Department of Immunology, Osaka Medical
Center for Cancer and Cardiovascular Diseases, Higashinari-ku,
Osaka 537-8511,1 Department of
General Biologics Control, National Institute of Health,
Musashimurayama, Tokyo 208-0011,3 and
Organization for Pharmaceutical Safety and Research, Tokyo
113,2 Japan
Received 13 September 1999/Returned for modification 29 October
1999/Accepted 25 November 1999
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INTRODUCTION |
The complement system is central to
innate immunity. It can directly recognize invading substances via the
alternative, or lectin, pathway and facilitate removal of infectious
organisms by phagocytes (9, 11). Deposition of the third
component of complement (C3) is a critical factor for host defense.
Several molecules of viral or microbial origin have been identified as activators of human complement (2, 32, 35).
Recently, we discovered a membrane-associated novel C3-activating
protein in human tumor cell lines (18-20). Based on the
genomic analysis, it was found to originate from Mycoplasma
fermentans (21). This protein, designated M161Ag, is a
palmitoylated protein with a molecular mass of 43 kDa (21).
It activates human complement via the alternative pathway, allowing the
deposition of C3b and C3bi on human cells infected by M. fermentans and thus overcoming the functions of the complement
regulatory proteins, CD46 and CD55, expressed on these cells (1,
18, 19). Interestingly, M161Ag efficiently promotes the
production of interleukin 1
(IL-1), tumor necrosis factor alpha
(TNF-
), IL-6, IL-10, and IL-12 in human peripheral blood monocytes
(21). Thus, M161Ag is a bifunctional protein which elicits
the innate immune responses via complement activation and stimulation
of monocytes.
M. fermentans is a mycoplasma species capable of infecting
humans and has been suggested to serve as a cofactor during the development of AIDS (3, 17). M. fermentans DNA
has been detected in the peripheral blood mononuclear cells of patients
with AIDS by PCR (8, 12). In addition, the products of
M. fermentans affect the host immune system via B- or T-cell
activation, monocyte/macrophage stimulation, and cytocidal ability
(6, 7, 25, 26, 28). However, its role as a cofactor in human
immunodeficiency virus disease is still unknown. Recent studies suggest
that AIDS-associated mycoplasma species, including M. fermentans, can invade host cells (30, 38), but direct
evidence for the latent infection of human cells by M. fermentans has not been found. Furthermore, the role of complement
in defense against M. fermentans infection has not been
elucidated. In this study, we established monoclonal antibodies (MAbs)
against M161Ag and demonstrated a rapid targeting of M. fermentans by human complement using MAbs as probes.
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MATERIALS AND METHODS |
Antibodies, cells and reagents.
MAbs against M161Ag (M161)
and CD46 (M177) were produced and purified in our laboratory as
described previously (19, 34). Anti-human C3b MAb (C5G) and
anti-CD55 MAb (IA10) were gifts from K. Iida (Takeda Chemical
Industries) and T. Kinoshita (Osaka University), respectively (10,
13). Mouse immunoglobulin G (IgG) was purchased from Sigma
Chemical Co. (St. Louis, Mo.). Fluorescein isothiocyanate (FITC)-labeled goat F(ab')2 anti-mouse IgG was from Cappel
(West Chester, Pa.), and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-labeled anti-rabbit IgG were from Bio-Rad Laboratories (Hercules, Calif.).
Gelatin veronal-buffered (GVB) saline containing 2 mM MgCl2
and 10 mM EGTA (Mg2+-EGTA-GVB) or 10 mM EDTA (EDTA-GVB) was
used in the C3 deposition assay. Normal human serum (NHS) was collected
from 20 healthy donors and stored in aliquots at 
70°C. Antibody to
M. fermentans was less than the detection limit (1 ng/ml) by
enzyme-linked immunosorbent assay in the pooled NHS (data not shown). A
1/20 volume of 40 mM Mg2+-200 mM EGTA (pH 7.4) or 200 mM
EDTA (pH 7.4) was added to NHS in the preparation of either
Mg2+-EGTA-NHS or EDTA-NHS.
