Microbiol.
Monoclonal Antibody fermentans-Specific
Immunol., 44(8), 695-702, 2000
against Mycoplasma Aminoglycoglycerolipid
Kazuhiro Matsuda*,4 4, Jin-Liang Li1, Shizuko Masaki Saito4, and Naoki Yamamoto'
Ichinose2,
Ryo Harasawa3,
1Department
of Microbiology, and 'Laboratory for Electron Microscopy; Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan, 3Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8654, Japan, and 4Virology and Glycobiology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan Received
November
18, 1999; in revised
form, May
16,2000.
Acceped
May
17,2000
Abstract: Previously, we reported that Mycoplasma fermentans has specific antigens (phosphocholinecontaining glycoglycerolipids: GGPL-I and GGPL-III) and discussed the possibility of their pathogenic role. In this paper, we report the characterization of a monoclonal antibody (MF-III-1) specific to GGPL-III (phosphocholine-containing aminoglycoglycerolipid) using methods of electron microscopy, immunofluorescence cell surface staining, laser scanning microscopy, immunoelectron microscopy, and thin-layer chromatography immunostaining. The MF-III-1 antibody specifically recognized M. fermentans attached to the surface of HTLV-I-infectedhuman helper T-cells,and it did not cross-react with other lipids nor with human T-cell antigens. Since MF-III-1 distinguishes GGPL-III from GGPL-I, the binding site may include a serinol (2-amino-1,3-propanediol)residue of GGPL-III. MF-III-1 is useful for the in vitro study of M. fermentans, and may also be useful as a tool for the study of the involvement of M. fermentans in human diseases. Key words: Monoclonal antibody, HTLV-I-infected noelectron microscopy
human helper T-cell, Mycoplasma fermentans,
Mycoplasma fermentans is suspected as a pathogen of rheumatoid arthritis (RA) and other human diseases (1, 4, 16, 27-29, 34, 35). M. fermentans is frequently isolated from the synovial fluid of rheumatoid arthritis patients (27, 29, 34, 35) and exhibits agglutinating activity toward erythrocytes. These observations suggest the possibility that M. fermentans has a role in the pathogenesis of rheumatoid arthritis. Recently, interest in M. fermentans has increased because it is frequently isolated from, or detected in, various tissues and blood of AIDS patients (1, 4, 12, 16) and has been implicated in the disruption of the immune system. M. fermentans also has high affinity for HTLV-I-infected T-cells (26). Autoimmune-like diseases are often seen in HTLV-Iinfected patients (11), therefore, it is suspected that M. fermentans may have some pathogenic' role in these patients. This, however, is still obscure because of the difficulty in detecting M. fermentans clinically. Previously, we reported that M. fermentans contained phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) (17), and determined the com-
plete
structures
of
GGPL-I
and
Immu-
GGPL-III
as
phocholine-ƒ¿-glucopyranosyl-(1'-3)-1, erol
and
respectively
structures
of
GGPL-I main
and
GGPL-III
lipid
that
(CRP)
of
the
increases
19,
M.
in
of
to a phosphocholine-containing
to
form
system the
a
and
chemical in Fig.
and
(2,
20).
inflam-
has
the
and
the
ability moi-
complement
cell
death.
Thus,
M. fermentans
have
the
and
have
a role
complexes
It has pro-
infection,
activates
might
are
C-reactive
CRP
1.
and
glycolipid
that
of
The
human to
9),
inflammation
antigens
immune
(8,
complex
induces
GGPL
form
damage
-
shown expressed
response
to ety
are
fermentans
amount
or
2-diacyl-sn
22-25).
specifically
mation bind
tissue
'-3)-1,
GGPL-III
are
antigens
reported
tein
(18,
GGPL-I
and
been
2 "-aminodihydroxypropane-
'-2-glucopyranosyl-(1
glycerol,
the
2-diacyl-sn-glyc-
1 "-phosphocholine,
3 "-phospho-6
6-O-phos-
ability in
to the
pathogenesis. In ization
this
paper, of
the
we mouse
report MoAb
establishment (MF-III-1)
and that
characterspecifically
Abbreviations: FBS, fetal bovine serum; GGPL, phosphocholine-containing glycoglycerolipid; MoAb, monoclonal antibody; PBS, phosphate-buffered saline; TLC, thin-layer chromatography. The abbreviations for gangliosides (GM, GM], GD1a and GD1b) are according to Svennerholm's nomenclature
*Address correspondence to Dr . Kazuhiro Matsuda, Virology and Glycobiology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Fax: 03-3543-2181. E-mail:
[email protected]
(31). 695
696
K. MATSUDA
Fig. 1. Structure of GGPL-III, GGPL-I, phosphatidylcholine,
ET AL
platelet-activating
recognizes GGPL-III. The results of electron microscopy, cell-surface staining and immune electron microscopic and thin-layer chromatography immunostaining analyses are presented. Also, GGPL-III availability in the in vivo or in vitro studies of M. fermentans is discussed.
factor (PAF) and sphingomyelin.
