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ELSEVIER
FEM.5 Immunology
and Medical Microbiology
12 (1995) 97-I
I2
IMMUNOLOGY AND
MEDICAL
MlCROE3lOLOGY
Cytokine-inducing glycolipids in the lipoteichoic acid fraction
from Enterococcus hirae ATCC 9790
Yasuo Suda a.*, Hidehito Tochio a, Kazuhisa Kawano a, Haruhiko Takada b,
Takeshi Yoshida ‘, Shozo Kotani d, Shoichi Kusumoto a
a Department of Chemistn,
’ Department
Facula of Science, Osaka University. I-l Machikane~ama, Townaka. Osaka 560. Japan
of Microbiology and Immunology, Kagoshima Universiv Dental School. Kagoshima X90, Japan
’ Toky Institute for Immunophannacology
Inc., Toshima-ku. Tok,vo 171, Japan
Osaka College of Medical Technology, Kita-ku. Osaka 530. Japan
Received 2 I April 1995; revised 3 July 1995; accepted 4 July 1995
Abstract
Five high molecular weight glycolipids capable of stimulating human peripheral whole-blood cell cultures to cause
interleukin 6 (IL-6) and tumor necrosis factor (TNF&
induction were isolated from one of the lipoteichoic acid fractions
(LTA-2) extracted from Enr~rococclas hirae ATCC 9790 (Tsutsui et al., (1991) FEMS Microbial. Immunol. 76, 21 I-21 8)
by a combination of hydrophobic interaction and anion-exchange chromatographies.
This purification procedure resulted in a
remarkable increase in the cytokine-inducing
activities on the weight basis of isolated glycolipids (a maximum of 36- and
17-fold increases of IL-6 and TNF-(w induction, respectively). The total yield of these bioactive glycolipids amounted to 6
wt% of the parent LTA-2 fraction, while the recovery rate in terms of the cytokine-inducing
activities was estimated to be
sufficient. The chemical composition
and the profile, using SDS-PAGE, revealed that all of the isolated bioactive
components were high molecular weight glycolipids, which were distinct from each other and from the parent LTA-2
fraction. These findings suggest that the IL-6 and TNF-o-inducing
activities previously noted in the parent LTA-2 fraction
are not attributable to a chemical entity, the structure of which had been proposed elsewhere (Fischer, W. (1990) in
Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M. ed.) pp. 123-234, Plenum Press, New York). but to the
other high molecular weight glycolipids described here.
Keywords:
hirae
Lipoteichoic
acid: Glycolipid;
Cytokine-inducing
activity; Tumor necrosis factor-a:
1. Introduction
A variety of compounds that can modulate host
defence functions
(biological
response modifiers,
BRMs) are located in the surface layers of bacterial
cells [Il. Representatives
are endotoxic lipopolysac-
* Corresponding author. Tel: + 81-6-850-5391;
850-5419:
Fax:
+ 81-6-
E-mail: f61425a@center,osaka-u.ac.jp
092%8244/95/$09.50
0 1995 Federation
SD! 0928-8244(95)00055-O
of European
Microbiological
Interleukin
6: Fractionation:
Entemcoccrrs
chat-ides (LPS) [2] from Gram-negative
bacteria and
muramyl peptide [3] which is a structural unit of the
cell wall peptidoglycan of most bacteria, irrespective
of Gram stainability.
Lipoteichoic
acids (LTA) [4] are cell surface
BRMs which are widely distributed among Grampositive bacteria. Yamamoto
et al. [5,6] first demonstrated in 1985 that a crude LTA fraction prepared
from Streptococcus pyogenes induced tumor necroSocieties.
All rights reserved
98
Y. Suds et al. / FEMS Immunology and Medical Microbiology I2 (1995) 97-112
sis factor (TNF) and this was followed by a series of
studies by our group on the TNF-inducing
and antitumor activities of S. pyogenes LTA [7,8]. Subsequently Tsutsui et al. [ 101 fractionated crude LTA
extracted from Enterococcus hirue ATCC 9790 according to the method of Fischer [9] into less hydrophobic LTA-1 and more hydrophobic LTA-2 by
hydrophobic
interaction
chromatography.
They
showed that the latter (LTA-2) exhibited various
immunobiological
activities such as the induction of
tumor necrosis factor-a (TNF-a), interleukin 1 and
interferon-a/@,
and regressive activity on the Meth
A fibrosarcoma
established
in BALB/c
mice in
combination with muramyl dipeptide (MDP) priming
1101.
Lipoteichoic acids are generally composed of two
main structural parts, glucose or D-alanine substituted polyglycerophosphate
and glycolipid [4]. The
structure has been proposed by Fischer et al. [4] for
LTAs from E. hirue and S. pyogenes. To investigate
the structure-activity
relationships of E. hirue LTA,
we synthesized the fundamental structures of LTA-1
and -2 [ 11,121 by mimicking the proposal of Fischer.
The oligoglucosides
or the D-alanine moiety, which
were assumed to be linked at the 2-position of the
glycerophosphate
part 141, were thereby excepted.
Neither cytokine-inducing
nor anti-Meth A tumor
activity, however, was noted with these synthetic
analogues [13]. These results either suggested that
substituting oligoglucosides
or D-alanine moieties on
the glycerophosphate
part is essential for the immunobiological
activities of an LTA-2 fraction, or
that the bioactivity noted with the LTA fraction is
attributable to an active component(s) which is contained in the fraction but differs from that proposed
by Fischer and which served as our synthetic target.
The second alternative seems to be supported by our
findings that the cytokine-inducing
potency considerably fluctuated among LTA preparations which were
obtained by the standard extraction procedure from
the same stock of E. hirue ATCC 9790 cells. Unknown active components might have been missed.
Consequently we attempted to characterize the bioactive component(s) of E. hirue LTA other than those
having Fischer’s structure [4]. In this study we isolated five high molecular weight glycolipids from an
LTA-2 fraction that powerfully induced TNF-(Y and
IL-6 in human peripheral whole-blood cell cultures.
All of these immunobiologically
active glycolipids
were composed of similar building blocks, but the
relative ratios of each component were distinct from
one another and from the parent LTA-2 fraction.