Human leukemia cell lines, P39 and CEM, were provided by the Japanese
Cancer Research Resources Bank. K562 (a chronic myelogenous leukemia
cell line) and Jurkat (a T-cell leukemia cell line) were gifts
from J. P. Atkinson (Washington University) and S. Nagasawa (Hokkaido University), respectively. The cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS) (CSL Ltd.,
Victoria, Australia) in the presence of antibiotics. M. fermentans-infected and M161Ag-expressed leukemia cell lines were denoted (+).
Preparation of MAbs against M161Ag.
MAbs were produced by
the method of Köhler and Milstein (15). M161Ag,
partially purified from P39(+) cell lysates using mouse IgG-Sepharose,
Q-Sepharose, and chromatofocusing columns as described previously
(19), was mixed with TiterMax (CytRx Co., Norcross, Ga.) and
injected subcutaneously into female BALB/c mice once every week for a
total of three times. After 1 week, P39(+) cells (8 × 106) were administered intraperitoneally as a final
booster. Three days later, the spleens were extracted and the cells
were fused with the mouse myeloma cell line NS-1. The supernatants of
hybridomas were screened by Western blotting and protein A rosette
assay using P39(+) and -() cells (19). Clones producing a
MAb that reacted with a 43-kDa protein in P39(+) cells but not in
P39() cells were established by limiting dilution. Three MAbs, MK53, MK5, and MK36, were purified from mouse ascites fluid by ammonium sulfate precipitation followed by protein G-Sepharose (Amersham Pharmacia Biotech).
Flow cytometry.
Cells (106) suspended in 50 µl
of Dulbecco's phosphate-buffered saline (DPBS) containing 0.5% bovine
serum albumin (BSA) and 0.1% NaN3
(BSA-NaN3-DPBS) were mixed with 50 µl of EDTA-plasma and
5 µg of mouse IgG or MAb and incubated for 30 min at 4°C. After
being washed with BSA-NaN3-DPBS, the cells were suspended in 90 µl of BSA-NaN3-DPBS and incubated with FITC-labeled
goat F(ab')2 of anti-mouse IgG at 4°C. After 30 min, the
cells were washed twice with DPBS and fixed with paraformaldehyde. The
samples were analyzed on an EPICS Profile II (Coulter Corp., Hialeah, Fla.). C3 deposition was assessed as described previously
(18). Briefly, 106 cells were incubated with
25% Mg2+-EGTA-NHS or EDTA-NHS for 30 min at 37°C.
After the cells were washed, C3 fragments bound to the cells were
detected with anti-human C3b or C3bi MAb followed by a FITC-labeled
secondary antibody.
Immunoblotting.
Cells (107) were lysed in 200 µl of lysis buffer (1% NP-40, 10 mM EDTA, 25 mM iodoacetoamide,
2 mM phenylmethylsulfonyl fluoride, DPBS) for 20 min at room
temperature. After centrifugation at 100 × g for 10 min, the supernatant was centrifuged again at 200,000 × g for 1 h at 4°C. Aliquots of 50 µl of the supernatant
were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (10% gel) under nonreducing or reducing
conditions. After electrophoresis, the resolved proteins were
transferred onto nitrocellulose sheets. The sheets were then blocked
with 10% skim milk for 1 h at 37°C and then overnight at 4°C
and sequentially incubated with MAb and HRP-conjugated goat anti-mouse
IgG, followed by staining with an ECL kit (Amersham Pharmacia Biotech).
Mycoplasmas grown in the growth medium were centrifuged at
16,000 × g for 30 min, and the cell pellets were
washed twice with PBS and resuspended in 500 µl of PBS. The cell
suspension was sonicated at 20 kHz for 3 min and used as the mycoplasma
cell lysate (31).
Immunoprecipitation.
Cell lysates (50 µl) were precleared
with protein G-Sepharose at 4°C for 1 h and incubated with 5 µg of mouse IgG or MAb at 4°C. After 1 h, protein G-Sepharose
was added to the mixtures and reacted overnight with rotation. The
Sepharose beads were washed three times with 0.2% NP-40, 25 mM
iodoacetoamide, 2 mM phenylmethylsulfonyl fluoride, and PBS (pH 7.4)
and incubated in 1% SDS, 0.2% NP-40, and PBS (pH 7.4) for 2 min at
100°C. Immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting.
Immunostaining.