MT-4 cells, and the purified GGPL-III have been described previously (17, 19, 22, 23). Lipids of various mycoplasma strains were extracted by the method of Bligh and Dyer (3).
Immunization of mice and hybridoma preparation. BALB/c
mice
purified
Materials
and Methods
at
37
C
culture.
in
a PPLO
Mich.,
U.S.A.)
serum
(FBS),
McLean,
5%
concentration) by
washed
red.
was
with
fetal
dextrose
mycoplasmas
10,000 •~
g
phosphate-buffered
and
for
(final
saline
har-
min,
and
(PBS),
pH
7.2. fermentans
obtained and
T.
the
same
had
been Lipid
fermentans
from
PG18 G.H.
Sasaki
Cassell
(Tokyo),
strain
as
cultured extraction GGPL
strains
(#1)
(Birmingham,
respectively. PG
strain
in
our
laboratories
strain
(#2) Ala.,
PG18
the
and
and
(#2),
but for
The
21,
the
was
PG18
(#3)
1 year.
Lipids
of
and
fermentans-infected
RPMI
M.
The FBS,
harvested and
detoxified
GGPL-III
Research
from
Immuno
at each
Strepto-
Chem
(20
pig)
injected
On
injections
according
Research
was
immunization. s.c.
Inc., from
Chem
endotoxin
(Ribi
PBS,
Tesque dimycolate
Immuno
performed
TLC
of
days
14
GGPL-III.
to the
for
staining.
Kohler
the
incubated
used
for
using
second
were
and
the
lipids
supplemented
were with
and
1 00
100
of
2
10%
Um]
three ill
of
M.
MT-4
for
with
washed
first
screening.
cultured
streptomycin cells
was
M. fermentans-infected
104/m1)
medium
( 1 4)
immunostaining
used
(30 •~
of
with
(Nacalai
GGPL-III
was
1640
emulsified
with
(10).
and
(17)
immunized
trehalose
received
using
100 ƒÊg/m1 the
mice
Cell-surface cells
of
R595
was
fermentans
of
of
method
ELISA
was
(Ribi
(s.c.)
all
Milstein
tg
emulsified
Hybridization
U.S.A.)
(#3)
purification. M.
were
mg
minnesota
screening, M.
50
subcutaneously and
were 30
50
were
adjuvant
phlei
and
coccus Inc.).
penicillin,
GGPL-III
Japan),
Inc.),
bovine
female)
incomplete
Mycobacterium
Laboratories,
of
percent
The at
grown Detroit,
(v/v)
(Flow Uml
One
added.
centrifugation
twice
10%
extract 1,000
were
Laboratories,
with
yeast
phenol
strains
(Difco
U.S.A.),
(w/v)
vested
broth
supplemented
Va.,
0.002%
Mycoplasma
weeks,
GGPL-III.
