Download Immunomodulating polysaccharides from the lichen Thamnolia

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Phytomedicine 14 (2007) 179–184
www.elsevier.de/phymed
Immunomodulating polysaccharides from the lichen Thamnolia
vermicularis var. subuliformis
Sesselja Omarsdottira, Jona Freysdottirb, Elin Soffia Olafsdottira,
a
Faculty of Pharmacy, University of Iceland, Hagi, Hofsvallagata 53, IS-107 Reykjavik, Iceland
NaturImm Ltd./Centre for Rheumatology Research, Landspitali-University Hospital, IS-101 Reykjavik, Iceland
b
Received 17 February 2006; accepted 19 April 2006
Abstract
Three heteroglycans Ths-4, Ths-5 and thamnolan and a b-glucan, Ths-2, isolated from the lichen Thamnolia
vermicularis var. subuliformis were tested for in vitro immunomodulating activities and shown to have various
influences on the immune system. All the polysaccharides except Ths-4 caused a stimulation of rat spleen cell
proliferation. In contrast, Ths-4 caused cell death early in the culture, probably due to over-stimulation. Moreover, the
galactofuranomannans, Ths-4, Ths-5 and the b-glucan Ths-2, induced rat spleen cells to secrete IL-10 significantly
above background levels. In addition, Ths-4 and Ths-5 stimulated significant TNF-a secretion by rat peritoneal
macrophages. The galactofuranomannans Ths-4 and Ths-5 have similar structures apart from the molecular weight.
Thus, it may be concluded that the molecular size might influence the potency but not the pattern of activity for Ths-4
and Ths-5. The galactofuranorhamnan thamnolan had less mitogenic effect than Ths-5 and Ths-2 and neither induced
IL-10 secretion by rat spleen cells nor TNF-a secretion by peritoneal macrophages to significant levels. This shows that
thamnolan with its unusual galactofuranorhamnan structure differs from the other Thamnolia polysaccharides in its
immunomodulatory activity.
r 2006 Elsevier GmbH. All rights reserved.
Keywords: Thamnolia vermicularis var. subuliformis; lichen; immunmodulating polysaccharides
Introduction
Thamnolia vermicularis (Sw.) Schaer. var. subuliformis
(Ehrh.) Schaer. (Purvis et al., 1992), previously named
Thamnolia subuliformis (Ehrh.) W. Culb. (Hale, 1974),
does not belong to a lichen family, but is classified in a
heterogeneous group of sterile lichen species named
Lichen imperfecti (Theler 1996). This lichen and a
chemical variant T. vermicularis var. vermicularis (Purvis
et al. 1992) have been used in traditional medicine as
Corresponding author. Tel.: +354 5255804; fax: +354 5254071.
E-mail address: [email protected] (E.S. Olafsdottir).
0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2006.11.012
health-promoting teas in Yunnan, China (Wang et al.
2001) and the latter one has been used for the treatment
of psychic disorders, high blood pressure and inflammatory conditions of the respiratory tract (Hansen and
Schädler, 1985).
T. vermicularis var. subuliformis has been investigated
to some extend for chemical constituents, and found to
contain two low molecular weight depsides, baeomycesic acid and squamatic acid (Asahina and Shibata 1973;
Ingólfsdóttir et al. 1997). Baeomycesic acid has shown
inhibitory effects on 5-lipoxygenase in vitro (Ingólfsdóttir et al. 1997). In addition, four polysaccharides
have been described from this lichen. Three hetero-
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glycans have been isolated from the aqueous extract; an
unusual cold water-soluble polysaccharide named thamnolan, consisting of galactofuranosyl chains and a
rhaman-rich core (Olafsdottir et al. 1999a), and two
heteroglycans, Ths-4 and Ths-5, which are similar and
consist of galactofuranosyl chains and a mannan core
but differ in molecular weight (19 and 200 kD,
respectively) (Omarsdottir et al. 2006). The fourth
polysaccharide is a lentinan-type gel-forming b-glucan
named Ths-2, which was isolated from a cold alkali
extract of the lichen. Ths-2 is the most abundant
polysaccharide of this lichen (Olafsdottir et al. 2003).
Polysaccharides from lichens have shown various
biological activities, such as anti-tumour and immunomodulating activity (Olafsdottir and Ingolfsdottir 2001).
However, many of these studies suffer from lack of purity
and homogeneity of the polysaccharides involved (Paulsen
et al. 2002). A few chromatographically purified polysaccharides from lichens have been shown to influence the
cells of the immune system, from both the adaptive and
the innate arm (Omarsdottir et al. 2005; Olafsdottir et al.