P39(+) cells (106) were labeled
with 1 µg of mouse IgG or MAb against M161Ag (MK53) in 100 µl of
RPMI supplemented with 10% FCS for 45 min at 4°C. After being washed
twice with medium, the cells were incubated with FITC-conjugated goat
anti-mouse IgG for 45 min at 4°C. The cells were washed three times
with medium and then immediately observed under a fluorescence
microscope. For double-staining of M161Ag and C3 fragments, P39(+)
cells were pretreated with 25% EDTA-NHS or Mg2+-EGTA-NHS
for 20 min at 37°C and then sequentially labeled with MAb against
human C3b (C5G), rhodamine-conjugated secondary antibody (TAGO Inc.,
Burlingame, Calif.), and MK53-FITC-conjugated secondary antibody. For
staining of Mycoplasma DNA, cells were fixed with 1%
glutaraldehyde for 30 min at room temperature. After being washed with
DPBS, the cells were stained with 0.17 mM Hoechst 33258 (Molecular
Probes Inc., Eugene, Oreg.) and subjected to fluorescence microscopy.
M. fermentans PG18, M. fermentans incognitus, and
Mycoplasma pulmonis were grown in a broth medium consisting
of 2.1% PPLO broth base (Difco), 10% horse serum, 0.002% phenol red,
and 0.25% glucose (31). The propagated mycoplasmas
(107 CFU/ml) were washed three times with DPBS and stained
with MAbs against M161Ag followed by rhodamine-conjugated anti-mouse
IgG. For the C3 deposition assay, the mycoplasmas were pretreated with 25% EDTA-NHS or Mg2+-EGTA-NHS for 20 min at 37°C and
then sequentially labeled with MAb against human C3b (C5G) and
rhodamine-conjugated secondary antibody.
Immuno-electron microscopy.
The method for preparing
electron microscopic specimens (for fixation, dehydration, and
embedding with Lowicryl K4M resin) was described previously
(37). The ultrathin sections of P39(+) cells cultured in
RPMI supplemented with 10% FCS or 25% fresh human serum (FS) for 5 days on nickel grids were incubated with MK53 or mouse ascites fluid as
a control. Next, they were reacted with colloidal gold (15-nm
diameter)-conjugated secondary antibody (Amersham Pharmacia Biotech)
and observed in a JEOL 100CX electron microscope operated at 80 kV.
PCR.
P39(+), CEM(+), and Jurkat(+) cells (107)
were washed with RPMI and suspended in 10 ml of RPMI supplemented with
10% FCS, 25% heat-inactivated human serum (HIS), or 25% FS. The
cells were cultured at 37°C in 5% CO2, and the medium
was changed every other day. After 5 days, the cells were washed twice
with RPMI, suspended in 10% FCS-RPMI, and maintained in culture.
Genomic DNAs were isolated from the cells using an Iso Quick nucleic
acid extraction kit (ORCA Research Inc., Chatsworth, Calif.). M161Ag
was amplified using forward (5'-TTGAGTCCTATTGCTGCTATT-3')
and reverse (5'-CACCAAATGCAACAACTCT-3') primers with
Taq-gold (Takara) and 1 µg of DNA template. PCR conditions were
95°C for 10 min followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s for denaturation, annealing,
and extension, respectively. M. fermentans was detected by
PCR using Mycoplasma genus-specific rRNA primers as
described previously (21). The PCR products were
electrophoresed on 1 or 3% agarose gels and stained with
ethidium bromide.
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RESULTS |
Development of MAbs against M161Ag.
We raised three MAbs
against M161Ag, MK53, MK5, and MK36, all of which were of the IgG1(
)
subclass. When used in Western blotting, these MAbs recognized a 43-kDa
molecule in M. fermentans and P39(+) (M. fermentans-infected cell line) cell lysates but not in P39()
(noninfected cell line) cell lysates in a fashion similar to that of
M161, a previously obtained MAb against M161Ag (Fig. 1A and
B). Unlike M161, MK53, MK5, and
MK36 immunoprecipitated the antigen in P39(+) cell lysates,
indicating that these MAbs efficiently reacted with native M161Ag (data
not shown). The immunoreactivity of each MAb to cell surface
M161Ag was then measured by flow cytometry using P39(+) and -()
cells. All MAbs reacted with cell surface M161Ag on P39(+) cells, while
none of the MAbs reacted with P39() cells (Fig. 1C).
