells by Mycoplasmas
Masakazu
KITA,*,1
Yasukazu
OHMOTO,2
Yoshikatsu
Blood
HIRAI,2
Nozomi YAMAGUCHI,1and Jiro IMANISHI1 Department of Microbiology,Kyoto Prefectural Universityof Medicine, Kamikyo-ku,Kyoto 602, Japan, and 2Cellular TechnologyInstitute, Otsuka Pharmaceutical Co., Ltd., Tokushima 771-01, Japan
1
(Accepted for publication, January
28, 1992)
Abstract Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasmapneumoniae, M. hyorhinis, M. arginini, M. salivarium,
M.
interleukin-1ƒÀ
orale,
M.
gallisepticum
(IL-1ƒÀ),
IL-2,
or IL-4,
A.
laidlawii
IL-6,
for
tumor
48
hr,
necrosis
and
the
factor-ƒ¿
activities
of
(TNF-ƒ¿)
and
interferon (IFN) in the supernatants were determined by ELISA or bioassay. mycoplasma
only duced
species
by M. pneumoniae. was
antigenically
plasma-stimulated
activities. kinds
immunological
IL-1ƒÀ,
IFN
was
confirmed cultures
did
Furthermore,
cells in human many
induced
PBMC
of cytokines
IL-6
induced to
not
and
be
IFN-ƒ¿.
experiments
by mycoplasma
IL-2
7 species, On
detectable
were induced
as well as by mycoplasma induced
although
by 5 of the mainly
contain
the cytokines
TNF-ƒ¿,
the
amounts
and
alone.
These
the
other of
by mycoplasmal
contamination
was
IFN
hand,
IFN-ƒÀ
All
induced
promyco-
and
IL-4
contaminating
results
suggest
in cell culture
that affect
in vitro.
Some mycoplasma species are found among the normal flora of the genital, urinary, and respiratory tracts. The major human pathogen in this group, Mycoplasma pneumoniae, is the causative agent of atypical pneumonia. In addition to their pathogenic properties, mycoplasmas are common contaminants of cell cultures. It has been reported that as much as 60 to 90% of all established cell lines may be contaminated with mycoplasmas (19). Mycoplasma has drastic effects on the physiology and the structural integrity of the infected host cells. Furthermore, a very disparate group of metabolic changes involving DNA, RNA and protein accompany mycoplasma infection (24). On the other hand, mycoplasma has a variety of effects on the immune system, including macrophage activation (11), enhancement of NK activity (6, 9, 10), an increase in T cell cytotoxicity (1, 8, 18) or the suppression of cytotoxic response (26), and the enhancement of B cell proliferation and maturation etc. (5, 12, 27). The proliferative response induced by M. pneumoniaein human lymphoid cell cultures reflects a mitogenic or an antigen-specific T cell response (3, 4). Mitogenic or 507
508
M.
antigenic which
stimulation regulate
Since caused blood
of lymphocytes
the
these
by
immune
effects
cytokine
of
cells
factor-ƒ¿
ET AL
in vitro
triggers
the
production
of
cytokines
response. mycoplasma
production,
mononuclear
necrosis
KITA
produce
(TNF-ƒ¿)
infection
we
immune or
1 ƒÀ (IL-ƒÀ,
interferon
(IFN)
MATERIALS
the
whether
interleukin-
and
on
examined
in
AND
system
not
IL-2,
response
be
peripheral
IL-4,
to
may
human
IL-6,
tumor
mycoplasmas
.
METHODS
Mycoplasmastrains and cultureprocedures. M. pneumoniaeMac, M. hyorhinisBST-7, Acholeplasmalaidlawii PG-8, M. arginini G-230, M. salivariumPG-20 , M. orale CH19299, and M. gallisepticumwere cultured for 2-4 days at 37 C in PPLO broth (Difco Laboratories, Detroit, Mich., U.S.A.) supplemented with a final concentration of 20% heat-inactivated horse serum, 10% fresh yeast extract (Flow Laboratories Inc .), 0.5% glucose or 0.5% arginine and 1,000 U/ml penicilln G. Mycoplasmas were harvested
by
buffered
centrifugation saline
at
(PBS),
and
7,000 •~
g for
stored
30
at -80
min,
washed
C until
use.
three All
times
with
phosphate-
mycoplasma
species
were
provided by Dr. Yamamoto of the National Institute of Animal Health. To determine colony-forming units (CFU) of mycoplasmas, the number of colonies of mycoplasmas in serial diluted samples was counted on PPLO agar plate after 7-day incubation at 37 C or the number of mycoplasmas was counted in a bacteriacounting
chamber
plasmas
was
under
dark-field
equivalent
to
microscopy.