2003, 1999a, b; Olafsdottir and Ingolfsdottir 2001; Ingólfsdóttir et al. 1994). Both thamnolan and Ths-2 from T.
vermicularis var. subuliformis have previously been shown
to be active in an in vitro classical anti-complementary
assay and thamnolan has induced phagocytosis in vitro
(Olafsdottir et al. 2003, 1999a).
Galactofuranose-containing molecules are absent in
green plants, but are widespread components of cell wall
glycoconjugates of bacteria, protozoa, and fungi.
Mammals do not produce galactofuranosyl-containing
molecules, and they are highly immunogenic in mammals (Jones et al. 2004; Sassaki et al. 2002; Levery et al.
1988; Suzuki et al. 1997; Barreto-Bergter and Gorin
1983).
The object of the present work was to investigate
immunomodulating effect of four previously described
polysaccharides Ths-4, Ths-5, thamnolan and Ths-2,
from the lichen T. vermicularis var. subuliformis. The
polysaccharides were tested for mitogenic activity on rat
spleen cells and their ability to stimulate production of
IL-10 by rat spleen cells, as well as TNF-a and IL-10
production by rat peritoneal macrophages.
Material and methods
The lichen material was identified by the lichenologist
Ph.D. Hordur Kristinsson and deposited in AMNH
(Icelandic Institute of Natural History, Akureyri,
Iceland) under No. LA-30040.
Lichen polysaccharides
The polysaccharides Ths-4, Ths-5, thamnolan and
Ths-2 were isolated from T. vermicularis var. subuliformis,
chromatographically purified and structurally determined as previously described (Omarsdottir et al.
2006; Olafsdottir et al. 2003, 1999a). Protein determination was performed with the Bio-Rad Protein Assay
based on the Bradford dye-binding procedure (Bradford
1976). The polysaccharides were dissolved in culture
medium.
Stimulation of rat spleen cells
The cultures of rat spleen cells in the presence of the
polysaccharides thamnolan, Ths-4, Ths-5 and Ths-2
were prepared as described before (Omarsdottir et al.
2005). The effect of the polysaccharides on the rat spleen
cells was determined by measuring cell proliferation and
cytokine secretion.
Stimulation of rat peritoneal macrophages
The cultures of peritoneal rat macrophages in the
presence of the polysaccharides thamnolan, Ths-4, Ths5 and Ths-2 were prepared as described before
(Omarsdottir et al. 2005). The effect of the polysaccharides on the rat macrophages was determined by
measuring cytokine secretion.
Spleen cell proliferation assay
The rat spleen cell proliferation was determined by
H-thymidine uptake by dividing cells after 72 h of
culture as previously reported (Omarsdottir et al. 2005).
The results represent means of four repeat experiments
and are expressed as stimulation index (SI), which was
calculated by dividing cpm of stimulated cells with cpm
of unstimulated cells.
3
Cytokine ELISA
The supernatant from the cell cultures was collected
after 72 h in culture and was used for investigating the
cytokine secretion following stimulation by the polysaccharides. IL-10 and TNF-a secretion by rat peritoneal macrophages and IL-10 production by rat spleen
cells was measured by sandwich ELISA as described
before (Omarsdottir et al. 2005) Each experiment was
run in duplicate and the results represent means of four
repeat experiments.
Statistical analysis
The data were presented as mean7standard deviations (SD) of four experiments. Statistical evaluation
was performed using Student’s t-test for unpaired
observations between a polysaccharide which did not
induce response, and the polysaccharides thamnolan,
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Results
In order to exclude the possibility of interference of
protein or lipopolysaccharide contamination on the
results, the protein content of the polysaccharides Ths-4,
Ths-5, thamnolan and Ths-2 was determined to be less
than 0.5% (Bradford 1976) and 1H-NMR spectra of the
four polysaccharides showed no signs of proteins or
lipopolysaccharides. Furthermore, LPS was used as a
positive control in the cytokine secretion assay of
peritoneal macrophages and the lichen polysaccharides
induced higher levels of cytokines (TNF-a) than the
positive control.
Immunological activity
12
Stimulation index
Ths-4, Ths-5 or Ths-2 for the rat spleen cell proliferation
assay, IL-10 secretion by spleen cells, and TNF-a and
IL-10 secretion by rat peritoneal macrophages in vitro.
