FEMS Immunology and Medical Microbiology 42 (2004) 155–166
www.fems-microbiology.org
The solubilization and biological activities
of Aspergillus b-(1 ! 3)-D -glucan
Ken-ichi Ishibashi a, Noriko N. Miura a, Yoshiyuki Adachi a, Hiroshi Tamura b,
Shigenori Tanaka b, Naohito Ohno a,*
a
Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science,
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
b
Tokyo Research Institute, Seikagaku Corporation, Tateno 3-1253 Higashiyamato-shi, Tokyo 207, Japan
Received 29 December 2003; received in revised form 3 April 2004; accepted 5 April 2004
First published online 12 May 2004
Abstract
We have recently demonstrated that the cell wall b-glucan of Candida albicans could be solubilized by sodium hypochlorite,
followed by dimethylsulfoxide-extraction (NaClO-DMSO method). In this study, applying this method to Aspergillus spp., we
prepared mycelial cell wall b-glucan and examined its physical properties and immunotoxicological activity. The acetone-dried
mycelia of Aspergillus spp. were oxidized by the NaClO-DMSO method. An analysis of 13 C NMR spectra revealed the preparations
to be composed of a-(1 ! 3) and b-(1 ! 3)-D -glucan. Also, the proportion of a-(1 ! 3) and b-(1 ! 3)-D -glucan varied. Futhermore,
a solubilized Aspergillus b-glucan (ASBG) was prepared from OX-Asp by urea-autocalve treatment. ASBG showed limulus activity
similar to Candida solubilized b-glucan (CSBG), and there was little difference in the activity of ASBG between various Aspergillus
spp. ASBG affected the production of IL-8 by human peripheral blood mononuclear cells (PBMC). ASBG should be useful for
analyzing the clinical role of b-glucan.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Aspergillus solubilized b-glucan; Limulus activity; Fungal infection
1. Introduction
The incidence of deep mycosis has been increasing
with improvements in chemotherapy for malignant diseases and the popularization of marrow transplant and
organ transplant medical care [1]. Moreover, changes in
the usage of immunosuppressive, antibacterial and antiviral drugs are affecting the incidence of the disease [2].
The number of aspergillosis in particular is increasing.
Most Aspergillus spp., as fungi causing deep mycosis,
have been detected at necropsy [1,3]. This tendency is
remarkable in organ transplant patients as especially
blood stem cell recipients [2,4]. Also, strains of Asper-
*
Corresponding author. Tel.: +81-426-76-5561; fax: +81-426-765570.
E-mail address: [email protected] (N. Ohno).
gillus fumigatus resistant to amphotericin B [5,6] and
itraconazole [7] are emerging.
Deep mycosis brings about a serious pathosis and
bad convalescence. The mortality rate reaches 50–60%
when invasive pulmonary aspergillosis occurs during
chemotherapy-induced neutropenia and can exceed 90%
in bone marrow transplantation [8]. To prevent this, it
has to be diagnosed quickly and treated as early as
possible. The diagnosis of deep mycoses is based on the
separation of the fungus or pathologic diagnosis. In
serious deep mycoses, it is not infrequently undiagnosed
until necropsy after death. A method that can diagnose
the infection from an early stage is needed, and serological methods which detect the protein and polysaccharide antigen of cell components are actually used in
the clinic.
b-(1 ! 3)-Glucan is a component of the fungal cell
wall but not found in bacteria. The limulus G test which
0928-8244/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsim.2004.04.004
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K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
using b-(1 ! 3)-glucan-sensitive factor G of the horseshoe crab coagulation enzyme is useful for the screening
of general fungal infections [9]. It is reported that patients with deep mycosis such as candidasis and carinii
pneumonia but not mucormycosis or cryptococcosis
show a positive reaction, and in sensitivity and specificity, the limulus G test is superior to detection systems
using CAND-TEC and mannnan antigen [10–12].
Moreover, patients and an animal model of aspergillosis
tested positive, and the measured values correlated with
clinical symptoms and pathological change [13–15].
Also, it was reported that b-(1 ! 3)-glucan showed
various biological activities triggering the activation of
complement and the production of inflammatory mediators such as leukotriene, TNF-a and so on [16,17]. It
is possible that b-(1 ! 3)-glucan has a some influence on
and can be a parameter which reflects the immune response and inflammatory reactions of the host. This
method of diagnosis method has become popular in
Japan because of its usability.
b-(1 ! 3)-Glucan shows a variety of structural and
physical properties such as degree of branching, conformation, molecular weight and solubility in water, and
these physical properties influence its biological and
immuno-pharmacological activities [18,19]. It was reported that the activation of limulus factor G was also
influenced by those physical properties [20–22]. Therefore, it is important clinically to compare the physical
properties of b-(1 ! 3)-glucan as a diagnostic parameter
of deep mycosis with the limulus factor G-specific activation. Considering these points, it should be used as a
standard material in diagnosis systems.