One ƒÊg/ml
approximately
2 •~
107
(wet
weight)
of
myco-
CFU/ml.
Mycoplasma-infected K-562 cells. In order to prepare the cell lines contaminated with mycoplasmas, mycoplasma-free K-562 cells were infected with each species of mycoplasma
at
doses
of
10 ƒÊg/ml.
After
a
few
passages,
these
cells
were
used
as
mycoplasma-infected cells. Mycoplasma infection of K-562 cells was determined by staining with bisbenzimide trihydrochloride (Hoechst 33258, Sigma Chemical Co., St. Louis, Mo., U.S.A.). Mycoplasma infecting K-562 cells were eliminated by treatment with MC210, a mycoplasmacidal agent (Dainippon Pharmaceutical Co.) for 1 week in vitro. Production of cytokines. Heparinized venous blood was obtained from healthy volunteers tested and proven to be serologically negative for IgM or IgG antibodies to M. pneumoniaeby determining with indirect immunofluorescence method. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-paque density gradient centrifugation. Following three PBS washed, PBMC were suspended at a
density
fetal
of
mycin. of 5% at
1 •~ 106
bovine
serum, Viable
at
15,000 •~
remove
for
10
min,
IL-1 ƒÀ
assay.
The
for
stored
IL-1ƒÀ
activities
U/ml
to 48
these
supplemented penicillin
PBMC
hr
in
a
at humidified
were
collected filter
100 ƒÊg/ml final
fully
membrane
with
and
cultures a
cultures
through
at -80
medium
100
added
from
filtrated
and
1640 and
incubated
Supernatants
mycoplasma,
RPMI
were
and
37 C. g
in
glutamine
mycoplasmas
0.01-100 ƒÊg/ml, CO2
cells/ml 1 mm
atmosphere
of
centrifugation
0.1 ƒÊm
porosity
to
C. in
the
supernatants
were
of
concentrations
by of
10% strepto-
measured
using
INDUCTION
a
human
IL-1ƒÀ
titration
ELISA
plates
kit
were
of PBS, pH 7.
OF
CYTOKINES
(Otsuka
coated
Pharmaceutical
with
Following
BY MYCOPLASMA
anti-IL-1ƒÀ
overnight
Co.,
Ltd.).
monoclonal
incubation
509
Briefly,
antibody
in
at 4 C, the wells were
micro-
100 ƒÊl/well
blocked
with
1% skim milk in PBS for at least 1 hr at room temperature and washed three times with
PBS
(100
pi)
4 C for
containing in
24
0.05%
0.1%
BSA-PBS
hr.
antibodies
The
were
the
plates
were
IgG
and
incubated
added
using
of
(Tween-PBS).
added
were
to
then
to each
washed,
incubated
100 ƒÊl
were
plates
23
well
C for
the
washed
and
supplemented
at
wells
and
three
standard
plate
2 hr
of
or
100 ƒÊl
for
100 ƒÊl
Finally,
the
times,
incubated with
2 hr.
Samples
was of
of
incubated
rabbit
at 23 C.
goat
enzyme
at
anti-IL-1ƒÀ
Subsequently,
POD-labeled
100 ƒÊl
IL-1ƒÀ
anti-rabbit
substrate
(1
mg/ml
in 0.1 Msodium citrate buffer, pH 5) was added to each well
O-phenylenediamine and
Tween-20
at
room
temperature
2 N H2SO4
a Titertek
to
for
each
Multiscan.
well This
5 min.
and kit
the
The
reaction
absorbance
at
is specific
for
IL-1ƒÀ,
was 492
and
stopped nm
does
by
was not
adding
determined
measure
other
cytokines. IL-2
assay.
IL-2
activity
was
also
measured
by
the
same
ELISA
as
IL-1ƒÀ,
using
anti-IL-2 monoclonal antibody (ANOC 101, Otsuka Pharmaceutical Co., Ltd.) and rabbit anti-IL-2 antibodies (OCT 113, Otsuka Pharmaceutical Co., Ltd.) instead of
anti-IL-1ƒÀ
assay
monoclonal
was
specific
antibody
for
IL-2,
and
and
did
rabbit
not
anti-IL-1ƒÀ
measure
antibodies.
other
This
ELISA
cytokines.