Incidentally, Leopold and Fischer [ 141 have reported
the heterogeneity
of a less hydrophobic LTA prepared from E. hirue ATCC 9790 strain which may
correspond to our LTA-I fraction.
2. Materials and methods
2. I. Chemical analysis
2. I. I. Phosphorus
Phosphorus levels were determined according to
the method of Bartlett [15] with a slight modification. The calibration was performed using sodium
dihydrogenphosphate.
2.1.2. Fatty acids
Fatty acids were analyzed according to Ikemoto et
al. [ 161 by gas chromatography (GC) using eicosanoic
acid as an internal standard. The apparatus and the
conditions for GC were as follows. The apparatus
used was a Shimadzu GC-14 gas chromatograph
equipped with a C-R7A data processor (Shimadzu,
Kyoto, Japan); column, 2% Silicone OV-1 on Uniport HP mesh 60/80 (GL Science, Tokyo, Japan) 3
mm diam. X 3 m; carrier gas, nitrogen (50 ml/min);
injection temperature, 250°C; column temperature,
190°C; detection, hydrogen flame ionization (FID).
2.1.3. Carbohydrates
The carbohydrate moieties of test fractions were
analyzed according to the alditol acetate method [ 171
by GC or gas chromatography-mass
spectrometry
(GC-MS). The conditions for GC were similar to
those described above except that the column contained 3% Silicone OV-225 on Uniport HP (mesh
60/80. GL Science) and the temperature was 190250°C. The GC-MS analysis was performed using a
QP-5000 (Shimadzu) with a fused silica capillary
column SPTM-2330 (0.25 mm diam. X 15 m. Supelco Inc., Bellefonte, PA, USA). The hexose was
quantified by the anthrone-sulfuric
acid method [ 181.
2.1.4. Glycerol
A test sample was hydrolyzed with 2 M HCl at
125°C for 48 h in a sealed tube under nitrogen.
Mannitol was added as an internal standard and the
mixture was extracted with hexane to remove fatty
acids. After removing
the HCI by repeated coevaporation with methanol, the hydrolyzed residue
was digested with alkaline phosphatase (source: Escherichia coli, Wako Pure Chemicals Co. Ltd., Osaka. Japan) at 37°C for 24 h in 0.04 M ammonium
carbonate
buffer at pH 9.0. After repeated coevaporation with methanol. the residue was trimethylsilylated
with
a mixture
of
1,I ,I .3,3.3hexamethyldisilazane/pyridine/trimethylsilyl
chloride (2/10/l.
v/v/v).
then analyzed by GC under
the conditions
similar to those described for the
qualitative analysis of carbohydrates
(column temperature: 8%200°C).
The content in each sample
was calculated from the calibration curve obtained
with glycerol (Wake Chemicals Co. Ltd) by CC.
2.1.5. SDS-PAGE
SDS-PAGE
was performed using an AE-6220
apparatus (ATTO. Tokyo. Japan) and a 15% gel
(PAGEL’ SPU-1%. ATTO) according to the method
of Schagger and Jagow [ 191. Samples (10 pg) were
dissolved in IO mM Tris-HCI buffer (pH 6.8) containing 1% SDS, I% 2-mercaptoethanol,
20% glycerol and 0.02% Coomassie brilliant blue and applied
to wells on the gel. Molecular weight markers (#80I 129-83. Pharmacia LKB. Uppsala. Sweden) were
also applied to the wells to compare the molecular
weights of individual fractions or components. The
electrophoresis
was run at a constant current (20
mAJ for 2 h. then the gel was stained with 0.5 wt%
Alcian blue in 50% methanol containing 2% acetic
acid according to Rice et al. [20] with a slight
or with 0.02 wt% CBB in 10%
modification.
methanol containing
10% acetic acid for visualization of marker proteins.
2.2. Bacterial cells
E. hircre ATCC 9790 was grown at 37°C for 6 h
in trypticase-tryptose-yeast
extract medium
[2 I].
Cells were harvested by centrifugation,
washed with
phosphate buffered saline (PBS) three times and
stored at - 30°C until use.
2.3. Estrrrction of the crude LTA ,fraction
Chloroform (500 ml) and methanol (1000 ml)
were added to a suspension of frozen E. hirue cells
(400 g, wet weight) in 500 ml of 0.1 M sodium
acetate buffer (pH 4.5). The mixture was stirred at
room temperature overnight. After centrifugation
at
3300 X %efor 20 min the solvent was decanted. The
residue was washed with methanol twice and dried
in vacua to give delipidated cells in which free. but
unbound. lipids were removed.
The delipidated cells (I20 .g) were mechanically
milled and suspended in 750 ml of 0. I M acetate
buffer (pH 4.5). A mixture of the above buffer
solution and phenol ( I /4. v/v. total 750 ml) was
added and the mixture was stirred at 65°C for 35
min. After cooling in an ice bath. the water phase
was separated by centrifugation at I5 000 X ,q for 20
min. To the residual mixture. 750 ml of 0.1 M
acetate buffer (pH 4.5) was added. and the mixture
was stirred at 65°C for 45 min. After centrifugation
under similar conditions to those described above.
the water phase was decanted. This process was
repeated once more, and the combined water phases
were thoroughly dialyzed against Toray endotoxinfree water (hereafter. referred to as water: see section
entitled Miscellaneous)
at 3°C for a few days (the
following dialysis was done under similar conditions). then lyophilized to give 12 g of crude LTA
fraction ( IO wt% of the delipidated cells).
A portion of the crude LTA fraction (X.0 g) was
dissolved in 160 ml of 0.1 M acetate buffer containing 5 mM MgSO, (pH 6.0). then digested with 7.3
mg of deoxyribonuclease
(from bovine pancreas.
Sigma Chemicals Co.. St. Louis. MO, USA) and
24.4 mg of ribonuclease
(from bovine pancreas.
Sigma Chemicals Co.) at room temperature for 24 h.
The reaction mixture was dialy/.ed and concentrated
to about 30 ml at 30°C in \acuo with a rotary
evaporator. After removing the precipitates by centrifugation. a IO ml portion of the resultant supcrnatant was applied to a column of Bio-Gel A-Sm
(Japan-Biorad
Lab.. Tokyo. Japan: 2.5 cm diam.