The Aspergillus mycelial cell wall is mainly composed
of glucan, chitin and galactomannan [23,24]. b-(1 ! 3)glucan is basically insoluble material in water and alkali
solutions and can not be solubilized easily. To examine
the structure and biological activities of the cell wall
b-(1 ! 3)-glucan in Saccharomyces cerevisiae, phosphate
and sulfate derivatization has been used [25,26]. However, the b-(1 ! 3)-glucan obtained using this protocol
has not been satisfactory for a precise structural determination. As mentioned above, solubilized b-(1 ! 3)glucan is present in the blood of the deep mycosis
patients. The host lacks a b-glucan catabolic enzyme
and it is thought that b-(1 ! 3)-glucan is solubilized by
the defense mechanism of the host or the metabolic
process of the fungus. We have demonstrated that the
majority of cell wall b-glucan was deposited in organs
over quite a long period of time with a gradual oxidative
degradation by phagocytes, which utilize non-specific
oxidation reactions involving O2 , H2 O2 and hypochlorous acid [27]. Another cell wall component, mannan, is
water-soluble and, thus, would be metabolized faster.
Based on this concept, we have recently demonstrated
that the cell wall b-glucan of Candida albicans could be
solubilized by the NaClO-DMSO method [28], and by
regulating the degree of NaClO-oxidation, we could
prepare both particulate and soluble forms of Candida
glucan having exactly the same primary structure that is
composed of slightly branched long b-(1 ! 6) glucan
and b-(1 ! 3)-glucan segment [29]. Also, regarding the
immunopharmacological and immunotoxicological activity of Candida solubilized b-glucan (CSBG) in vivo
and vitro, we have found augmentation of lipopolysaccharide-mediated TNF-a and nitrogen oxide production
by macrophages, activation of an alternative pathway of
complement, enhanced vascular permeability, an adjuvant effect on antibody production, synthesis of IL-6 by
macrophage, and so on [30].
In this study, we tried to prepare solubilized Aspergillus cell wall glucan by applying the NaClO-DMSO
method. Also, we examined the physical properties and
biological activities of this glucan as a limulus factor
G-specific activator.
2. Materials and methods
2.1. Materials
All strains of Aspergillus spp. (A. fumigatus IFO
30870, IFO 4400, Aspergillus niger IFO 6342, Aspergillus
oryzae 30103) and C. albicans IFO 1385 purchased from
the Institute for Fermentation (Osaka, Japan) were
maintained on Sabouraud agar (Difco, USA) at 25 °C
and transferred once every three months. A Sodium
hypochlorite solution and sodium hydroxide (NaOH)
were purchased from Wako Pure Chemical Industries,
Ltd. Distilled water (DIW) was from Otsuka Co., Ltd.
(Tokyo, Japan).
2.2. Media
A C-limiting medium originally described by Shepherd
and Sullivan [31] was used to grow all strains of yeast
unless stated otherwise. The medium contained (per liter):
sucrose, 10 g; (NH4 )2 SO4 , 2 g; KH2 PO4 , 2 g; CaCl2 2H2 O, 0.05 g; MgSO4 7H2 O, 0.05 g; ZnSO4 7H2 O, 1
mg; CuSO4 5H2 O, 1 mg; FeSO4 7H2 O, 0.01 g; biotin, 25
lg; final pH, 5.2. Five liters of medium was placed in the
glass jar of a microferm fermentor (New Branswick Scientific Co., Inc., USA) and cultured at 27 °C with 5
L min1 of aeration and stirring at 400 rpm.
2.3. Carbohydrate analyses
Carbohydrate content was determined by the phenol–
sulfuric acid method. Component sugars were identified
by capillary gas–liquid chromatography (Ohkura Riken
Co. Ltd., Tokyo) of alditol acetate derivatives after
complete hydrolysis with 2 M trifluoroacetic acid. A
capillary column of fused silica (J&W Scientific, Inc.,
K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
CA, 30 m 0.262 mm, liquid phase; DB-225, 0.25 mM)
was used at 220 °C. The molar ratio of glucose and
mannose and galactose, was calculated from the peak
area of each component.
2.4. Preparation of Aspergillus cell wall glucans
The acetone-dried mycelia of Aspergillus spp. (2 g)
was suspended in 200 mL of 0.1 M NaOH with NaClO
of various available chlorine concentrations for 1 d at
4 °C. After the reaction was completed, the reaction
mixture was centrifuged to collect the insoluble fraction.