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FIG. 1.
Establishment of MAbs against M161Ag. (A) Western blot
of M. fermentans PG18 with MAbs against M161Ag. Ten
microliters of mycoplasma cell lysate (1.0 mg/ml) was applied to each
lane. SDS-PAGE was performed under reducing conditions. Lane 1, Coomassie blue staining of M. fermentans PG18 proteins.
Samples were transblotted onto nitrocellulose sheets and detected with
the indicated MAbs (lanes 2 to 5). Mouse IgG was used as a control
(lane 6). Molecular mass markers are shown to the left. (B) MAbs
recognized the 43-kDa protein in P39(+) cells but not P39() cells.
P39(+) and -() cell lysates were subjected to SDS-PAGE (10% gel)
under reducing or nonreducing conditions and transferred onto
nitrocellulose sheets. Each sheet was blotted with M161, MK53, MK5, or
MK36 and then with HRP-conjugated goat anti-mouse IgG. NR, nonreducing
conditions; R, reducing conditions. The positions of molecular mass
markers are shown on the right. (C) Flow cytometric analysis of M161Ag
on the P39 sublines. Cell surface M161Ag was assessed using flow
cytometry with each MAb followed by a FITC-labeled anti-mouse IgG using
P39(+) and -() cells.
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C3 deposition on M. fermentans-infected cells.
In
human leukemia cell lines infected by M. fermentans,
expression of M161Ag was examined by Western blotting and flow
cytometry using M161 and MK53. In the T-cell lines Jurkat(+) and
CEM(+), M161Ag was detected with MK53 but not with M161, while in the myeloid cell lines, P39(+) and K562(+), M161Ag expression was detected
with both antibodies (Fig. 1C and
2; blotting data not shown). An
isoform-typing study of M161Ag suggested that P39(+) and K562(+) cells
had M161Ag-1 (His139), Jurkat(+) cells had M161Ag-2
(Tyr139), and CEM(+) cells had M161Ag-3
(Tyr139 with Ala285 insertion) (20).
Since M161 MAb reacts with both recombinant M161Ag-1 and M161Ag-2 (M. Matsumoto and T. Seya, unpublished data), M161 epitope may be hidden in
Jurkat(+) and CEM(+) cells. In contrast, MK53 efficiently detected
M161Ag in any infected cells, a reaction profile shared with the other
MAbs, MK5 and MK36 (data not shown).
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FIG. 2.
C3 deposition induced on M. fermentans-infected cell lines. Jurkat(+), CEM(+), K562(+),
P39(+), and P39() cells were analyzed for M161Ag expression and C3
deposition by flow cytometry. C3 deposition was assessed after the
cells were treated with 25% Mg2+-EGTA-NHS or EDTA-NHS at
37°C for 20 min, using anti-human C3b MAb and FITC-labeled secondary
antibody. The levels of complement regulatory proteins, CD46 and CD55,
in each cell line are also shown. Of note, CEM(+) cells lack CD55
expression. The P39() cells (M. fermentans-uninfected cell
line) do not express M161Ag and do not induce C3 deposition.
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Homologous C3 deposition was induced on these cells but not on P39()
cells after treatment with Mg2+-EGTA-NHS, indicating that
the cells infected by M. fermentans are targets for human
complement via the alternative pathway regardless of their M161Ag
isoforms. However, the extent of C3 deposition varied among cells with
equivalent levels of M161Ag, and there was no relation between the
levels of complement inhibitors (CD46 and CD55) and C3 deposition (Fig.
2). It is possible that C3-binding molecules expressed on the affected
cells and/or mycoplasma may participate in C3 deposition. The
C3-bearing cells were not lysed because of the presence of CD59, which
protects cells from homologous complement-mediated lysis
(16).
We next analyzed the relationship between M161Ag expression on M. fermentans and complement activation via the alternative pathway
in the absence of host cells. M161Ag was shown to be expressed on the
cell surface of M. fermentans PG18 (Fig.
3B) and incognitus (data not shown) by
immunostaining using MK53. After treatment with 25%
Mg2+-EGTA-NHS, C3 fragments bound directly to
the mycoplasmas (Fig. 3D).
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FIG. 3.
