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Immunomodulating polysaccharides from the lichen Thamnolia vermicularis var. subuliformis.

Abstract

Three heteroglycans Ths-4, Ths-5 and thamnolan and a [beta]-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 [beta]-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-[alpha] 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-[alpha] 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.

[c] 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 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 Schadler, 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; Ingolfsdottir et al. 1997). Baeomycesic acid has shown inhibitory effects on 5-lipoxygenase in vitro (Ingolfsdottir et al. 1997). In addition, four polysaccharides have been described from this lichen. Three heteroglycans 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 [beta]-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; Ingolfsdottir 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-[alpha] 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, Ths-5 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 [.sup.3.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.

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-[alpha] 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 mean [+ or -] standard 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, Ths-4, Ths-5 or Ths-2 for the rat spleen cell proliferation assay, IL-10 secretion by spleen cells, and TNF-[alpha] and IL-10 secretion by rat peritoneal macrophages in vitro.

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 [.sup.1.H]-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-[alpha]) than the positive control.

Immunological activity

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

The polysaccharides Ths-5, thamnolan and Ths-2 caused a dose-dependent proliferation of the rat spleen cells, with the highest concentration tested (100 [micro]g/ml) giving the highest stimulation index for all three polysaccharides (Fig. 1). These mitogenic polysaccharides, at all concentration levels tested (11, 33 and 100 [micro]g/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 [micro]g/ml, which was used as a positive control, induced a substantial proliferation with SI of 28.6[+ or -]10.6 (data not shown).

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 [micro]g/ml, with Ths-5 at both 100 and 33 [micro]g/ml and with Ths-2 at 100 [micro]g/ml, as compared with rat spleen cells cultured with lichenan at the same concentrations. PHA at 7.5 [micro]g/ml, used as positive control, induced IL-10 secretion of 547[+ or -]379 pg/ml above background levels (data not shown).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

TNF-[alpha] and IL-10 secretion by rat peritoneal macrophages

TNF-[alpha] 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 peritoneal macrophages to secrete TNF-[alpha] levels above background levels (negative control). The TNF-[alpha] levels were significantly higher when the rat peritoneal macrophages were cultured with either Ths-4 or Ths-5 at 100 [micro]g/ml as compared with rat peritoneal macrophages cultured with lichenan at 100 [micro]g/ml. LPS at 10 [micro]g/ml was used as positive control and induced TNF-[alpha] levels of 111[+ or -]44pg/ml above background levels (data not shown). None of the four polysaccharides induced any IL-10 secretion by the rat peritoneal macrophages.

[FIGURE 3 OMITTED]

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-[alpha] 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-[alpha] 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 200kDa for Ths-5 (Omarsdottir et al. 2006). These polysaccharides had very similar effect on both IL-10 secretion by rat spleen cells and TNF-[alpha] 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 [beta]-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-[alpha] 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 [beta]-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-[alpha]. 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-[alpha] 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.

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-[alpha] 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 three-dimensional 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 Bergporu and porsteins Scheving Thorsteinssonar fund are gratefully acknowledged.

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Sesselja Omarsdottir (a), Jona Freysdottir (b), Elin Soffia Olafsdottir (a,*)

(a) Faculty of Pharmacy, University of Iceland, Hagi, Hofsvallagata 53, IS-107 Reykjavik, Iceland

(b) NaturImm Ltd./Centre for Rheumatology Research, Landspitali-University Hospital, IS-101 Reykjavik, Iceland

Received 17 February 2006; accepted 19 April 2006

*Corresponding author. Tel.: +354 5255804; fax: +354 5254071.

E-mail address: elinsol@hi.is (E.S. Olafsdottir).
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Author:Omarsdottir, Sesselja; Freysdottir, Jona; Olafsdottir, Elin Soffia
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Drug overview
Geographic Code:4EXIC
Date:Feb 1, 2007
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