X85 cm) and eluted with distilled water or 0.1 M
acetate buffer (pH 4.5) at 4’C at a rate of’ 20 ml/h.
Fractions of 6 ml were collected and monitored by
phosphorus
determination
(in terms of the ahsorbance at a wave length of 675 nm). High molecu-
100
Y. Suds et al. / FEMS Immunology and Medical Microbiology
lar weight fractions were combined and concentrated
to about 10 ml at 30°C in vacua, and the concentrate
was applied to hydrophobic interaction chromatography on an Octyl-Sepharose
CL4B column (2.5 cm
diam. X40 cm, Pharmacia LKB, Uppsala, Sweden)
equilibrated
with 0.1 M acetate buffer (pH 4.5)
containing 15% (v/v) I-propanol. The column was
eluted with a linear gradient of I-propanol (15 to
70%, v/v) to give three pooled fractions (NA, LTA- 1
and LTA-2; Fig. I> by monitoring the phosphorus
content. Each fraction was dialyzed and lyophilized.
The yields of LTA-1 and -2 were 6 and 8% of the
applied crude LTA fraction, respectively.
2.5. Ion-exchange
LTA-2 fraction
column
chromatography
of the
The LTA-2 fraction (11.7 mg) was dissolved in 2
ml of 0.1 M acetate buffer (pH 4.5) containing 0.1%
Triton X- 100, applied to a column of DEAE-Sep
hacel (1 cm diam. X 17 cm, Pharmacia LKB) equilibrated with the same buffer, then eluted with a linear
gradient of sodium chloride (O-O.5 M) at 3.5 ml/h
(Fig. 2a). The three subfractions (D- 1, -2 and -3)
which were separated by monitoring the phosphorus
content were pooled, dialyzed and lyophilized. The
lyophilized fractions were suspended in 1.5 ml of
ethanol, vortexed thoroughly,
then centrifuged
at
12 000 X g for 20 min at 4°C to remove the supernatant containing Triton X-100. This procedure was
repeated several times until the absorbance of the
12 ( 1995) 97- I12
supematant at 260 nm, which corresponded to the
remaining Triton X-100, became undetectable. The
washed
powder
was dissolved
in water and
lyophilized.
2.6. Ion-exchange membrane
LTA-2 and its subfractions
chromatography
of the
2.6.1. DEAE-Men Sep 1000
The LTA-2 fraction (26 mg) was dissolved in 5
ml of 0.1 M acetate buffer (pH 4.5) containing 35%
(v/v) I-propanol and applied to a DEAE-Mem Sep
1000 (20 mm diam.; Japan Milipore Ltd. Osaka,
Japan) equilibrated with the same buffer. Membrane
chromatography
was performed by elution with a
stepwise gradient of sodium chloride (O-O.8 M) at a
flow rate of 2 ml/min.
Each 6 ml elute was collected and monitored by determination
of the phosphorus and hexose contents (Fig. 3a). Three subfractions (mD-1, -2, and -3) were separated, dialyzed
and lyophilized.
2.6.2. QMA-Mem Sep 1000
The biologically active fraction obtained by anion-exchange
membrane chromatography
(mD- 1 in
Fig. 3a; 8 mg) was dissolved in 2 ml of 0.01 M
acetate buffer (pH 4.5) containing 35% (v/v) l-propanol and applied to a QMA-Mem Sep 1000 (20 mm
diam.; Japan Millipore Ltd.) equilibrated with the
same buffer. The membrane was eluted with a linear
gradient of sodium chloride (O-O.8 M). The eluates
100
Tube
number
Fig. I. Elution profile of hydrophobic interaction chromatography
on Octyl-Sepharose
CL-4B. A crude lipoteichoic acid fraction from the
deoxyribonuclease
and ribonuclease-treated,
hot phenol-water extracts of delipidated Enterococcus hirue ATCC 9790 was partially purified.
Fractions of 4 ml (flow rate: 30 ml/h) were monitored by measuring the phosphorus content. NA: a fraction derived from nucleic acids.
Y. Sudu
et al. / FEMS
Immunology
und
were combined into 4 subfractions (mDQ-1, -2, -3,
and -4; Fig. 4a) by monitoring the hexose content,
then dialyzed and lyophilized. Under similar conditions, the parent LTA-2 fraction was directly applied
to anion-exchange
chromatography
to separate the
subfractions, mQ-I, -2. -3. -4 and -5 (Fig. 5a).
of
2.7. Hydrophobic
interaction
chromatograph_v
bioactil>e subfractions of the LTA-2
The bioactive fractions (mDQ-1 and -2 in Fig. 4a;
mQ-1 and mQ-2 in Fig. 5a) obtained by ion-ex-
r
Medical
Microbiology
12 f 19951
97-I
I2
101
change chromatography with a QMA-Mem Sep 1000
were combined. The resultant fraction (37 mg in
total) was dissolved in 1 1 ml of 0.1 M acetate buffer
containing
15% (v/v)
I-propanol and applied to a
Octyl-Sepharose
CL-4B column (2.5 cm diam. X 30
cm) equilibrated with the same buffer. Five fractions
(FOS- 1, -2. -3. -4 and -5: Fig. 6) were separated by
elution with a linear gradient of 1-propanol (I 5-60%‘.
v/v) in terms of the phosphorus content. Each fraction was dialyzed and lyophilized. Separation of the
three fractions, FOS-3. -4 and -5. was incomplete, so
they (4-6 mg) were separately applied once again to
a
60
40
20
0
Tube number
800
,
LTA-2
Fig. 2. (a) Anion-exchange
presence of 0.1%
column chromatography
Triton X-100.
D-l
of LTA-2
D-2
(I 1.7 mg) with a DEAE-Sephacel
Fractions of 0.7 ml (3.5 ml/h)
were pooled into 3 subfractions (D-I.
phosphorus content. The recovery rates were 9.2. 12. I and 65.2%. respectively (86.5%
subfractions (D-l,
D-3
column (I cm diam.