Insoluble fractions were dried by washing with ethanol
and acetone (NaClO-treated Aspergillus, OX-Asp). OXAsp suspended in 8 M urea was autoclaved (120 °C, 20
min) and the resulting solutions were centrifuged (12,000
rpm, 20 min) and divided into sup and ppt. Each fraction was dried with ethanol and acetone.
2.5. Measurement of b-(1 ! 3)-D -glucan using Fungitec
G-test MK
The activation of factor G (limulus reactivity) by b(1 ! 3)-D -glucans was measured with a chromogenic
method using a b-(1 ! 3)-D -glucan-specific reagent
(Fungitec G-test MK, Seikagaku Corp., Tokyo), which
eliminates factor C. The glucan sample was dissolved
NaOH (1 mg mL1 ) and diluted with 0.01 M NaOH.
Usually, the dilution was done with 0.01 M NaOH, and
the sample solution was used directly for the limulus
reaction without neutralization. Dilute NaOH was
confirmed to be usable for the limulus reaction because
of the high buffer action of the reagent. Reactions were
performed in a flat-bottomed 96-well Toxipet plate 96F
(Seikagaku Corp.) as follows. Samples (50 lL) were
placed in the wells, and the Fungitec G-test MK reagent
(50 lL) was added to each well. The plate was incubated
at 37 °C, and during incubation the absorbance at 405
nm (reference: 492 nm) was measured kinetically using a
microplate reader (Wellreader SK601, Seikagaku
Corp.). Disposable plastic materials for tissue culture or
clinical use were employed, and all glassware was sterilized at 260 °C for 3 h. All operations were performed in
triplicate under aseptic conditions.
2.6. NMR analysis
Solubilized fractions and authentic materials were
dissolved in Me2 SO-d6 , and the 13 C NMR spectra were
recorded at 70 °C. A Bruker DPX 400 equipped with
‘XWIN-NMR’ software was used.
2.7. Zymolyase digestion
Glucan sample (20 mg) suspended in acetate buffer
(50 mM, pH 6.0) was mixed with 100 lg/mL of zymol-
157
yase 100T (Seikagaku Corp.). After incubation overnight at 45 °C, the reaction mixture was boiled for 3 min
to inactivate the enzyme. The resulting solution was
mixed with ethanol (1:4) and soluble and insoluble
fractions were prepared. The ratio was monitored using
the phenol-H2 SO4 method.
2.8. Cell culture
Peripheral blood mononuclear cells (PBMC) were
obtained from the peripheral blood of healthy donors.
The blood was centrifuged (3000 rpm at 4 °C for 10 min)
to obtain a leukocyte-rich fraction. The fraction was
mixed well with an equal volume of phosphate-buffered
saline (PBS) and centrifuged on 6mL of HISTOPAQUE
(density 1.077; Sigma Chemical Co., USA) in a 15-mL
centrifuge tube (Falcon 352196, Becton–Dickinson,
Lincoln Park, NJ) at 2500 rpm for 25 min at room
temperature. After centrifugation, isolated PBMC were
washed three times with PBS and PBMC (2 106 cells
mL1 ) were cultured in polypropylene-tubes (IWAKI
GLASS, Japan) in 500 lL of RPMI 1640 medium
(Sigma Co., USA) supplemented with gentamicin sulfate
(Sigma Co., USA) (5 lg/ml) containing 10% autologous
plasma in culture tubes at 37 °C in humidified 5% CO2 .
The IL-8 in the supernatants was detected using an
ELISA method.
2.9. Assessment of helix conformation using Congo Red
The change in the absorption maximum of Congo
Red (Wako Pure Chemical Co., Ltd.) in the presence or
absence of polysaccharide preparations was measured.
An equal volume of polysaccharide preparations (1
mg mL1 ) and Congo Red solution (2 105 M) was
mixed in sodium hydroxide (final concentration, 0.1 or
0.35 M) and the absorption maximum of the resulting
solution was measured immediately.
2.10. ELISA for IL-8
A 96-well Nunc plate was coated with capture antibody for mouse anti-human IL-8 mAb (Pharmingen
Co., USA) in 5 lg mL1 of Na2 HPO4 buffer (pH 9.0) by
incubation at 4 °C overnight. The plate was washed with
PBS containing 0.05% Tween 20 (Wako Pure Chemical
Co. Japan) (PBST) and blocked with 0.5% bovine serum
albumin (BSA: Sigma Co. USA) (BPBST) at 37 °C for
40 min. After a wash, the plate was incubated with rhIL-8 (Pharmingen Co., USA) or 50 lL of test sample,
obtained by the above procedure, at 37 °C for 40 min.