Immunostaining of M161Ag and C3 fragments deposited on
M. fermentans. M. fermentans PG18 was reacted with mouse
IgG1 (a and A) or MK53 (b and B) and then with rhodamine-conjugated
secondary antibody. For the C3 deposition assay, the organisms were
incubated with 25% EGTA-NHS (c, C, d, and D) or 25% EDTA-NHS (e and
E). After the cells were washed, C3 fragments deposited on the
organisms were stained with anti-human C3b MAb and rhodamine-labeled
secondary antibody (D and E). Mouse IgG1 was used as a control antibody
(C). The mycoplasmas were observed under a phase-contrast microscope (a
to e) or a light fluorescence microscope (A to E; the same field as in
panels a to e). Magnification, ×1,000.
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Clearance of M. fermentans by human complement.
To
understand the role of complement in defense against M. fermentans infection in vivo, infected cells [P39(+) and
Jurkat(+)] were cultured in RPMI in the presence of either 25% FS,
25% HIS, or 10% FCS. After 1 day, M161Ag was completely depleted from
the cells cultured in FS (Fig. 4A). HIS did not affect M161Ag
expression in P39(+) cells. In Jurkat(+) cells, the surface M161Ag
level was considerably decreased in HIS culture, but the total protein expression levels were unaffected (Fig.
4B). Immunostaining with MK53 confirmed
the absence of M161Ag on P39(+) cells cultured in FS (Fig.
5G). Interestingly, the decrease in
M161Ag expression was associated with destruction of M. fermentans by complement in culture. Membrane-attached organisms
could not be stained with Hoechst 33258 (Fig. 5E), which was further
demonstrated by immuno-electron microscopy (Fig.
6B).
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FIG. 4.
Disappearance of M161Ag from M. fermentans-infected cells after treatment with FS. (A) Flow
cytometric analysis of M161Ag expression. P39(+) and Jurkat(+) cells
were cultured in RPMI supplemented with 10% FCS, 25% HIS, or 25% FS
for 5 days, and the medium was changed every other day. After 5 days,
the cells were washed, resuspended in 10% FCS-RPMI, and maintained in
culture. The cells were harvested at each indicated time point, and
M161Ag expression was assessed by flow cytometry using MK53. As a
control, nonimmune mouse IgG was used. (B) Immunoblotting analysis of
M161Ag. The cells were cultured in 10% FCS-RPMI (lanes 1), 25%
HIS-RPMI (lanes 2), or 25% FS-RPMI (lanes 3) for 1 day and lysed in
the lysis buffer as described in Materials and Methods. The cell
lysates were analyzed by SDS-PAGE followed by immunoblotting with MK53.
Cells in which M161Ag was diminished were transferred to 10% FCS-RPMI
and maintained in culture for 8 days (lanes 4) or 15 days (lanes 5).
M161Ag expression was assessed as described above. The arrowhead
indicates M161Ag.
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FIG. 5.
Double staining of M161Ag and C3 fragments on P39(+)
cells. After treatment of P39(+) cells with Mg2+-EGTA-NHS,
M161Ag and C3 fragments on the same cells were sequentially stained
with MK53-FITC-conjugated anti-mouse IgG and anti-C3b
MAb-rhodamine-conjugated anti-mouse IgG. M. fermentans cells
attached to the membranes of P39(+) cells were stained with Hoechst
33258. (Left) P39(+) cells cultured in 10% FCS-RPMI. (Center) P39(+)
cells cultured in 25% FS-RPMI for 1 day. (Right) P39() cells
cultured in 10% FCS-RPMI. (a to j) Phase-contrast photomicrographs of
P39(+) and -() cells. (A, E, and I) Hoechst staining. (B and F)
Immunostaining with control mouse IgG. (C, G, and J) Immunostaining of
M161Ag with MK53. (D and H) Immunostaining of C3 fragments.
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FIG. 6.
Clearance of M. fermentans by complement in
P39(+) cell culture. P39(+) cells cultured in 10% FCS-RPMI or 25%
FS-RPMI for 5 days were subjected to immuno-electron microscopy. (A)
Electron micrograph of P39(+) cells cultured in 10% FCS-RPMI. (B)
Electron micrograph of P39(+) cells cultured in 25% FS-RPMI. (C)
Postembedding immuno-electron micrograph of P39(+) cells cultured in
10% FCS-RPMI. (D) Magnification (10-fold) of the region indicated
with an arrow in panel C. The mycoplasma particles are decorated with
immunogold reacting with M161Ag.