X 17 cm) in the
-2 and -3) by monitoring the
in total). (b) The IL-6.inducing
activity of the LTA-2
-2 and -3) by stimulating human peripheral whole-blood cell cultures. Donor of the blood sample: Y.S. In this and the
following assays. the human peripheral whole-blood cell cultures were stimulated with test samples or a reference LPS at the indicated doses
in RPM1 1640 medium. The levels of induced cytokines were assayed by means of duplicate ELISA
The data are expressed as the mean f S.D.
determinations (triplicate cell cultures).
01 1l:B4
an Octyl-Sepharose
CL-4B column (2.5 cm diam.
X 6 cm) equilibrated
with 0.1 M acetate buffer (pH
4.5) containing
30% (v/v> I-propanol and eluted
with a linear gradient of I-propanol (30-60%, v/v)
to give FOS-3R, -4R and -5R, respectively.
using an LPS specimen from
(Sigma) as a reference standard.
2.8. Limulus assay
The reaction mixture consisting of test sample (in
25 ~1 of saline) and heparinized human peripheral
whole-blood (25 ~1) collected from an adult volunteer in RPM1 1640 medium (75 ~1; Flow Laboratories. Irvine, Scotland. UK), was incubated in tripli-
2.9. Cytokine induction
blood cell cultures
Limulus activity of the final products (FOS-1. -2,
-3R, -4R and -5R) was measured by means of the
Endospecy Test ’ (Seikagaku Kogyo. Tokyo, Japan)
E.
coli
in human peripheral
whole-
620 nm for Hexose
2
*
675 nm for Phosphorus (x10)
-------
NaCl (M)
20
Tube number
2000
lOO~g/ml
q
10 ng/ml
q
lOpg/ml
q
1 @ml
n
1500
0
mD-1
LPS
Fig. 3. (a) Anion-exchange
I-propanol
in 0.
I
membrane chromatography of the crude LTA-2
mD-2
mD-3
(26 mg) with DEAE-Mem
Sep loo0 in the presence of 35%
M sodium acetate buffer (pH 4.5). The membrane was eluted at a flow rate of 2 ml/min
sodium chloride in the buffer. Fractions of 6 ml were pooled into 3 subfractions (mD-I.
with a stepwise gradient of
-2 and -3) by monitoring the contents of phosphorus
and hexose. The recovery of each subfraction was 22.7. 10.5 and 52.8%. respectively (X6% in total). (b) The IL-6 inducing activity of each
subfraction (dose: I - 100 pg/ml)
isolated ah shown in (a).
Y.S.
donated the blood sample. Dose of LPS: 0. I-IO
ng/ml.
cate in a 96well plastic plate (#25850-96,
Coming
Lab. Sci. Company, Coming, NY, USA) at 37°C in
5% CO,. After 24 h, the plate was centrifuged at
300 X g for 2 min and cytokines were assayed in the
supematant as follows. Both the dose of test samples
and the level of induced cytokines were expressed as
the final concentration in the above reaction mixture
(,ug/ml
and pg/ml, respectively).
Among periphera1 whole-blood
samples from different donors,
small but discernible differences were evident regarding the susceptibility
to cytokine induction by
test samples. However, all of the data presented in
each table or figure were obtained from a same day
0.8
assay with a whole-blood sample drawn from one
donor as one set of experiment, and so are completely comparable. An LPS specimen prepared by
Westphal method from E. co/i 01 I l:B4 (Sigma
Chemicals Co.) was used as a positive control to
check the responsiveness
of whole-blood cell cultures to IL-6 and TNF-(Y induction by test materials
throughout the present study.
2.9. I. IL-6 n.wq
The levels of IL-6 induced by stimulating human
peripheral whole-blood cell cultures with test samples were measured by means of an enzyme-linked
0.8
a
z
0.4
5
z”
0
0.0
-1”“”
100 @ml
q
10 ngtml
q
15000
mDQ-1
Fig. 4. (a) Anion-exchange
mDQ-2
membrane chromatography of the bioactive LTA-2
1000 in IO mM sodium acetate buffer (pH 4.5) containing 35% (v/v)
I-100
kg/ml)
respectively (89.7%
subfraction (mD-I
I-propanol.
gradient of sodium chloride in the buffer solution were pooled into mDQeach subfraction was 10.1. 11.8. 45.9 and 16.98,
mDQ-4
mDQ-3
I. -2, -3.
LPS
in Fig. 5a; 8.2 mg) with QMA-Mem
The 6 ml fractions eluted at 2 ml/min
Sep
with a linear
and -4 by monitoring the hexose content. The recovery of
in total). (b) The IL-6 inducing activity of each subfractions (dose:
obtained in (a). H.T. donated the blood sample. Dose of LPS: 0.1 -IO
n&/ml.
104
Y. Suds et al. / FEMS Immunolog! and Medical Microbiology
immunosorbent
assay (ELISA). Briefly, 200 ,ul of
goat anti-IL-6 antiserum (a gift from Prof. T. Kishimoto at Osaka University School of Medicine) dihued to l/lo6
in 0.1 M sodium bicarbonate buffer
(pH 9.6) was placed in each well of a 96-well ELISA
plate (SUMILON MS-8596F, Sumitomo Bakelite Co.
Ltd., Tokyo, Japan) and incubated at 4°C overnight.
After washing the wells with PBS containing 0.05%
Tween 80 (PBS-T), 250 ~1 of 1% bovine serum
albumin (BSA) in PBS containing 0.1% NaN, was
added and incubated at 4°C for 8 h. After 5 washes
with PBS-T, 20 ~1 of the supematant from stimulated whole-blood cell cultures and 80 ~1 of PBS
1.2
containing 0.1% BSA and 0.1% NaN, were added
and incubated at 4°C overnight. After a thorough
wash with PBS-T, 80 ~1 of anti-IL-6 monoclonal
antibody conjugated with horseradish peroxidase (a
gift from Fuji Rebio Co., Tokyo, Japan) were added
and the plate was incubated at 25°C for 60 min.