The plate was washed with PBST and then treated with
antibody for biotinylated mouse anti-human IL-8 mAb
(Pharmingen Co., USA) in BPBST. Next, the plate was
treated with peroxidase-conjugated streptavidine
(ZYMED Laboratories Inc.) and developed with a
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K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
tetramethylbenzidine (TMB) substrate system (KPL
Inc., MD). Color development was stopped with 1 M
phosphoric acid and the optical density was measured at
450 nm.
2.11. ELISA for TNF-a
Immune plates (Nunc 442404, F96 Maxisorp) were
coated with capture antibody for anti-human TNF-a
monoclonal antibody (Pharmingen Co., USA) in 0.1 M
Carbonate buffer (pH 9.6) by incubation at 4 °C overnight. The plates were washed with PBST and blocked
with PBST containing 10% heat-inactivated fetal calf
serum (FCS) at room temperature (RT) for 1h. They
were then washed, incubated with recombinant human
TNF-a (Pharmingen Co., USA) or 50 lL of sample at
RT for 3 h, washed with PBST, and treated with biotinylated anti-human TNF-a monoclonal antibody
(Pharmingen Co., USA) and Avidin-horseradish peroxidase conjugate, and developed with a TMB substrate
system (KPL Inc., MD). Color development was stopped with 1 M phosphoric acid and the optical density
was measured at 450 nm.
Fig. 1. 13 C NMR spectra of OX-Asp of Aspergillus spp. in Me2 SO-d6
OX-Asp and CSBG dissolved in Me2 SO-d6 and measured by 13 C
NMR as described in Section 2.
3. Results
3.1. Physical properties of the NaClO-oxidized cell wall
of Aspergillus spp.
We first applied the NaClO-DMSO method to acetone-dried mycelia of Aspergillus as well as Candida. By
analysing the 13 C NMR spectrum of the preparation
(Fig. 1). Twelve spectra were observed. Six signals attributable to b-(1 ! 3)-D -glucan were common to OXAsp and CSBG which composed of slightly branched
long b-(1 ! 6)-glucan and b-(1 ! 3)-glucan segment
[28]. The other six signals were identified as a-(1 ! 3)glucan from agreement with the spectra of purified a(1 ! 3)-glucan derived from Hypsizygus marmoreus
(HmCAER-8MUP) [32], Saccharomyces pombe [33].
This result shows that OX-Asp was mainly composed of
a-(1 ! 3)-glucan and b-(1 ! 3)-glucan.
Moreover, acetone-dried Aspergillus mycelia were
oxidized with various concentrations of NaClO solution
containing various amount of available chlorine. The
yield and properties of OX-Asp are shown in Table 1.
As the NaClO concentration increased, the yield decreased and the proportion solubilized by DMSO rose,
suggesting that the mycelial cell wall was resolved by
oxidation. Also, the nitrogen content which reflected the
quantity of chitin, protein and nucleic acid decreased
and the glucose composition ratio increased accompanying the decrease in the ratio of galactose and mannose. The glucan of Aspergillus was relatively resistant
to oxidation compared to other cell wall component and
was gradually purified. However, although we could
purify b-glucan of Candida by the NaClO-DMSO
method, under intensive oxidation conditions, b-(1 ! 3)glucan, the zymolyase-sensitive part of Aspergillus, was
resolved with other cell wall components and not purified absolutely.
We next oxidized the acetone-dried mycelial cells of
various Aspergillus spp. and compared 13 C NMR spectra of each OX-Asp (Fig. 2). It was found that the ratio
of a-(1 ! 3)-glucan and b-(1 ! 3)-glucan was quite different among Aspergillus spp. Notably, the cell wall of
A. fumigatus contained a-(1 ! 3)-glucan abundantly.
Hence, it was suggested that the content of b-(1 ! 3)glucan was different among Aspergillus spp.
3.2. Preparation of Aspergillus cell wall and b-1,3-D glucan (ASBG)
Although we applied the NaClO-DMSO method to
the Aspergillus mycelical cell wall, OX-Asp was obtained
as a complex of a-(1 ! 3)-glucan and the b-(1 ! 3)glucan contained nitrogen. We could only partially purify the cell wall b-glucan. Hence, to refine the ASBG in
one step, we suspended oxidized OX-Asp in 8 M Urea
and autoclaved it at 120 °C and 20 min (Fig. 3). ASBG
was obtained in the urea-sup fraction (Fig. 4). Moreover, we compared the physical properties of urea-sup
fraction derived from various Aspergillus spp. (Table 2).
