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Effects of lichen heteroglycans on proliferation and IL-10 secretion by rat spleen cells and IL-10 and TNF-[alpha] secretion by rat peritoneal macrophages in vitro.

Abstract

Four polysaccharides Pc-1, Pc-2, Pc-3 and Pc-4 were isolated from water and alkali extracts of the lichen Peltigera canina using ethanol fractionation, gel filtration and preparative HP-GPC. The monosaccharide composition was determined by methanolysis and GC and showed mannose and galactose as the predominating structural units. The mean [M.sub.r] was determined by HP-GPC. The heteroglycans were tested for in vitro immunomodulating activities and showed mitogenic activity in rat spleen cell proliferation assay and stimulated IL-10 secretion. In rat peritoneal macrophages, the heteroglycans stimulated TNF-[alpha] secretion, but not IL-10 secretion. These results indicate that the polysaccharides influence cells of the immune system both from the innate and the adaptive systems.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Lichens; Polysaccharides; Peltigera canina; Spleen cell proliferation; IL-10; TNF-[alpha]; Immunomodulating activity

Introduction

The foliose lichen Peltigera canina (L.) Willd. belonging to the family of Peltigeraceae (Kristinsson, 1964), is a lichen species that grows circumpolar in the northern hemisphere, arctic to temperature but is scattered and rare in the southern hemisphere (Vitikainen, 1994).

Powdered P. canina mixed with black pepper and warm milk was used in traditional medicine for the treatment of rabies (Moberg and Holmasen, 1982; Hale, 1974) and this lichen is also eaten in India as a remedy for liver ailments and it has been suggested that the alleged curative power may be contributed to its high content of the amino acid methionin (Hale, 1974; Saklani and Upreti, 1992).

Previous investigations for chemical constituents indicate that P. canina contains glycosidic compounds of allo-murolic acids (Rezanka and Guschina, 2000), 18-hydroxy-protoconstipatic acid (Rezanka and Guschina, 2001a) and 18S-hydroxy-neodihydroprotolichesterinic acid (Rezanka and Guschina, 2001b; Krog et al., 1994; Vitikainen, 1994; Martinez et al., 1997). The presence of tenuiorin in P. canina has been claimed by Solberg (1975) and several other authors (Culberson, 1969); however this may be due to mixed or misidentified specimens as suggested by Vitikainen (1994) and needs to be confirmed. A galactomannan has been described from a related species Peltigera aphthosa (Gorin and Iacomini, 1985), but the polysaccharide content of P. canina has not been reported before.

Many naturally occurring polysaccharides from plants, fungi and lichens have been found to have immunomodulating activities (Wagner and Proksch, 1985; Wagner, 1991; Olafsdottir and Ingolfsdottir, 2001) as well as anti-tumour (Fukuoka et al., 1968; Shibata et al., 1968; Maeda and Chihara, 1999) and anti-viral (Stubler and Buchenauer, 1996) activities. However, these studies often suffer from lack of purity and homogeneity of the polysaccharides involved (Paulsen et al., 2002). A few chromatographically purified homogenic lichen polysaccharides, including two heteroglycans have been shown to induce phagocytosis and/or anti-complementary activity (Ingolfsdottir et al., 1994; Olafsdottir et al., 1999a, b; Olafsdottir et al., 2003). However, in vitro studies on the effects of lichen polysaccharides on cell proliferation and cytokine secretion have not been reported before.

The aim of the present work was to isolate and chromatographically purify both water- and alkali-soluble polysaccharides from the lichen P. canina, to characterise their structures to some extend and to investigate their effect on rat spleen cells and peritoneal macrophages in vitro. The polysaccharides were tested for mitogenic activity in rat spleen cell proliferation assay, and their ability to stimulate in vitro production of IL-10 by rat spleen cells and TNF-[alpha] and IL-10 production by rat peritoneal macrophages was also investigated.

Materials and methods

Plant material

The lichen P. canina (L.) Willd (Peltigeraceae) was collected in Fljotshlid in south-western Iceland. The lichen was identified by S. Baldursdottir, lichenologist, Prokaria Ltd., Reykjavik, Iceland and by Dr. Hordur Kristinsson, Director of the Icelandic Institute of Natural History, Akureyri, Iceland. A voucher specimen (catalogue no. AMNH L-28941) is deposited at the Icelandic Institute of Natural History, Akureyri, Iceland.

General methods

Spectra/Por dialysis tubing (MWCO 6-8 kD) from Serva (Serva, Heidelberg, Germany) was used for the dialysis. GC of TMS-derivatives of the methanolysis products was performed on a DB-5 capillary column (30 m X 0.32 mm i.d.) in Carlo Elba 6000 Vega Series 2 gas chromatograph as described earlier (Olafsdottir et al., 1999a; Paulsen et al., 2002). [.sup.1]H-NMR spectra were recorded in [D.sub.2]O on a 250 MHz Bruker instrument referenced to internal C[H.sub.3]CN set to 2.06 ppm. Protein determination was performed with the Bio-Rad Protein Assay based on the Bradford dye-binding procedure (Bradford, 1976).