After removing the supematant and careful washing
with PBS-T, 100 ~1 of a substrate solution consisting of 2,2-azinobis-(3-ethylbenzothiazoline-6sulfonic acid) diammonium
salt (ABTS, 60 mg dissolved in 100 ml of 0.1 M NaH,PO,)
and H,O,
(30%, 35 ~1 in 100 ml of ABTS solution) was
added. After an incubation at 25°C for 60 min. 1 M
a
mQ-4
.n
10
a
mDQ-1
______________
20
Tube
Fig. 5. (a) The parent LTA-2
12 (I 995) 97- II2
mDG2
30
number
mDQ3
mDQ-4
LPS
fraction (10.8 mg) was fractionated by anion-exchange membrane chromatography with QMA-Mem
under conditions similar to those described in the legend to Fig. 6a without
Sep 1000
prior DEAE chromatography.
Fractions of 6 ml (2 ml/mitt)
were combined into 5 portions, mQ-I, -2, -3, -4 and -5 by monitoring the hexose content. The recovery of each subfraction were 3.8. 2.3,
16.2, 18.9 and 35.4%, respectively (76.6% in total). (b) The IL-6-inducing activity of the subfractions (dose: l-100 pg/ml)
separated in
(a). H.T. donated the blood sample. Dose of LPS: 0. I-10 ng/ml.
Y. Sudo et al. / FEMS Immunology and Medical Microbiology
sulfuric acid was added to stop the enzymatic reaction. The absorbance at 415 nm of each well was
measured using a microplate reader (MTP-32, Colona
Electric., Ibaragi, Japan). The IL-6 levels (pg/ml) in
the test culture supematant samples were calculated
by reference to the calibration curve obtained by
rHuIL-6 (a gift from Prof. T. Kishimoto) [22,23].
2.9.2. TNF-a assay
The concentration
of TNF-(Y in the test culture
supematant (pg/ml) was determined using a TNF-a
ELBA kit (PredictaTM, Genzyme Co., Cambridge,
MA, USA) according to the manufacturer’s instructions. The induced TNF-o levels were calculated in
comparison with the calibration curve obtained with
rHuTNF-a (Genzyme Co.).
e
2
::
0
.c
12 f 1995) 97-I
105
12
2.10. Miscellaneous
We prevented contamination
of test materials and
instruments with extraneous bacterial endotoxins. For
example, endotoxin-free
water prepared with Toray
Pure LV-308 (TORAY, Tokyo, Japan) was used
throughout the study and it was further distilled for
use in chromatography.
All test specimens for cytokine assays were kept at -20°C until use.
3. Results
3.1. Fractionation
of the crude LTA fraction
hydrophobic interaction chromatography
by
Fig. 1 shows the elution profile of a crude LTA
fraction by hydrophobic interaction chromatography
0.09
0.07
P
i
0.05
5
2
0.03
fD
u)
$
0.01
20
0.00
FOS3R
FOS4R
FOS-SR
Fig. 6. Hydrophobic interaction chromatography
of the bioactive subfractions separated by QMA-Mem Sep IO00chromatography
(mDQ- I,
mDC-2, mQ-1 and mQ-2; 37 mg in total) on Octyl-Sepharose
CL-4B. Fractions of 6 ml (flow rate: 24 ml/h) eluted by a linear gradient of
I-propanol (15 to 60%, v/v) in 0.1 M acetate buffer were pooled into 5 portions, FOS-1, -2. -3, -4, and -5 relative to the phosphorus
content. The recovery of each subfraction was 16.0, 10.4, 12.6, 13.0 and 18.2%. respectively (70.2% in total). FOS-3. -4 and -5 subfractions
were further purified by a repeated chromatography
to yield FOS-3R. -4R and -5R with recovery rates of 57.7, 68.6 and 36.1%,
respectively.
Octyl-Sepharose
CL-4B. Three major peaks, nucleic acid (NA), less hydrophobic LTA-I and more
hydrophobic LTA-2 fractions, were separated as reported by Tsutui et al. [ 101. In agreement with their
results, TNF-a
and IL-6 inducing activity of the
distinct LTA-2 fraction was observed by stimulation
of the human peripheral whole-blood cell cultures.
Since the broad peak of the LTA-2 fraction (a principal starting material for fractionation experiments in
this study) suggested the presence of several subfractions, this fraction was again eluted through an
Octyl-Sepharose column. However. no better separation was effected in terms of the absorption at 675
nm in the phosphorus determination and the biological activities (data not shown).
on
3.2. Fractionation
change
of the LTA-2 ,fraction by anion-ex-
chromatography
The LTA-2 fraction was then subjected to further
fractionation by anion-exchange
chromatography
in
the presence of a detergent, Triton X-100, since
components
of the fraction are amphipathic.
The
chromatogram
of the parent LTA-2 fraction on a
DEAE-Sephacel column (Fig. 2a) showed that it was
separable into three subfractions (D-I, D-2 and D-3)
by increasing the sodium chloride concentration
in
the elution buffer. The IL-6 induction assay indicated
that only the fraction which was eluted at lower
concentrations of sodium chloride, namely D- 1, possessed distinct IL-6-inducing activity (Fig. 2b).
0
FOS-1
FOS2
FOS-3A
FOS-4R
FOS-SR
LTA-2
400
LPS
10 q/ml
E
200
FOS-1
FOS-2
FOS-3R
FOS-4R
FOSSR
LTA-2
Fig. 7. The cytokine-inducing
activity of purified components. FOS- I, -2. 3R. AR. and -5R by stimulating
cell cultures. IL-6 (a) and TNF-a (b) induction. H.T. donated the blood sample.
LPS
human peripheral
whole blood
Y. Suda r~ al. / FEMS Itnmunolog~ and Medical Microhiolo~y
Separation of the LTA-2 fraction by DEAE-Sephacel chromatography in the presence of 0.1% Triton
X- 100 was reproducible in the elution profile. However. the ethanol extraction to remove the detergent
was troublesome and time-consuming,
and tended to
reduce the recovery of the target components. Therefore, we separated the fraction by means of ion-exchange chromatography
using a buffer containing
I -propanol instead of Triton X- 100. We also used a
new commercial membrane for ion-exchange
chromatography.