Freund's Kyoto,
Mycoplasma
(4
times
MF-III-1
of
days (v/v)
in FBS,
penicillin. in PBS-1% hybridoma
a
MONOCLONAL
ANTIBODY
culture supernatant liquid ( X 10) at 37 C for 1 hr. The cells were then washed three times in PBS-1% FBS, and incubated with 100 ill of fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG (heavy and light chains) polyclonal antibodies (Bethyl Laboratories Inc., Montgomery, Ala., U.S.A.) ( X 200) at 37 C for 1 hr. The cells were then washed three times with PBS-1% FBS. Localization of GGPL-III antigen was detected by fluorescence microscopy or by a laser scanning microscope, LSM-GB200 (Olympus, Tokyo). Electron microscopy. M. fermentans-infected MT-4 cells were layered over a glass cover slide pretreated with poly-L-lysine (50 14/m1) for 2 hr at 37 C, and washed 3 times with PBS. The cells were fixed for 2 hr in 2.5% glutaraldehyde in 0.2 M PBS, stained with 1% 050, in 0.2MPBS at 4 C for 2 hr. Cells were dehydrated by exposure to increasing concentrations of ethanol. For the preparation of samples for scanning electron microscopy, the cells were dried at a critical point and sputter-coated with a thin layer of gold palladium. Samples were examined in a Hitachi S-700 (Hitachinaka, Japan) at an accelerating voltage of 15 kV. Micrographs were recorded on a Polaroid type 55 positive/negative film. For the preparation of samples for transmitted immune electron microscopy, the cells were processed for conventional Epon embedding. Ultra-thin sections were cut and transferred to nickel grids. The sections were then treated with 0.5% H20, in water at 25 C for 5 min. The grids were incubated with PBS-1% FBS for 30 min to block the non-specific attachment of antibodies to residual glutaraldehyde. After washing three times with PBS-1% FBS, samples were reacted with anti-GGPL-III MoAb or a control solution at 4 C overnight and then washed three times with PBS-1% FBS. The grids were incubated with a gold-conjugated goat anti-mouse IgG and IgM antibody (the gold particle size was approximately 20 nm) (Biocell Research Laboratories, Cardiff, U.K.) ( X 10). The samples were washed extensively with PBS-1% FBS three times and finally washed with distilled water. The sections were stained with saturated aqueous uranyl acetate for 10 min and washed with distilled water. The sections were then 'viewed with a Hitachi H-600 electron microscope at an accelerating voltage of 75 kV. Thin-layer chromatography (TLC). The developing solvent was a mixture of chloroform:methano1:0.2% aqueous CaC1, (50:45: 10, by volume). Lipids were separated on a high-performance TLC (HPTLC)-plate (Merck, Darmstadt, Germany). For the detection of lipids, the following spray reagents were used: orcinol reagent (30) for glycolipids and Dittmer's reagent (5) for
AGAINST
GGPL-III
697
phospholipids. Immunostaining of lipids on an HPTLC-plate was performed as described previously (21). Horseradish peroxidase (HRPO)-conjugated goat anti-mouse IgG (heavy and light chains) (Bethyl Laboratories Inc.) was used for the second antibody. An antigen band was visualized with a Konica immunostaining HRP kit (Konica Co., Ltd., Tokyo). Results
and Discussion
We established a monoclonal antibody MF-III-1 against the phosphocholine-containing aminoglycoglycerolipid (GGPL-III) purified from M. fermentans. To demonstrate the specificity and usefulness of the MF-III1, we carried out the following studies. The scanning electron microscopy shows the M. fermentans attached to the surface of HTLV-I-infected human helper T-cells (MT-4 cells) (Fig. 2A). The attached microorganisms were round, rod, or four-leafclover shaped (Fig. 2B). However, it is difficult to distinguish M. fermentans from the microvilli of the MT-4 cell. Then, the MF-III-1 was used to stain the MT-4 cells (Fig. 3). The staining intensity of each M. fermentansattached cell varied (Fig. 3C), and this difference may be attributed to the amount of M. fermentans attached to the MT-4 cell surface. M. fermentans-infected MT-4 cells were not stained by the preimmune mouse IgG fraction (Fig. 3B). M. fermentans-free MT-4 cells were not stained (data not shown). The location of the GGPL-III antigen on the cell was analyzed using laser scanning microscopy, which showed that GGPL-III antigens were stained along the cell surface (Fig. 3C). Scattered or accumulated M. fermentans bodies can be seen. On the right side, the localization of M. fermentans is shown schematically. The result of confocal microscopy suggested that M. fermentans were attached to the MT-4 cell surface. To confirm this, immunoelectron microscopy was carried out. This MF-III-1 antibody could also detect M. fermentans (Fig. 3, D and E). The gold particles show the existence of GGPL-III antigens. The gold particles accumulated on the M. fermentans attached to the surface of an MT-4 cell. However, there are no particles on the MT-4 cell cytoplasm and nucleus. The result clearly shows that the MF-III-1 antibody recognizes M. fermentans specifically. Figure 3, C and D shows a different area. Figure 3C shows the locus where the M. fermentans membrane appears to attach to the MT-4 cell membrane. Figure 3D may correspond to the clover or rod shape (detail in Fig. 2B). A few gold particles could be seen on the surfaces of MT-4 cells. We investigated a chemical structure specific to the
K. MATSUDA
698
A
FT AL
B
Fig. 2. Scanning electron microscopy (SEM) of a M.fermentans-infected MT-4 cell. (A) Many mycoplasma attached to the surface of MT-4 cells. Bar= 1 pm. (B) Microorganisms with round, rod or four-leaf-clover shapes can be seen. Bar=l pm.