33 µg/ml
10
8
11 µg/ml
**
6
**
**
4
**
2
**
*
** *
** ** **
0
Ths-5
Thamnolan
Ths-2
Polysaccharides
Fig. 1. Effect of the polysaccharides Ths-4, Ths-5, thamnolan
and Ths-2 at various concentrations, on rat spleen cell
proliferation in vitro. The stimulation index is calculated by
dividing count per minute (cpm) of stimulated cells with cpm
of unstimulated cells. Each experiment was run in triplicate
and the results were represented as mean7standard deviation
of four separate experiments. Students’s t-test for unpaired
observations between the polysaccharides and a polysaccharide that did not stimulate cell proliferation, was carried out to
identify statistical differences. *po0.05 and **po0.01 was
considered statistically significant.
100µg/ml
400
350
*
*
33 µg/ml
*
11 µg/ml
300
IL-10 pg/ml
The polysaccharides Ths-5, thamnolan and Ths-2
caused a dose-dependent proliferation of the rat spleen
cells, with the highest concentration tested (100 mg/ml)
giving the highest stimulation index for all three
polysaccharides (Fig. 1). These mitogenic polysaccharides, at all concentration levels tested (11, 33 and 100
mg/ml), induced significantly higher SI levels than the
polysaccharide lichenan, which did not cause proliferation, as analysed by Student’s t-test. Interestingly, rat
spleen cells cultured with the polysaccharide Ths-4 died
quite early during the culture and this was seen for all
three concentrations tested. In contrast, PHA at 7.5 mg/
ml, which was used as a positive control, induced a
substantial proliferation with SI of 28.6710.6 (data not
shown).
100 µg/ml
**
Ths-4
The effects of the polysaccharides from T. vermicularis var. subuliformis on rat spleen cell proliferation and
cytokine secretion by rat spleen cells and peritoneal
macrophages were analysed in vitro.
Rat spleen cell proliferation
181
250
*
200
150
100
50
0
Ths-4
Ths-5
Thamnolan
Ths-2
Fig. 2. IL-10 secretion of rat spleen cells stimulated with Ths4, Ths-5, thamnolan and Ths-2 at various concentrations. The
cytokine secretion was measured by sandwich ELISA. Each
experiment was run in duplicate and the results were
represented as mean7standard deviation for four separate
experiments. Student’s t-test for unpaired observations between the polysaccharide and a polysaccharide that did not
stimulate IL-10 secretion, was carried out to identify statistical
differences. *po0.05 was considered statistically significant.
IL-10 secretion by rat spleen cells
The IL-10 secretion by rat spleen cells cultured with
the four polysaccharides was measured by sandwich
ELISA (Fig. 2). All four polysaccharides induced the rat
spleen cells to secrete IL-10 above background levels
(spleen cells cultured with medium alone). Significantly
higher IL-10 secretion was observed for rat spleen cells
cultured with Ths-4 at 100 mg/ml, with Ths-5 at both 100
and 33 mg/ml and with Ths-2 at 100 mg/ml, as compared
with rat spleen cells cultured with lichenan at the same
concentrations. PHA at 7.5 mg/ml, used as positive
control, induced IL-10 secretion of 5477379 pg/ml
above background levels (data not shown).
TNF-a and IL-10 secretion by rat peritoneal
macrophages
TNF-a and IL-10 secretion by rat peritoneal macrophages cultured with the four polysaccharides was
measured by sandwich ELISA (Fig. 3). All the
polysaccharides except thamnolan induced the
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*
500
TNF-α pg/ml
400
100 µg/ml
33 µg/ml
*
11 µg/ml
300
200
100
0
-100
Ths-4
Ths-5
Thamnolan
Polysaccharides
Ths-2
Fig. 3. TNF-a secretion of rat peritoneal macrophages
stimulated with Ths-4, Ths-5, thamnolan and Ths-2 at various
concentrations. The cytokine secretion was measured by
sandwich ELISA. Each experiment was run in duplicate and
the results were represented as mean7standard deviation for
four separate experiments. Student’s t-test for unpaired
observations between the polysaccharide and a polysaccharide
that did not stimulate IL-10 secretion was carried out to
identify statistical differences. *po0.05 was considered statistically significant.
peritoneal macrophages to secrete TNF-a levels above
background levels (negative control). The TNF-a levels
were significantly higher when the rat peritoneal
macrophages were cultured with either Ths-4 or Ths-5
at 100 mg/ml as compared with rat peritoneal macrophages cultured with lichenan at 100 mg/ml. LPS at
10 mg/ml was used as positive control and induced TNFa levels of 111744 pg/ml above background levels (data
not shown). None of the four polysaccharides induced
any IL-10 secretion by the rat peritoneal macrophages.