After preliminary
trials with various
solvent systems, we optimized the experimental conditions and chromatographically
separated LTA-2
Table
107
I2 ( 19951 Y7- I I2
with DEAE-Mem Sep in the presence of 35% I-propanol instead of Triton X-100 (Fig. 3a). The parent
LTA-2 fraction was separated into three subfractions
(mD-I, -2 and -3) by monitoring
the contents of
phosphorus and hexose. The assay for IL-6 induction
by each subfraction revealed that the mD-1 fraction
that eluted at zero sodium chloride had significantly
higher activity than other subfractions (Fig. 3b).
3.3. Isolation
qf bioactive gl_vcolipid.s
Since the biologically active fraction (D-l in Fig.
2a and mD-I in Fig. 3a) was eluted at a very low
I
Biological
activities and chemical compositions of bioactive glycolipids
separated by a combination of hydrophobic and ion-exchange
chromatography
Separatedbioactiveglycolipids
L-t-A-2
FOS-1
FOS-2
FOS-3R
FOS-4R
FOS-SR
100
1.9
1.2
0.9
1.1
0.8
U-6 induction”
1
31
36
9
21
19
TNF-a induction’)
1
17
17
3
7
5
Limulus Activity’)
40
7
60
30
1 x 104
10
Phosphorus (wt.%)
5.4
3.6
6.1
5.3
5.1
2.2
Glycerol (wt.%)
20.0
7.7
13.0
8.0
13.1
6.4
44.43)
56.64’
63.03’
52.1”
50.43’
21.63’
3.0
8.1
4.5
4.2
5.4
3.2
24.4
12.2
1.8
3.5
3.2
1.0
14.6
0.3
15.3
0.4
9.1
0.3
4.0
0.3
0.;
1.0
0.9
1.0
1.0
1.8
1.0
0.6
0.;
1.0
0.2
0.5
1.0
0.3
0.3
0.8
1.0
0.1
0.2
1.1
0.2
0.2
Y&d
(M.96)
Hexose (wt.%)
Amino Acid (wt.%)
Ala
Asp
CYS
GlU
GUY
Leu
LYS
Se1
Thr
Val
1.0
1.0
0.1
0.3
1.1
0.2
0.2
FanY Acid (wt.%)
35.0
.. . . _........____...__._............
I.?._.....__.._._.
239...._____
..?.?.____
__
.__._..
4:!!___..._.___.._
8.t__..___._..
1.00
1.00
l.Gil
1.00
1.00
1.00
0.23
0.21
0.15
0.14
0.09
0.42
I.;1
0.12
0.52
1.32
“;:
0.72
0.92
0.87
0.23
’ Relative cytokine levels induced by stimulation of the human peripheral whole-blood cell culture at a dose of 100 pg of test sample per
ml. The levels of IL-6 and TNF-u
’
induced by parent LTA-2
were 480 and 41 pg/ml.
Equivalent to a reference standard LPS derived from E co/i 01
’
Exclusively glucose according to CC-MS.
’
Glucose
’
Not detected.
I I:B4
and rhamnose were detected at a molar ratio, 3: I. by GC-MS
respectively.
(Sigma Chem. Co.) in ng/mg.
108
Y. Suds et al. / FEMS Immunology and Medical Microbiology
concentration of sodium chloride, fractions D-l and
mD-1 were combined and further purified with a
quaternary amine grafted QMA-Mem Sep using a
buffer of lower ionic concentration
(10 mM) as
shown in Fig. 4a. The IL-6-inducing
activity was
exclusively
recovered in the mDQ-1 and mDQ-2
fractions which were eluted at a lower concentration
of sodium chloride in the buffer (Fig. 4b). Similar
procedures were applied to direcfy separate bioactive fractions (mQ-1 and -2) from the parent LTA-2
fraction which had not undergone
the preceding
DEAE-Sephacel
or DEAE-Mem
Sep chromatography (Fig. 5a).
All of the bioactive fractions obtained by anionexchange chromatography
(mDQ- 1, mDQ-2, mQ- 1
and mQ-2) were combined and applied on an OctylSepharose CL-4B column to separate 5 subfractions,
FOS-1, -2, -3, -4 and -5 (Fig. 6). Three incompletely
separated fractions (FOS-3, -4 and -5) underwent a
repeat of this procedure and FOS-3R, -4R and -5R
fractions were isolated (Fig. 6). Fig. 7 shows that all
of the final products obtained induced IL-6 and
TNF-a in human peripheral whole-blood cell cul-
12 f 1995) Y7- I I2
tures. Thus, efficient separation of several components capable of inducing IL-6 and TNF-(r was
achieved by a combination
of anion-exchange
and
hydrophobic interaction chromatography
of the parent LTA-2 fraction (Fig. 61, in contrast with the
incomplete separation by direct hydrophobic interaction chromatography
of the parent LTA-2 fraction
(cf. Fig. 1).
3.4. Cytokine-inducing
activities and the chemical
properties of glycolipids as final products
Table 1 summarizes the IL-6 and TNF-a-inducing
activities and the chemical composition of the high
molecular weight glycolipids that were separated by
the final hydrophobic
interaction
chromatography
presented in Fig. 6. All of the test fractions, particularly FOS4R, had detectable Limulus activity, but
there was no correlation between this and cytokine
induction among these fractions. This indicates that
the cytokine-inducing
activities are inherent in the
test components,
and are not due to extraneously
contaminating
LPS. Fatty acid analysis by GC-MS
17kDa
+
c-
17kDa
14kDa
+
t
14kDa
+
t
8kDa
8kDa
2.5 kDa e
f-
2.5 kDa
Fig. 8. SDS-PAGE profiles of the final bioactive glycolipids, parent LTA-2 fraction and reference compounds synthesized by mimicking the
proposed structure of LTA-1 and LTA-2 (SLTA-1 and SLTA-2, respectively; [ 11,121).
Test samples (10 kg) were applied to Pagel”
SPU-15 (AlTO, Japan) and electrophoresis was performed at a constant current (20 mA) for 2h. The FOS-1 to -5R, LTA-2, SLTA-I and
SLTA-2 were visualized by staining with 5% Alcian blue. The marker proteins (MP) were stained with 0.02% CBB. The gels were scanned
and the data were processed using Macintosh Quadra 800 (Apple Japan Inc., Tokyo, Japan) equipped with a ScanJet IIcx (Hewlett Packard,
San Diego, CA, USA). The software was Adobe Photoshop 2.5J (Adobe Systems Japan, Tokyo, Japan).