GGPL-III by using TLC-immunostaining methods to determine the specificity of the MF-III-1 antibody. Purified GGPL-III was developed on TLC and stained as phospholipids and glycolipids (Fig. 4A). The phospholipid (lane 1) and glycolipid (lane 3) patterns of the M. fermentans GGPL strain show that GGPL-I and GGPLIII are the main phospholipids and glycolipids. The GGPL-III band could not be detected by Dittmer's staining (Fig. 4D, lane 4). However, MF-III-1 could detect GGPL-III (Fig. 4E, lane 4). GGPL-III was not detected in M. arthritidis (Fig. 4E, lane 8). A few faint bands that were also detected by MF-III-1 may have the same antigenic determinant as GGPL-III (GGPL-III-related antigens). The lipid extract (Fig. 4B, lane 1 and 3) and the purified GGPL-III (Fig. 4B, lane 2 and 4) were used as antigens. The MF-III-1 antibody recognized the GGPLIII band and purified GGPL-III (Fig. 4B, lane 1 and 2). However, normal mouse serum did not react with any lipids of M. fermentans (Fig. 4B, lane 3 and 4). To determine whether the MF-III-1 MoAb crossreacted with human cell lipids, we analyzed the neutral lipid fraction (containing phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, etc.) and the acidic lipid fraction (containing phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, etc.) of M. fermentans-free MT-4 cells. The acidic lipid fraction also contained glycolipids including GM1a,GM2 and GM, (21). They did not react with MF-III-1 (data not shown). Then, the neutral (Fig. 4C, lane 1 and 3) and acidic (Fig. 4C, lane 2 and 4) lipids of M. fermentans-infected
MT-4 cells were analyzed. The phospholipid components were visualized by Dittmer's staining (Fig. 4C, lanes 1 and 2), and MF-III-1 specifically recognized the GGPL-III band (Fig. 4C, lane 3). Trace bands were also recognized (Fig. 4C, lane 4). We have previously determined the structure of GGPL-III (18, 23, 25). Although GGPL-III has a structural similarity to phosphatidylcholine, sphingomyelin, platelet-activating factor (PAF) and GGPL-I (Fig. 1), the MF-III-1 MoAb does not cross-react with these compounds or with the glycolipids of human helper Tcells. All of the above contain phosphorylcholine residues which have hydrophilic head groups, while the ceramide or diacylglycerol (DAG) groups form hydrophobic tail groups. Thus, the amino group of GGPL-III is important because it is recognized by MFIII-1 MoAb. Because the recognition of MF-III-1 is highly specific, the minor bands recognized by the MFIII-1 MoAb may have the same epitope as GGPL-III. Recently, M. fermentans has been shown both to have oncogenic activity (32, 37) and to activate the hypothalamo-pituitary adrenal axis (33). This microorganism contains a large amount of unique phosphocholine-containing glycophospholipids (20). We call attention to its choline residue which has the potential to interact with cholinergic neurons or with the choline metabolic pathway (e.g., sphingomyelin pathway) and act as a pathogen. M. fermentans may also be involved in the pathogenesis of AIDS dementia (36). MF-III-1 may be a useful tool for confirming our hypothesis that the presence of this
MONOCLONAL
ANTIBODY
AGAINST
699
GGPL-III
A
B
C
E
D
Fig.
3. Immunofluorescence
1 MoAb
(A)
and
ic representation in
M. fermentans-infected
a normal of the
staining mouse manner MT-4
of M. IgG
of
the cells
fermentans.
fraction
(B).
attachment (D
and
Optical Confocal of M.
E).