IL-4 assay. IL-4 activity was measured using Intertest-4 (Human interleukin-4 ELISA test kit; Genzyme). IL-6
assay.
IL-6
activity
was
also
measured
by
the
same
ELISA
as
IL-1ƒÀ,
using anti-IL-6 monoclonal antibody (ANOC 602, Otsuka Pharmaceutical Co., Ltd.) and rabbit anti-IL-6 antibodies (OCT 601, Otsuka Pharmaceutical Co., Ltd.) in
place
This
of
assay
TNF-ƒ¿
by
anti-IL-1ƒÀ
ELISA
using
assay.
two
specific
IFN
for
TNF-a
FL
was
and
and also
did
rabbit
not
measured
antibodies
anti-IL-1ƒÀ
measure by
the
(ANOC
and
human as
did
not
activity effect
ammion
in
other
culture
inhibition cells
previously
measure
same
705
was
performed
as indicator
described
cells
(13).
cytokines. ELISA
and
as
IL-1ƒÀ,
706.
Otsuka
This ELISA assay
cytokines.
supernatants
assay
antibodies.
other
Co., Ltd.) to recognize the different epitope. Antiviral
virus
IL-6,
monoclonal
cytopathic
or WISH
challenge
for
activity
TNF-ƒ¿,
assay.
conventional
antibody
specific
anti-TNF-a
Pharmaceutical was
monoclonal
was
and
Briefly,
determined
in microtiter vesicular
5 •~ 104
by
plates
stomatitis
WISH
cells
virus in
a
with
0.05
as ml
were incubated with 0.05 ml of serial diluted samples or standard preparation in microtiter plates at 37 C in a 0.5% CO2 humidified atomosphere. After 24 hr incubation, the cells were challenged by VSV, and incubated for 18 to 24 hr. The viable cells were stained with 1% neutral red, then the dye extraction solution (0.1 sodium phosphate : ethanol =1 : 1) was added. The absorbance at 540 nm was determined 69/19) which
using and
IFN-ƒÁ
is expressed Sheep
antiserum
a Titertek (Gg as
Multiscan.
203-901-530) international against
The were units
Sendai
international
used per
ml
virus-induced
to
calibrate
references units
IFN-ƒ¿ of
IFN
(MRC activity,
(IU/ml). HuIFN-ƒ¿
(provided
from
Japan
510
M.
Chemical from
Research
Toray
Co.,
Industries,
Ltd.), Inc.)
KITA
ET AL
monoclonal and
antibodies
monoclonal
against
antibodies
HuIFN-ƒÀ
against
(provided
HuIFN-ƒÁ
(provided
from Midori Juji Co., Ltd.) were used to characterize the antigenic properties of antiviral activity in culture supernatants. They contained more than 3,000 neutralizing units/ml. Equal volumes of antibodies and samples were mixed and incubated for 1 hr at 37 C. The samples were then assayed for residual IFN activity as described above. RESULTS
IFN Production in PBMC Cultures by M. pneumoniae PBMC from 2 healthy volunteers with no previous at
exposure
doses
Fig.
of
1.
to M. pneumoniae,
10 ƒÊg/ml.
Kinetics
The
of IFN
were
culture
serologic
cultured
in the presence
supernatants
production
in human
indication
collected
PBMC
at
culture
of recent
or
of M. pneumoniae various
by M. pneumoniae.
time
were
Human
PBMC were cultured in the presence of M. pneumoniaeat a dose of 10 itgiml. The culture supernatants
collected
Table
1.
at various
Induction
times
of IFN cells
were
assayed
in human
culture
for
peripheral
by mycoplasmas
IFN
activity.
blood
mononuclear
INDUCTION
OF
assayed for IFN activity. peaked at 48 hr (Fig. 1). To
determine
cultures
at
the
doses
of
to
of
IFN,
and
the
mycoplasma
100 ƒÊg/ml,
in culture were measured. ml
BY MYCOPLASMA
511
IFN activity was first detected after 6 hr in culture and
optimal
0.01
CYTOKINES
dose,
and
IFN
In this experiment,
optimal
dose
was
7 species
activities
were
in
added
to PBMC
supernatants
after
M. pneumoniae induced
48
hr
22 to 150 IU/
10 ƒÊg/ml.