Y. Suda et al. / FEMS Immunology and Medical Microbiology 12 ( I9951 97- I12
showed that 3-hydroxy fatty acids were undetectable
with any of the five test components, indicating that
LPS contamination
was below the detection limit of
the analysis, namely less than 10 ng per mg (data not
shown). This suggested that even the fairly high
Limulus activity detected with FOS-4R may be due
to its inherent activity independent of LPS contamination [24].
In agreement with the principle of separation by
hydrophobic interaction chromatography,
the order
of fatty acid contents of the test glycolipids increased
in the order of elution: FOS-2 through FOS-3R and
-4R to FOS-SR excepting FOS- 1, and the fatty acid
content of FOS-SR was as high as 35% (Table 1).
Fraction FOS-1, which was eluted first from the
Octyl-Sepharose column possessed a distinctly higher
fatty acid content than the other subfractions,
but
lacked octadecenoic acid (Cl8: 1) which is a fatty
acid common to all from FOS-2 to FOS-SR. It also
contained rhamnose, in addition to glucose, unlike
the other four glycolipids in which the sole hexose is
glucose. The reverse order was generally true of the
hexose content, to a lesser extent with the content of
phosphorus and amino acids (mainly alanine) among
the FOS-2 to FOS-5R fractions. FOS-1 was also an
exception in this respect. No correlation was noted
among the order of elution from hydrophobic chromatography. the glycerol content and the IL-6 or
TNF-a-inducing
activity.
The molecular weight of biologically active glycolipids was examined by SDS-PAGE (Fig. 8). All
of FOS-2, -3R, -4R and -5R glycolipids gave broad
bands visualized by Alcian blue staining in a molecular weight range from 8000 to 17 000 Da. This was
in contrast to the narrower bands given by synthetic
LTA analogues
(SLTA- 1 and SLTA-2 [ 11,12]),
which migrated in a manner compatible with their
molecular weights.
The migration pattern of the final products in
SDS-PAGE together with their chemical composition suggested that components FOS-2, -3R, -4R and
-5R were glycolipids with wide ranges of molecular
weight. In accordance with the behavior in SDSPAGE, no distinct peaks were observed with any of
FOS-2 to FOS-5R in the laser desotption-time
of
flight mass spectrometry (data not shown). FOS-1
again behaved differently
in SDS-PAGE
and no
band was visualized other than staining in the stack-
109
ing gel, probably reflecting a very high molecular
weight or aggregation under SDS-PAGE conditions
containing 1% SDS.
4. Discussion
We identified five glycolipids, FOS-1 to FOS-SR,
which induced IL-6 and TNF-a in human wholeblood cell cultures. These glycolipids were isolated
by a combination
of hydrophobic
interaction and
ion-exchange column or membrane chromatography
of a parent LTA-2 fraction extracted from delipidated E. hirue ATCC 9790 cells by means of hot
phenol-water followed by ribonuclease/nuclease
digestion. The increases in the IL-6-inducing
potency
resulting from purification
procedures
were estimated to be 31-, 36-, 9-, 21- and 19-fold for FOS-1,
-2, -3R, -4R and -5R respectively, in terms of the
levels induced by a test dose of 100 pg/ml
of the
reaction mixture compared with the potency of the
parent LTA-2 fraction. A roughly parallel but less
marked (3- to 17-fold) increase was noted with
TNF-(-u induction (Table 1). The recovery of these
bioactive glycolipids on a weight basis was less than
10% of the parent LTA-2 fraction in all. The recovery rate of cytokine-inducing
activities, on the other
hand, was provisionally
and roughly estimated in
terms of the sum of [the relative potency of each
glycolipid] X [the weight recovery (%) of each fraction]. The approximate value was calculated to be
150% with the IL-6-inducing
activity and 70% with
TNF-a-induction.
The Limulus activity observed for the bioactive
components is assumed to be inherent in them, but
not attributable
to the contaminating
LPS as described in Results. If the activity, particularly that of
FOS4R,
was due to contamination,
higher IL-6
induction (at least 10 to 100 times) would be observed as is expected from the dose dependency of
the control LPS (Fig. 7a). The presence of 3-hydroxyl fatty acids would also have to be detected in
the hydrolysate of FOS4R. This explanation is also
supported by the fact that the Limulus gelation is not
completely specific to LPS. Some acidic phospholipids were shown to activate factor C, which is a
key enzyme in the gelation cascade [24]. Nevertheless, the Limulus
activity seems to have an ex-
110
Y. Sudu et al. / FEMS Immunolug~ and Medial Microbiology 12 ClYY5l Y7- I12
tremely high dependency on the chemical or physical
properties of substrate: there is no other rational
explanation at the moment with regards to the fact
that only the particular component, FOS4R, exhibits
10’ to lo3 times higher activity man the others
(Table 1).
All of the 5 final products and the parent LTA-2
fraction were composed of essentially the same major constituents as described for LTA by Fischer [4]
but their relative contents differed from one another
and from that of Fischer’s proposal. The LTA-2
fraction used as the starting material was quite different in both chemical composition and IL-6 and
TNF-o-inducing
potency from the corresponding
fraction which had been used in a study by Takada et
al. [ 131, although both samples were prepared from
the same stock of E. hirue ATCC 9790 culture. The
efficient separation of bioactive glycolipids from the
parent LTA-2 may be attributed to the fact that
biologically inactive components which amounted to
over 90% of the parent LTA-2 fraction were removed by anion-exchange
chromatography
in the
presence of 0.1% Triton X-100 or 35% 1-propanol
prior to the final hydrophobic interaction chromatography. Owing to the amphipathic
nature of most
components in the LTA-2 fraction, bioactive glycolipids may exist as aggregates with bio-inactive components
during Octyl-Sepharose
chromatography.
This may explain the insufficient separation by direct
hydrophobic chromatography
of the LTA-2 fraction.