In every fraction, the percentage of nitrogen was less
than 1% which suggests further purification. In Asper-
K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
159
Table 1
Yield and properties of NaClO-oxidized cells (OX-Asp)
Concentration of
available chlorine (%)
Aspergillus
0.25
0.5
0.75
1
1.25
1.5
Candida
0.5
1
a
b
Yield (mg)a
N content (%)b
Man/Gal/Glc
Solubilized ratio by
DMSO (%)
% of zymolyasesensitive part
1066
735
696
561
515
353
3.61
2.25
1.95
1.41
1.35
1.30
13.2/20.2/100
8.6/8.7/100
7.9/7.2/100
6.7/5.8/100
5.6/3.0/100
2.6./0.8/100
25.4
80.1
81.1
88.5
92.0
92.0
49.7
57.4
55.6
55.6
44.7
41.9
517
310
1.49
0.44
3.6/0/100
2.4/0/100
99.0
99.0
61.0
64.0
From 2 g of acetone-dried mycelia.
N content determined by elemental analysis.
NaClO-Oxidized
cell (OX-Asp)
Suspended in 8M Urea
Autoclaved (120˚C, 20min)
Centrifuged 12k rpm, 20min
Ext
Residue
EtOH
Acetone, dry
Urea sup
Urea ppt
Fig. 3. Preparation of Aspergillus solubilized b-glucan.
Fig. 2. 13 C NMR spectra of OX-Asp of various Aspergillus spp. in
Me2 SO-d6 . Each OX-Asp was dissolved in Me2 SO-d6 and measured
by 13 C NMR as described in Section 2. (a) A. niger 6342, (b) A. fumigatus 30870, (c) A. fumigatus 4400, (d) A. oryzae 30103.
gillus niger and Aspergillus oryzae, Aspergillus cell wall
b-(1 ! 3)-glucan was obtained as ASBG in this fraction.
However, in A. fumigatus, the yield was small and it is
difficult to separate a-(1 ! 3)-glucan and b-(1 ! 3)-glucan on the 13 C NMR spectrum (data not shown).
We next examined the physical properties of ASBG.
The molecular weight of ASBG was analyzed by gel
filtration with 0.3 N NaOH (Fig. 5). It showed wide
molecular weight distribution and an average molecular
weight of about 30,000. ASBG is smaller than CSBG,
whose average molecular weight is 106 Da. Also, the
conformation of ASBG was examined by Congo Redinduced metachromasy, which is a well-known property
of high molecular weight and gel forming 1,3-b-glucan
[34,35]. The absorption maximum of ASBG as well as
CSBG returned to a shorter wavelength in 0.3 N
NaOH (Fig. 6). This result suggested that the conformation of ASBG could be single helix or random coil
(see Table 2).
3.3. Limulus reactivity of ASBG
In Fig. 7, we examined the limulus reactivity to factor
G of ASBG. Each glucan was dissolved in 0.3 N NaOH
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K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
Fig. 4. 13 C NMR spectra of urea-treated fraction of OX-Asp in Me2 SO-d6 Each preparation was dissolved in Me2 SO-d6 and measured by 13 C NMR
as described in Section 2.
1.4
Dextran T500
ASBG
Dextran T10
1.2
Abs (OD 492)
1
0.8
low concentration. Also, comparing with it of urea-sup
fractions derived from various strains, there was little
difference in this activity. Therefore, it was suggested
that ASBG could be a standard for the limulus G-test,
like CSBG.
3.4. Activation of leukocytes by ASBG in vitro
0.6
0.4
0.2
0
20
30
40
Fraction No.
50
Fig. 5. Gel-filtration chromatography of ASBG from a column of
HW65. ASBG dissolved in 0.3 M NaOH was applied to a column of
Toyopearl HW-65F (1 45 cm) equilibrated with 0.3 M NaOH and
fractionated. Eluted fractions (200 lL) were collected and monitored
using the phenol–H2 SO4 method.
and diluted in order to fit the single helix conformer,
which is the conformer responsible for the limulus Gtest. ASBG showed activity as did CSBG at 1 pg/mL, a
b-(1 ! 3)-Glucan usually shows immunopharmacological and immunotoxicological activities, and CSBG
shows inflammatory cytokine productivity, enhanced
vascular permeability and activation of an alternative
pathway of complement.
We estimated the level of inflammatory cytokine
production by human PBMC stimulated with ASBG.
The activity was tested in the presence of autologous
plasma under nonadherent condition and compared
with related materials. We examined the dose-response
and kinetics of ASBG on IL-8 production (Fig. 8).
ASBG brought about the maximum effect on IL-8
production in 10 lg mL1 , and suppressed in higher
dose. Also, IL-8 appeared in the culture supernatant as
K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
0.3
161
120
Blank
100
0.1
mAbs/min
0.2
0.1N
0.35N
0
0.3
80
60
A. oryzae 30103
A. niger6342
A. fumigatus 30870
A. fumigatus 4400
C. albicans 1385
40
ASBG
Absorbance
0.2
20
0.1
0
× 1010
0
0.3
× 108
dilution ratio
× 107
× 106
Fig. 7. Limulus activity of urea-sup fraction derived from various
Aspergillus spp. Urea-sup fraction derived from various Aspergillus
spp. was dissolved in 0.3 M NaOH and 10-fold dilutions were prepared
using distilled water. Fungitec G-test MK reactivities of these solutions
were determined as described in Section 2.