We determined if other mycoplasma species were capable of inducing IFN. The results showed that M. pneumoniae,M. salivarium, M. orale, M. gallisepticumand A. laidlawii induced IFN production, although M. arginini and M. hyorhinisdid not (Table 1). Characterizationof IFN Inducedby Mycoplasmas The antiviral activity induced by mycoplasmas was characterized by a neutralizing
test
using
The M.
by
IFN-ƒ¿
and
were
These
not
to
results
not
by
was neutralized
reduced
demonstrated
mycoplasma
supernatants
neutralized
On the other
was
antibodies.
culture
Accordingly,
M. salivarium.
mycoplasmas
to
mostly
IFN-ƒÁ.
-r
in PBMC
M. salivarium
and
sponse
-ƒÀ and
activities
gallisepticum
induced
of
anti-IFN-ƒ¿,
IFN
infection
by the
IFN-ƒÁ
hand,
was
exposure
that
the
was
partially
IFN
mainly
by
induced
anti-IFN-ƒÀ
produced antigenically
M.
that
amounts
M.
of
pneumoniae
by all the species
antibodies
in
orale and
whereas
to equal
induced
activity to
by
antibody,
antibodies
the IFN
after
induced
anti-IFN-ƒ¿
PBMC identical
(Table
cultures to
2).
in
IFN-ƒ¿,
rebut
IFN-ƒÀ.
Inductionof TNF-cc in PBMC Culture by Mycoplasmas In
order
to
study
mycoplasmas, TNF-a species of mycoplasmas As more
shown
than
in
whether
or
not
PBMC
produce
TNF
activities in PMBC culture supernatants were determined by ELI SA.
Table
3,
TNF-a
was
induced
by
of IFNs
induced
all
mycoplasma
0.1 ƒÊg/ml.
Table
2.
Characteristics
by mycoplasmas
in
the
presence
stimulated species
of
by at
doses
7
512
M.
Table
Table
4.
Table
3.
Induction
5.
induction
of
Induction
KITA
ET AL
of TNF-cr by various mycoplasma in human PBMC culture
TNF-a,
IL-1ƒÀ,
of cytokines
K-562 cells infected
IL-2,
IL-4
and
in co-culture
with various
IL-6
by
of human
mycoplasmasa)
species
mycoplasmasa)
PBMC
and
INDUCTION
OF
CYTOKINES
BY MYCOPLASMA
513
Inductionof Interleukinsin PBMC Culture by Mycoplasma As mycoplasmas possess the ability to induce human PBMC to produce IFN and TNF, it is possible that mycoplasmas induce the production of other cytokines. IL-1ƒÀ,
IL-2,
IL-4
mycoplasma The
and
were results
IL-6
activities
therefore
in
determined
demonstrated
that
PBMC
culture
supernatants
stimulated
by
by ELISA. IL-1 ƒÀ
and
IL-6
were
induced
by
all
myco-
plasma species, whereas IL-2 was induced by only one, M. pneumoniae. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IL-4 activity (Table 4). Inductionof Cytokinesin Co-Cultureof Human PBMC and Mycoplasma-InfectedK-562 Cells In order to confirm the induction of cytokines by mycoplasma-contaminated cells, mycoplasma-infected K-562 cells were tested for their ability to induce the production of cytokines. The results demonstrated that cytokines were induced by K-562 cells infected with various mycoplasmas in co-culture with human PBMC in the same manner as that by mycoplasma alone (Table 5). DISCUSSION
The present study demonstrated that in vitro cultures of human PBMC are capable of producing many cytokines in response to infection with mycoplasmas. Our results showed that human PBMC produced significant levels of IFN in response to M. pneumoniae, M. salivarium, M. orale, M. gallisepticum and A. laidlawii, and that the IFN
induced
by these mycoplasmas
was mainly
the a-type.
Cole et al (7) have reported that IFN was induced by M. pneumoniaeand M. synoviein human lymphocyte cultures, but not by M. salivarium, M. orale, M. gallisepticumand A. laidlawii. They observed a marked variation in the levels of IFN induced by mycoplasmas in lymphocytes from the same donor on different occasions and from different donors in the same experiment. However, we did not observe a marked variation in IFN production in PBMC from the same donor. On the other hand,
it was
natants
of
reported M.
that
arthritidis
IFN-ƒÁ (14,
was
20).
induced
However,
by we
M.
arginini
could
not
and detect
by
culture
IFN-ƒÁ
superactivity
supernatants of cells culture stimulated by M. arginini. This discrepancy due to the differences in species of mycoplasma and animals used. Human used
in
the
PBMC present
produced study.