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These mycoplasma-cleared cells were not targets for human complement
(Fig. 5H). On the other hand, cells infected by M. fermentans carrying M161Ag were tagged with C3. Double staining of
M161Ag and C3 fragments (Fig. 5C and D) and a previous study using
blocking antibodies to complement inhibitors (18) showed
that C3 fragments were deposited on the organism itself and on further
host cell membrane around the organism.
Persistent infection of M. fermentans in human
cells.
Next, we studied whether M. fermentans
persistently infected human cells by using an in vitro model system.
P39(+) or Jurkat(+) cells cultured in FS for 5 days were washed,
resuspended in RPMI supplemented with 10% FCS, and maintained in
culture. At timed intervals, M161Ag expression was analyzed by flow
cytometry, Western blotting, and PCR using genomic DNA isolated from
cells. At the same time, in order to detect the organisms, genomic
sequences of M. fermentans rRNA were amplified by PCR. As
shown in Fig. 4, M161Ag was not expressed on day 8 but appeared on day
15. Interestingly, both M161Ag and M. fermentans were
detected by PCR in the P39(+) and Jurkat(+) cells with diminished
M161Ag cultured in FS for 5 days (Fig. 7, lane
3), indicating the presence of the
organism at low titer in these cells. Both were amplified to a greater degree after the cells were transferred into 10% FCS-RPMI and cultured for 8 days (Fig. 7, lane 6), whereas the M161Ag protein could
not be detected by either flow cytometry or immunoblotting (Fig. 4). On
day 15, M161Ag protein expression was detectable by flow cytometry and
Western blotting, suggesting that M. fermentans escaped from
complement attack, regrew, and expressed detectable M161Ag after the
removal of complement.
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FIG. 7.
PCR analysis of M161Ag. Genomic DNA was isolated from
P39(+) cells (left) or Jurkat(+) cells (right). M161Ag (top), genomic
sequence of M. fermentans rRNA (center), and -actin
(bottom) were amplified using specific primers. The solid arrowheads
indicate M161Ag, and the open arrowheads indicate rRNA of M. fermentans. To confirm the mycoplasma species amplified by PCR,
nested PCR was performed (data not shown). Jurkat(+) cells have a
correct-size PCR fragment in addition to a smaller PCR fragment, which
overlap (center, lanes 6). M, DNA marker; N, negative control. Lanes 1, 2, and 3, cells cultured for 5 days in FCS, HIS, and FS, respectively;
lanes 4, 5, and 6, cells cultured for 5 days in FCS, HIS, or FS were
transferred to FCS and cultured for 8 days; lanes 7, 8, and 9, cells
cultured for 5 days in FCS, HIS, or FS were transferred to FCS and
cultured for 15 days.
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We then performed immuno-electron microscopy using MAb against M161Ag
(MK53) to detect intracellular organisms. M. fermentans was
detected in close proximity to P39(+) cells and also intracellularly at
low frequency. Both extra- and intracellular mycoplasma particles were
nonuniformly decorated with immunogold, which recognized M161Ag
(Fig. 6 and 8).
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FIG. 8.
Immuno-electron micrograph of M. fermentans
in cultured P39(+) cells. Immunogold staining shows extra- and
intracellular M. fermentans. An intracellular mycoplasma
particle decorated with 15-nm-diameter immunogold is indicated by the
arrow. The arrowhead indicates a representative extracellular
mycoplasma. Bar = 500 nm.
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DISCUSSION |
In this study, we focused on the role of complement in defense
against M. fermentans infection in vivo by developing MAbs against the mycoplasma lipoprotein M161Ag. The findings from this study
can be summarized as follows. (i) MAbs against M161Ag are useful tools
to detect M. fermentans. (ii) The cells infected by M. fermentans were targeted by human complement via the alternative pathway, and homologous C3 deposition was rapidly induced on the cells.
(iii) M. fermentans was cleared from the surface of infected cells by human complement, but low-grade infection persisted in human
tumor cell lines.