CSBG
0.2
0.1
0
0.3
Dextran
0.2
0.1
0
400
× 109
450
500
550
600
Wavelength (nm)
Fig. 6. Metachomasy of Congo Red in polysaccharide fractions. Absorption of Congo Red in the presence of polysaccharide fractions at
0.1 and 0.35 M NaOH was measured as described in Section 2.
early as 4 h culture and gradually increased over 24 h.
PBMC responded to ASBG, and the IL-8 productivity
was enhanced by ASBG.
Next, we compared with IL-8 production of ASBG
and CSBG on appropriate condition (Fig. 9(a) and (b)).
PBMC stimulated with ASBG showed the advance of
IL-8 production, however it was less than CSBG,
suggesting the influence of heterogeneity of the
b-(1 ! 3)-D -glucan structure. It is of note that in the
presence of heat-inactivated autologous plasma, both
soluble b-(1 ! 3)-D -glucan decreased activity.
Compared with the other substances, ASBG was less
potent than OX-Asp as insoluble materials (Fig. 9(b)).
Solubilized OX-Asp (sOX-Asp), the DMSO extracted
preparation of OX-Asp showed active pattern as well as
ASBG. On the other hands, insoluble/particulate fractions were active regardless of heat-inactivated autologous plasma. In TNF-a production, OX-Asp showed
intensive activity. But soluble glucan not showed in the
presence of normal plasma (Fig. 9(c)). These results
suggested that soluble and particulate materials use
different molecular mechanisms.
We further examined the relation of b-(1 ! 3)-D glucan with the activity of OX-Asp using b-(1 ! 3)glucanase treatment (OX-Asp Z) (Fig. 10). Although
b-(1 ! 3)-glucan was excluded from OX-Asp, the strong
activity of OX-Asp Z was maintained. These results
suggested that the b-(1 ! 3)-glucan of OX-Asp participated this activation however, other cell wall components, mainly a-(1 ! 3)-glucan could, also induce strong
inflammatory cytokine production.
Table 2
Yield and properties of OX-Asp and ASBG of various Aspergillus spp.
OX-Asp
A.
A.
A.
A.
a
oryzae 30103
niger 6342
fumigatus 30870
fumigatus 4400
Urea-sup
Yield (%)a
N content (%)
% of zymolyase
sensitive part
Yield (%)a
N content (%)
% of zymolyase
sensitive part
26.3
33.0
18.5
18.9
2.89
1.41
2.17
2.33
54.6
43.8
27.6
22.0
9.5
13.6
4.4
4.2
0.36
0.37
0.38
0.45
72.6
67.2
48.2
30.5
From acetone-dried mycelia.
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K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
30000
30000
25000
25000
20000
20000
pg/mL
pg/mL
control
15000
15000
10000
10000
5000
5000
0
0
Nil
(a)
ASBG
5
10
50
ASBG dose (µg
100
m-1)
4
200
(b)
12
24
h
Fig. 8. Dose dependency and kinetics of IL-8 production by human PBMC stimulated with ASBG. (a) Dose dependency of IL-8 production by
human PBMC stimulated with ASBG. PBMC obtained from the peripheral blood of healthy donors were adjusted to a concentration of 2 106 cells
ml1 in RPMI1640 medium containing 10% normal or heat-inactivated autologous plasma and cultured with ASBG for 12 h in a 5% CO2 incubator.
Subsequently, the culture supernatants were collected, and IL-8 was measured as described in Section 2. (b) Kinetics of IL-8 production by human
PBMC stimulated with ASBG. PBMC obtained from the peripheral blood of healthy donors were adjusted to a concentration of 2 106 cells ml1 in
RPMI1640 medium containing 10% normal or heat-inactivated autologous plasma and cultured with ASBG (10 lg ml1 ) for 24 h in a 5% CO2
incubator. Subsequently, the culture supernatants were collected, and IL-8 was measured as described in Section 2.
4. Discussion
The deep mycosis, specifically aspergillosis, candidasis has become a clinical problem. b-(1 ! 3)-glucan is
one of the main components of the fungal cell wall and
is used as a parameter for the serological diagnosis of
deep mycosis. b-(1 ! 3)-glucan also shows immunopharmacological and immunotoxicological activities. It
is not easy to solubilize fungal cell wall b-(1 ! 3)-glucan.