TNF-a Murine
in response spleen
cells
to all the released
species TNF-ƒ¿
in
may
be
of mycoplasma when
they
were
incubated with mycoplasmas or with mycoplasma-infected L929 cells (11, 16). Moreover,
it was reported
that
TNF-a
was induced
by a various
species
plasmas by a protein kinase C-independent pathway (2, 22, 23, 25). were coincident with these.
of myco-
Our results
Several B cell stimulatory factors inducing B cell growth and differentiation have been characterized and molecularly cloned, including IL-1, IL-2, IL-4 and IL-6. Among them, IL-1 and IL-6 possess a number of biological important activities in immune function and the inflammation response. All species of mycoplasmas
514
M.
KITA
ET AL
shown here induced the production of IL-1 and IL-6 in culture of human PBMC. Quentmeier et al (21) have reported that M. fermentans-derived high-molecularweight material induced IL-6 release in cultures of murine macrophages and human monocytes. However, there have been no reports indicating that IL-6 is produced by various mycoplasma species. Preliminary results showed that heat-killed mycoplasmas also induced the production of cytokines. Therefore, some substances other than mycoplasma-derived factors in culture media may exist. We did not identify a substance from mycoplasmas which induces the production of cytokines. However, the study to identify a substance which induces the cytokines is in progress, and the results will be reported in separate papers. Makhoul et al (17) have reported that M. pneumoniaemembranes lack the ability to induce IL-2 production in human PBMC. However, low levels of IL-2 were induced only by M. pneumoniae. A few factors were considered as the cause of the conflicting results from these experiments as follows : 1), the different strains of mycoplasma used (M. pneumoniaeEaton FH and M. pneumoniaeMac) ; 2), a difference in the IL-2 assay sensitivity (a bioassay using the IL-2-dependent T cell line and ELISA) and 3), different culture conditions. Since a low level of IL-2 was detected in this study and since rat lymph node cells produced IL-2 in response to M.pulmonis membranes (15), the largest cause of the discrepancy seems to be a difference in sensitivity. Yoshida et al have reported that the rate of mycoplasmal contamination in cell lines maintained in Japan might be higher than 26% and the mycoplasmas detected by an immunoblot were M. hyorhinis, M. fermentans, M. orale, M. hominis and A. laidlawii (28, 29). In most cases the number of viable mycoplasmas in contaminated cell lines ranged from 105 to 108 CFU/ml. This number of mycoplasmas was equivalent
to
approximately
0.01-10 ƒÊg/ml
in
this
study.
Furthermore,
we
examined
whether or not human PBMC produce cytokines in co-culture with mycoplasmainfected cells. As cytokine activities were detected in the co-culture supernatants in the same manner as the previous results, human PBMC produced the same cytokines when they were incubated with mycoplasma or with mycoplasma-contaminated cells. These contamination
results affect
suggest the
that results
many
kinds
of
cytokines
of immunological
induced
experiments
by
mycoplasma
in vitro.
REFERENCES
1) Aldridge, K.E., Cole, B.C., and Ward, J.R. 1977. Mycoplasma-dependent activation of normal lymphocytes: induction of a lymphocyte-mediated cytotoxicity for allogenic and syngeneic mouse target cells. Infect. Immun. 18: 377-385. 2) Arai, S., Furukawa, M., Munakata, T., Kuwano, K., Inoue, H., and Miyazaki, T. 1990. Enhancement of cytotoxicity of active macrophages by mycoplasma : role of mycoplasma-associated induction
3) 4)
of tumor
necrosis
factor-ƒ¿
(TNF-ƒ¿)
in
macrophages.
Microbiol.
Immunol.
34:
231-243.
Biberfeld, G., and Gronowics, E. 1976. Mycoplasma pneumoniaeis a polyclonal B-cell activator. Nature 261: 238-239. Biberfeld, G. 1977. Activation of human lymphocyte subpopulations by Mycoplasma pneumoniae. Scand. J. Immunol. 6: 1145-1150.
INDUCTION
5) 6)
7) 8)
9) 10) 11) 12) 13) 14)
15) 16)
17) 18)
19) 20)
OF
CYTOKINES
BY MYCOPLASMA
515
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