As previously reported, M161Ag purified from P39(+) cell lysates
activates human complement via the alternative pathway and allows the
deposition of C3 fragments on itself (19), which has been
proven with the use of a recombinant protein (M. Nishiguchi, M. Matsumoto, and T. Seya, unpublished data). Our data revealed that
M161Ag could induce C3 deposition not only on mycoplasma particles but
also on mycoplasma-infected cells. Mycoplasma lipoproteins are known to
undergo rapid phase and size variation (39). Although the
possibility that the level of M161Ag reflects its expression states
could not be excluded, most mycoplasma particles and cell-attached organisms expressed M161Ag (Fig. 3 and 6). Recently, Calcutt et al.
(4) reported the differential posttranslational processing and intraspecies variation for a major surface lipoprotein of M. fermentans (MALP-404) which is identical to our M161Ag
(21). They also demonstrated that most M. fermentans strains express a 41-kDa protein (4).
A previous report indicated that several species of mycoplasmas could
activate complement and be rapidly removed from contaminated cells by
the addition of fresh animal serum (42). Unlike the results
presented here, they showed complete removal of mycoplasmas by the
addition of fresh serum and activation of the classical complement
pathway. In our study, low-grade infection persisted in the cells even
after treatment with FS (Fig. 4 and 7). Immuno-electron microscopic
analysis suggested the presence of intracellular M. fermentans, which could explain the persistent infection by the organism. However, the possibility still remains that complement treatment selects organisms that do not express M161Ag and that a few
adapted organisms may still be replicating outside the human tumor
cells. This possibility is presently under investigation.
Among the mycoplasma strains, M. fermentans may be a unique
strain which possesses the alternative complement activator, invades human cells (38), and stimulates monocytes/macrophages.
Although our results showed the intracellular persistence of this
organism in human tumor cell lines, whether this also occurs in normal peripheral blood cells remains to be examined. Preferential target cells for M. fermentans invasion may exist in the presence
of human serum, since cell-surface M161Ag expression was largely diminished in Jurkat(+) cells by HIS but not in P39(+) cells (Fig. 4A).
Our findings, together with previous observations, demonstrated that in
the bloodstream, both M. fermentans-infected cells and the
organism itself are rapidly tagged with C3 as nonself cells and release
the C5a chemotactic factor (18). The C3b and C3bi molecules
on the cells enhance the phagocytic activity of complement receptor
(CR1 and CR3)-bearing cells and facilitate the elimination of nonself
cells by phagocytes (5, 36). In addition to the destruction
of cell surface M. fermentans, infected cells may be cleared
from the host (27, 41). However, all of the M. fermentans cells (PG18) were not killed by complement during 1 or
2 h of incubation in FS at 37°C (data not shown), suggesting
that the organisms may escape complement attack by their rapid invasion
of the cells and tissues and persistently infect host cells.
As M161Ag and related products elicit innate immune responses, such as
inflammatory cytokine production and nitric oxide synthesis, by
monocytes/macrophages at low concentrations similar to
lipopolysaccharide (21, 22, 26), liberated M161Ag serves
locally as a potent modulator of the host immune system. Recently,
Toll-like receptors (TLRs), which provoke innate immune responses, were
identified in humans (23, 29). TLR type 2 was identified as
a receptor for lipopolysaccharide of gram-negative bacteria (14,
40) and peptidoglycan and lipoteichoic acid of gram-positive
bacteria (33). These receptors are thought to recognize
pathogen-associated molecular pattern (24). Interestingly, a
characteristic motif in M161Ag structure is shared with specific
lipoproteins in a variety of bacterial genera, including
Borrelia, Listeria, Mycoplasma, and
Treponema, all of which possess immune modulatory activity (22). Thus, these lipoproteins may be akin to
pathogen-associated molecular pattern recognized by TLRs. The most
important structural feature of M161Ag for complement or
monocyte activation, as well as the identification of its receptor, are
under investigation in our laboratory using deletion mutants of M161Ag
and MAbs as probes.
We are grateful to H. Akedo (Osaka Medical Center) for support
of this work and to M. Nomura, N. A. Begum, S. Tsuji, M. Kurita-Taniguchi, and K. Shida in our laboratory for thoughtful
discussions. Thanks are also due to T. Kinoshita and K. Iida for
providing MAbs.
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Abstract
Introduction