But we recently found that Candida b-(1 ! 3)-glucan
could be efficiently solubilized by the NaClO-DMSO
method. In this study, we applied this method to Aspergillus mycelial cell wall and attempted the solubilization of b-(1 ! 3)-glucan.
We first examined the physical properties of
OX-Asps in various oxidized conditions. On the
strengthening of the degree of oxidization, galactomannan and chitin were gradually excluded, and a
complex of a-(1 ! 3)-glucan and b-(1 ! 3)-glucan was
mainly found. The results suggested that a-(1 ! 3)glucan and b-(1 ! 3)-glucan are more resistant to oxidation than the other cell wall components. It is
possible that the Aspergillus mycelical cell wall was
resolved by a similar mechanism of resolution in the
host. b-(1 ! 6)-glucan which plays an important role
in the inner and outer cell wall component in yeast
fungi such as C. albicans [36–38] was not identified in
Aspergillus spp. Because disruption of the gene
encoding Bgl2p, a 1,3-b-glucanosyltransferase which
introduces intrachain 1,6-b linkages into 1,3-b-glucan
in A. fumigatus, did not result in a phenotype distinct
from the parental strain. It was suggested that
b-(1 ! 6)-glucan did not play a significant role [39,40].
Even though the NaClO-DMSO method was applied
to Aspergilus mycelial cell wall, we could not easily
refine b-glucan. b-(1 ! 3)-Glucan and chitin are part
of a complex in the alkali insoluble fraction of
Aspergillus mycelial cell wall [23,41]. Also, this binding
was strong and covalent and not broken by the nitrous
acid treatment. Hence, under the strong oxidized
conditions, nitrogen was not excluded absolutely.
Interestingly, on comparing OX-Asps derived from
various Aspergillus strains, it was found that the ratio of
a-(1 ! 3)-glucan and b-(1 ! 3)-glucan was different in
this study. Notably, in the case of A. fumigatus with high
virulence in aspergillosis patients, it was composed of a(1 ! 3)-glucan. This result suggested between that for
the fungus and b-(1 ! 3)-glucan. Mori and Matsumura
[12] and Yoshida [42] pointed out in aspergillosis, the
sensitivity is worse in candidasis and plasma b-(1 ! 3)glucan did not significantly become positive in the early
phase of aspergillosis. Further examination is necessary.
But this result is important for clinical diagnosis and a
reviewal of the cutoff value for diagnosis will be necessary. Also, recently, echinocandins, a new class of
antifungal agents that act on the fungal cell wall by inhibiting b-(1 ! 3)-glucan synthesis, have attracted attention [43,44]. It is known that the echinocandins have
activity against Candida and Aspergillus spp. but not
Fusarium spp.and the Mucorales spp. which not do
contain b-(1 ! 3)-glucan in the cell wall. It was reported
that Aspergillus spp. are not killed and acted fungal-
K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
40000
Normal
Heat-inactivated
pg/mL
30000
20000
10000
0
(a)
control
ASBG
CSBG
140
Normal
120
Heat-inactivated
ng/mL
100
80
60
40
20
0
(b)
control
CSBG
ASBG
sOX-Asp
OX-Asp
control
CSBG
ASBG
sOX-Asp
OX-Asp
2500
pg/mL
2000
1500
1000
500
0
(c)
Fig. 9. IL-8 production by human PBMC stimulated with Aspergillus
cell wall preparations. (a) Comparison of IL-8 production by ASBG
and CSBG. PBMC obtained from the peripheral blood of healthy
donors were adjusted to a concentration of 2 106 cells ml1 in
RPMI1640 medium containing 10% normal or heat-inactivated autologous plasma and cultured with ASBG or CSBG (10 lg ml1 ) for 12
h in a 5% CO2 incubator. Subsequently, the culture supernatants were
collected, and IL-8 was measured as described in Section 2. *p < 0:01
vs. control, **p < 0:01. (b) IL-8 production and (c) TNF-a production
by human PBMC stimulated with Aspergillus cell wall preparations
PBMC obtained from the peripheral blood of healthy donors were
adjusted to a concentration of 2 106 cells ml1 in RPMI1640 medium
containing 10% normal or heat-inactivated autologous plasma and
cultured with ASBG and related materials (100 lg/ml) for 12 h in a 5%
CO2 incubator. Subsequently, the culture supernatants were collected,
and IL-8 and TNF-a was measured as described in Section 2.
statically toward echinocandins, and in animal models
of aspergillosis, survival is improved by echinocandins
in an impressive fashion, but organ cultures remain
163
positive [45–47]. These results also may be influenced by
the content of b-(1 ! 3)-glucan.