Discussion
The results of this study show that the four
polysaccharides derived from the lichen T. vermicularis
var. subuliformis, thamnolan, Ths-4, Ths-5 and Ths-2,
have various influences on the immune system. For all
the polysaccharides except Ths-4, stimulation of rat
spleen cells proliferation was observed. Ths-4, Ths-5 and
Ths-2 induced rat spleen cells to secrete IL-10 above
background levels and Ths-4 and Ths-5 stimulated
TNF-a secretion by rat peritoneal macrophages.
Interestingly, spleen cells cultured with the galactofuranomannan Ths-4 were dead or dying after three
days in culture. However, substantial amount of IL-10
was detected in the supernatant collected from the
spleen cells after 72 h in culture with Ths-4, revealing
that the cells were active at some stage in the culture,
with death occurring at a later stage. Furthermore,
Ths-4 induced rat peritoneal macrophages to produce
TNF-a above background levels, indicating that the
spleen cells were not dying because of impurities within
the Ths-4 product. The reason for the massive spleen cell
death is unknown and needs to be investigated further,
but over-stimulation of the cells may be an explanation.
Ths-4 and Ths-5 are heteroglycans with similar
structure, apart from the molecular size, which is
19 kDa for Ths-4 and 200 kDa for Ths-5 (Omarsdottir
et al. 2006). These polysaccharides had very similar
effect on both IL-10 secretion by rat spleen cells and
TNF-a secretion by rat peritoneal macrophages, thus
showing similar pattern of activity apart from death of
the rat spleen cells caused by Ths-4. Thus, it may be
concluded that the molecular size might influence the
potency but not the pattern of activity for Ths-4 and
Ths-5. Molecular size-immunomodulatory activity relationship for polysaccharide has been reported previously, although for different polysaccharide structures
(Falch et al. 2000; Im et al. 2005).
Lentinan and other similar b-glucans have been used
as prescription drugs for the treatment of cancer in
Japan (Mizuno 1999). Considering the effect of the
lentinan-like Ths-2 in inducing TNF-a secretion by rat
peritoneal macrophages, and its previously reported
anti-complementary activity (Olafsdottir et al. 2003) it
will be worthwhile to study the immunological activity
of this b-glucan in more detail.
The polysaccharides Ths-4, Ths-5 and Ths-2 induced
IL-10 secretion by rat spleen cells but not by peritoneal
macrophages. These polysaccharides on the other hand,
induced the peritoneal macrophages to secrete TNF-a.
Therefore, it can be suggested that the IL-10 production
of the spleen cells did not originate from monocytes/
macrophages, but most likely from T cells; however, the
origin of the IL-10 secreting cells within the spleen cell
culture remains to be determined. The polysaccharides
Ths-2 and Ths-5 showed similar enhancement on rat
spleen cell proliferation and cytokine secretion,
although the structure of these polysaccharides are
different. The same effect has been shown previously for
four galactomannans isolated from the lichen P. canina
(Omarsdottir et al. 2005). These findings do not show
any obvious link between the structure and the
immunologcial effects of these lichen polysaccharides.
In addition, many complex polysaccharides from plants
and fungi have been shown to influence the lymphocyte
proliferation and cytokine secretion (Bao et al. 2001;
Han et al. 2001; Yamada and Kiyhara 1999) although
the assays used were different. To be able to compare
the activity of natural polysaccharides on cell proliferation and cytokine secretion, the assays would need to be
standardized.
The galactofuranorhamnan thamnolan had less mitogenic effect than Ths-5 and Ths-2 and did neither induce
IL-10 secretion by rat spleen cells nor TNF-a secretion
by peritoneal macrophages to significant levels. This
shows that thamnolan with its unusual galactofuranorhamnan structure differs from the other Thamnolia
polysaccharides in its immunomodulatory activity.
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In conclusion, it may be suggested on the basis of the
presented data that the Thamnolia polysaccharides
affect the components of the adaptive immune system
due to their stimulation of proliferation and IL-10
secretion of the rat spleen cells. In addition, the
galactofuranomannans Ths-4 and Ths-5 also affect the
components of the innate immune system by increasing
TNF-a secretion by peritoneal macrophages. The
knowledge of structural details of lichen polysaccharides
has increased in recent years, and it would be of great
interest to investigate further the effects of polysaccharides with well characterised structures, involving considerations on the effect of molecular size and threedimensional structure, on their potency and pattern of
immunomodulating activity.
Acknowledgements
The supporting grants from Icelandic Council of
Science and University of Iceland Research Fund, The
Icelandic Research Fund for Graduate Students, as well
as The Berg“óru and –orsteins Scheving Thorsteinssonar fund are gratefully acknowledged.
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