Bar
=
microscopy laser
fermentans 5 ƒÊm
to (A,
microorganism has a role in these human diseases. Phosphatidylcholine, sphingomyelin and platelet-activating factor (PAF) have important roles in cell signal transduction. Because of their molecular similarity, it is
of
microscopic
B
MT-4
and
M.
fermentans-infected
image
C).
of an
cell
(right)
Bar
=0.5 ƒÊm
MT-4
intensively
(C).
stained
Immune (D
and
electron
cells cell
stained (left)
microscopy
with and
the of
MF-IIIschematGGPL-III
E).
postulated that GGPLs have biological functions affecting signaling systems. Previously, we reported that GGPLs have strong antigenicity and species-specific antigens of this mycoplasma (20) and that they are active
700
K. MATSUDA
ET AL
D
A
E B
C
Fig. 4. (A) Thin-layer chromatography of lipids from M.fermentans GGPL strain (lanes 1 and 3) and purified GGPL-III (lanes 2 and 4). Development was performed with a mixture of chloroform:methano1:0.2%CaCl2(55:45:I0, by volume). Developed lipids were visualized with Dittmer's (lanes I and 2), and orcinol reagent (lanes 3 and 4). (B) TLC immunostaining of M. fermentans lipids using an antiGGPL-III monoclonal antibody (MF-III-1). Lipids from M.fermentans GGPL strain (lanes I and 3) and purified GGPL-III (lanes 2 and 4) were developed with a mixture of chloroform:methanol:0.2% CaC12(55:45:10, by volume). Lipids were detected with anti-GGPLIII MoAb (MF-III- 1) (lanes I and 2) and non-immunized rabbit serum (lanes 3 and 4). (C) TLC immunostaining of M.fermentans-infected human helper T-cell lipids using anti-GGPL-III monoclonal antibody (MF-III-1). Neutral (lanes 1 and 3) and acidic (lanes 2 and 4) lipids extracted from M.fermentans-infected MT-4 cells were developed with a mixture of chloroform:methanol:0.2% CaCl2 (55:45:10, by volume). Developed lipids were visualized with Dittmer's (lanes 1 and 2) and TLC immunostaining using the MF-III-1 antibody (lanes 3 and 4). (D) TLC of lipids of various mycoplasma strains. Lipids were extracted from: M. fennentans GGPL strain (lane I), M.fermentans incognitus strain (lane 2), M.,fermentans F7 strain (lane 3), M.fermentans PG18 strain (#1) (lane 4), M.fennentans PG18 (#2) (lane 5), M. fermentans PGI8 (#3) (lane 6), M. fermentans F1 strain (lane 7) and M. arthritidis strain (lane 8). Developed lipids were visualized by Dittmer staining. (E) TLC immunostaining of lipids of various mycoplasma strains using MF-III-1 antibody. Lipids were extracted from: M. fennentans GGPL strain (lane 1),M.fermentans incognitus strain (lane 2), M.fermentans F7 strain (lane 3), M.fermentans PG18 strain (#1) (lane 4), M.fermentans PG18 (#2) (lane 5), M.fermentans PG18 (#3) (lane 6), M.fermentans Fl strain (lane 7), and M. arthritidis strain (lane 8).
in modulating interleukin 6 production. We have evidence that lipids of M. fermentans can induce HIV from latently HIV-infected macrophages. It was found that MF-III-1 antibody can recognize and neutralize this activity (paper in preparation). M. fermentans has also been reported to be capable of fusing with T-cell and peripheral lymphocytes (7, 13, 15). Our hypothesis for pathogenesis is as follows: M. fermentans invades the lesion and contributes to the acute inflammation. The GGPLs and other antigens may form immune complexes which persist and continue to stimulate the immune response resulting in more tissue damage. RNA or DNA are digested with RNAase or DNAase; therefore, PCR or isolation methods cannot be used to detect these microorganisms. There is also a report that primers used for the detection of M. fermentans in AIDS was not specific (6). On the other hand, GGPL-III antigen is stable and specific to GGPL-III structure. As a result, the
MF-III-1 MoAb may be more useful for the histochemical or immunoelectron microscopic analysis of pathological tissues. MF-III-1 may be an important tool in helping to determine the mechanisms underlying these processes. The fact that the structure of the GGPL-III antigen is well characterized is an advantage for the further study of molecular mechanisms. We would like to thank Tsuguo Sasaki (Department of Safety Research on Biologics, National Institute of Health, Tokyo) for M. fermentans Fl and F7 strains and Shyh-Ching Lo (Division of Molecular Pathobiology, Department of Infectious and Parasitic Disease Pathology, Armed Forces Institute of Pathology, Washington, D. C.) for the M. fermentans incognitus strain. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, Japan, 3rd Field of Science and a grant from Seikagaku Co., Ltd.
MONOCLONAL
ANTIBODY
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