In Aspergillus spp., a-(1 ! 3)-glucan and b-(1 ! 3)glucan could not be separated as mentioned above as
Candida spp. Hence, to refine Aspergillus cell wall bglucan in one step, we applied the urea-autoclave
treatment to OX-Asp. ASBG contained little nitrogen in
the urea-sup fraction. ASBG showed no less limulus
activity than CSBG. Also, there was not a remarkable
difference among the strain of Aspergillus. In series of
previous studies, we have already found that the highly
branched b-(1 ! 3)-glucan obtained from a medicinal
mushroom, as grifolan from Grifola frondosa [35], AgCAE from Agaricus blazei [48] and Sonifilan from
Schizophyllum commune [49,50] showed lower specific
activity toward limulus factor G. Therefore in structure,
ASBG would have greater similarity to CSBG than to
mushroom b-(1 ! 3)-glucans. However, even with this
method, in A. fumigatus with a low b-glucan content, the
cell wall b-(1 ! 3)-glucan could not be refined absolutely. Further improvement as a genetic technique will
be necessary. Three kits for measuring the b-(1 ! 3)glucan concentration in blood are used in Japan today.
As a standard material for these kits, b-glucan derived
from a nonpathogenic fungus, Pachyman refined from
basidiomycete and carboxymethylated curdran originated Alcaligenes genus, which is a gram negative bacterium, are used. Therefore, because the standard
material differs among the kits, the cut off value of each
kit is different. This causes confusion. Also, because
limulus activation depends on the physical properties
and structure of b-glucan, it is natural that the standard
material be derived from a pathogenic fungus. ASBG
was able to be applied as a pathogenic fungal material
the same as CSBG in this study.
It is reported that inflammatory cytokine is strongly
produced in Aspergillus infected mice or on stimulation
with the hyphae fragment in the whole blood of normal
subjects and chronic granulomatous disease patients ex
vivo [51,52]. We and other researchers have reported
that b-(1 ! 3)-glucan shows various immunopharmacological and immunotoxicological activities. Therefore,
in this study, we examined the inflammatory cytokine
production by human PBMC stimulated with ASBG in
vitro. In the presence of normal plasma, ASBG showed
IL-8 production. On the other hand, in heat-inactivated
serum, it decreased. This tendency of IL-8 production
was like that of CSBG. It suggested that the cytokine
production by soluble glucan in vitro was induced
through a complement system. We previously reported
that b-(1 ! 3)-glucan activated the alternative and
classical pathways of complement dependent on the
physical state of the glucan [53]. Therefore, it is thought
that the difference in b-(1 ! 6)-glucan side chain and
molecular weight between CSBG and ASBG influenced
cytokine production. On the other hand, OX-Asp, a
164
K. Ishibashi et al. / FEMS Immunology and Medical Microbiology 42 (2004) 155–166
2500
80
2000
60
pg/mL
ng/mL
1500
40
1000
20
500
0
0
(a)
control
ASBG
OX-Asp OX-Asp Z
(b)
control
ASBG
OX-Asp OX-Asp Z
Fig. 10. Effect of zymolyase digestion of OX-Asp on activation of leukocytes by human PBMC stimulated with OX-Asp. Zymolyase digestion of OXAsp as described in Section 2. PBMC obtained from the peripheral blood of healthy donors were adjusted to a concentration of 2 106 cells ml1 in
RPMI1640 medium containing 10% normal or heat-inactivated autologous plasma and cultured with Aspergillus cell wall preparations (100 lg ml1 )
for 12 h in a 5% CO2 incubator. Subsequently, the culture supernatants were collected, and IL-8 and TNF-a was measured as described in Section 2.
insoluble preparation, showed strong inflammatory cytokine production. Our previous reports also showed
that an insoluble/particle glucan strongly induced inflammatory cytokine production [29,54]. Although b(1 ! 3)-glucan was excluded from OX-Asp, the strong
activity of OX-Asp was maintained. These results suggested that another cell wall component, mainly a(1 ! 3)-glucan, could also induce inflammatory cytokine
production. It was recently reported that the Toll-like
receptor is involved in the response of leukocytes to
Aspergillus [55,56], and that the dependence on MyD88,
which is a downstream adapter molecule of these receptors, varies in each opportunistic pathogen [57]. The
ligand candidates that induced these responses may be a
component of OX-Asp Z.
In this study, we prepared a solubilized Aspergillus b(1 ! 3)-glucan, ASBG, from mycelial cell wall and provided clinically useful information such as the limulus
specific activity. Further examination of the composition
of the cell wall of Candida and Aspergillus, is excepted to
reveal the role of cell wall b-glucan in deep mycosis.
Acknowledgements
The authors express sincere thanks to Mr. Wataru
Ochiai for technical assistance.
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