In fact, one of the final products, FOS-1 seemed to
have a unique chemical property as well as a strong
tendency to self-aggregate.
This situation may explain why FOS-1 had less affinity to Octyl-Sepharose than that expected from its high fatty acid
content. The possibility that FOS-1 is a simple mixture of four other components can be excluded by
the fact that this fraction lacks octadecenoic
acid
(Cl 8: 1) which is a fatty acid residue common to
FOS-2 to FOS-SR. Among the amino acids detected
with the five bioactive components,
the alanine
residue as a principal amino acid is presumed to
mainly covalently link with a polyglycerophosphate
moiety as shown by Fischer for LTAs from some
Gram-positive bacteria [4]. The nature of other minor
amino acids remains unknown, but the possibility
that these amino acids are derived from extraneous
contamination
with free peptides may be excluded in
view of the hydrophilic
nature of most of these
amino acids. Such hydrophilic peptides, even if present in the parent LTA fraction, should be removed
during chromatographic
separation, particularly by
Octyl-Sepharose chromatography.
In a series of studies into the immunobiological
activities of streptococcal lipoteichoic acids, we have
almost exclusively used mice in both in vitro and in
vivo assays as a target host for stimulation. Following our pioneering studies [5-81 a number of investigators have shown that LTAs from various Grampositive bacterial species, particularly enterococcal
LTAs, could induce IL-l and TNF in human peripheral monocyte cultures [25-281. Bhakdi et al. [27]
prepared two LTA fractions from enterococcal LTA
by Octyl-Sepharose
column chromatography,
which
contained two and four acyl residues and which
probably corresponded to our LTA-1 and -2 fractions, respectively.
They found that there was no
significant difference in the bioactivities
of these
fractions on human monocyte cultures. On the other
hand, Tsutsui et al. [ 101 showed that cytokine-inducing and antitumor activities were mainly located in
the LTA-2 fraction in a murine assay systems. In this
study, we examined the IL-6 and TNF-a induction
by stimulating
human peripheral whole-blood
cell
cultures with LTA-2 and its subfractions. This system had the advantages of high reproducibility
and
technical simplicity as compared with that of murine
peritoneal macrophage cultures or the in vivo system
with primed mice. In addition, the human peripheral
whole-blood cell system seemed to be more prevalent in the studies of the immunobiological
activities
of LTAs than murine systems, and consequently
is
more convenient
for comparing the assay results
among different investigators,
since various assay
systems, especially with several animal species may
give quite discordant results in terms of the immunobiological activities of the same test sample. Our
preliminary study suggests that human and murine
mononuclear
cells differ each other regarding the
susceptibility to immunobiological
activities of LTA
preparations (details to be published elsewhere).
The four bioactive glycolipids (FOS-2, -3R, -4R
and -5R) might share structural characteristics which
are compatible
with Fisher’s proposal. But these
products have not yet been completely purified on
the molecular level. However, it is likely that the
Y. Suds er al. / FEMS Immunoio~~
and Medics1 Microhiolo,qy
heterogeneity of our final products is mainly due to
the presence of congeners or homologues with different number of repeating units like many other
naturally occurring glycoconjugates.
We thus anticipate that further purification to complete homogeneity is not possible. Structural analyses of these bioactive. high molecular weight glycolipids by means of
selective chemical and enzymatic degradation are in
progress to elucidate the manner of linkage among
their constituents.
12 ( IYY5J 97- 1 I2
III
colipids (Kates, M., Ed.) pp. 123-234.
Plenum Press. New
York.
151Yamamoto.
Hamada.
A.. Usami.
H., Nagamuta.
S.. Yamamoto.
Kotdni. S. (1985)
M..
Sugawara,
T., Kato. K.. Kokeguchi.
Y..
S. and
The use of lipoteichoic acid (LTA)
from
Streptococcus pyogenes to induce a serum factor causing
tumor necrosis. Br. J. Cancer 5 I. 739-742.
[61 Yamamoto,
A.. Nagamura,
Watanabe.
M..
Usami. H., Sugawara.
N., Niitsu. Y. and Urushizaki,
Y..
I. (198.5) Produc-
tion of cytotoxic factor into mouse peritoneal fluid by OK432, a streptococcal preparation. Immunol. Lett. I I, 83-88.
[71 Usami. H.. Yamamoto. A.. Sugawara, Y.. Hamada. S., Yamamoto, T.. Kato. K., Kokeguchi, S.. Takada, H. and Kotani.
S. (1987)
A
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Acknowledgements
181Usami,
The authors are grateful to Prof. Tadamitsu Kishimoto at Osaka University School of Medicine for
supplying rHuIL-6 and anti-IL-6 antiserum, and Mr.
Hiroshi Miyasaka of Fuji Rebio Co. (Tokyo, Japan)
for HRP-conjugated
anti-IL-6 monoclonai antibody,
respectively. The authors would also like to thank
Prof. Toshihide Tamura and Dr. Tomoko Hayashi at
Hyogo College of Medicine for their kind advice on
human peripheral whole-blood cell assay. This work
was supported in part by Grants-in-aid for Scientific
Research (No. 05403035 to S.K.) and Scientific Research on Priority Areas (No. 05274102 to Y.S.)
from the Ministry of Education, Culture and Science,
Japan and by a grant from Chugai Pharmaceutical
Co. Ltd.. Tokyo. Japan.
H., Yamamoto,
A., Yamashita.
Hamdda.
S..
Yamamoto.
T.,
Ohokuni.
H.
and Kotani,
W..
Kato.
S. (1988)
K..
Sugawara.
‘I’..
Kokeguchi.
S..
Antitumor
effects of
streptococcal lipoteichoic acids on Meth A lihrosarcoma. Br.
J. Cancer 57, 70-73.
[91Fischer. W..
preparation
Koch.
H.U.
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R. (1993)
Improved
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523-530.
II01 Tsutui.
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Kokeguchi.
S., Matsumura,
T.
and Kato,
K.
t 1991) Relationship of the chemical structure and immunobiological activities of lipoteichoic
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FEMS
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biol. Immunol. 76. 21 I-218.
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Fukase. K.. Matsumoto, T.. Ito. N.. Yoshimura.
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