Isolation of polysaccharides

The dried plant material (197 g) was first extracted in a Soxhlet apparatus with three different organic solvents (light petroleum, acetone and methanol), followed by extraction with hot (95[degrees]C) distilled water for 2 h and hot filtration. The precipitate formed upon cooling was removed by centrifugation, and the supernatant repeatedly treated by freezing, thawing and centrifugation and then processed further according to previously described fractionation process (Olafsdottir et al., 1999a, b; Paulsen et al., 2002) to give 252 mg of precipitate from EtOH (1:1) (Fraction II) and 1273 mg of precipitate from EtOH (1:4) (Fraction III).

The plant residue from the water extraction was then extracted with 0.5 M aqueous NaOH according to Caldes (Caldes et al., 1981) and kept at 4[degrees]C overnight. The mixture was centrifuged and the supernatant was processed further according to previously described fractionation process (Caldes et al., 1981; Paulsen et al., 2002) to give 4.77 g of precipitate from EtOH (1:4) (Fraction V).

Anion-exchange chromatography

Fraction III was dissolved in [H.sub.2]O and applied to DEAE Sepharose fast flow anion exchange column (Amersham Biosciences, Uppsala, Sweden) (2.6 X 40 cm, acetate form) in two runs (300 mg each time). The columns were kept at 4[degrees]C and first eluted with [H.sub.2]O giving 116 mg of fraction III-a (100-260 ml), followed by a gradient of 0-1 M NaCl (800 ml) giving 266 mg of fraction III-b. The elutes were monitored by the phenol sulphuric acid assay (Dubois et al., 1956). Fractions containing polysaccharides were collected, dialysed and lyophilised.

Purification by gel permeation chromatography

The crude fractions III-a and III-b were dissolved separately in 3.0 ml of 0.2 M NaCl and purified further by gel filtration on 1.6 X 85 cm Sephacryl S-400 column (Amersham Biosciences) and eluted with 0.2 M NaCl. The resulting fractions, Pc-1 (100-270 ml) from III-a, and Pc-2 (70-120 ml) and Pc-3 (130-180 ml) from III-b, were dialysed and lyophilised to give 96 mg of Pc-1, 60 mg of Pc-2 and 89 mg of Pc-3, correlating to a yield of 0.12%, 0.06% and 0.10% of dry plant material, respectively.

One hundred and fifty milligrams of the alkali-soluble fraction V were dissolved in 3 ml of 0.2 M aqueous NaOH and purified further on a 1.6 X 85 cm Sephacryl S-400 column and eluted with the same solvent. The fractions (100-200 ml) were dialysed and lyophilised to give 101 mg of Pc-4 (1.6% of dry plant material).

Purification by preparative high-performance gel permeation chromatography

Further purification of Pc-2 and Pc-3 was performed with preparative HP-GPC on a PL-aquagel-OH 60 10 [micro]m column (Polymer Laboratories Ltd., Shropshire, UK), eluted with [H.sub.2]O using refractive index detection. The samples were applied as 1% solutions, and the injected volume was 900 [micro]l. Seven injections of Pc-2 gave 39 mg of pure Pc-2 and ten injections of Pc-3 gave 48 mg of pure Pc-3.

Determination of mean [M.sub.r] and homogeneity

Homogeneity and mean [M.sub.r] of Pc-1, Pc-2, Pc-3 and Pc-4 was determined by HP-GPC on a Superose 6 HR 10/30 column (Amersham Biosciences) eluted with 0.05 M sodium phosphate buffer pH 6.0, containing 0.15 M NaCl, with a flow rate of 0.4 ml/min, using refractive index detection (HP 1047A RI detector). The samples were applied in 1% solutions in the mobile phase, and the injected volume was 20 [micro]l. For the [M.sub.r]-estimation, calibration was performed using dextrans of known [M.sub.r] (T10, T40, T70, T250, T500 and T2000, Amersham Biosciences).

Methanolysis and determination of sugar composition

The monosaccharide composition of the purified water-soluble polysaccharides was determined by GC of the trimethylsilylated derivatives of the methyl glycosides obtained by methanolysis of 1 mg of polysaccharide in 1 ml of 4 M HCl in methanol with mannitol as an internal standard (Reinhold, 1972; Barsett and Paulsen, 1992).

Stimulation of rat spleen cells

Single spleen cell suspension was prepared by mincing the spleen from female Lewis rats (6 months old) through a 70 [micro]m nylon cell strainer (Falcon, Becton Dickinson, Erembodegem, Belgium). The cells were washed twice with PBS containing 1% foetal calf serum (FCS; Gibco, Invitrogen, Paisley, UK) (PBS-FCS) and erythrocytes in the cell mixture were destroyed by the rapid addition of nine volumes of [H.sub.2]O, followed by one volume of 10 X PBS. The cells were then suspended in culture medium (RPMI-1640, Gibco, supplemented with 10% FCS and antibiotics (Pen/Strep), Gibco) at a concentration of 1 X [10.sup.6] cells/ml. The cells were cultured in 96 well plates (Nunc, Invitrogen) in the presence of the polysaccharides Pc-1, Pc-2, Pc-3 and Pc-4 and for comparison nigeran from Stereocaulon alpinum (Olafsdottir and Ingolfsdottir, 2001) (concentration range between 11 and 100 [micro]g/ml) in 0.2 ml of culture medium for 3 days at 37[degrees]C, 5% [CO.sub.2] and 100% humidity. Phytohemagglutinin (PHA; Sigma, Deisenhofen, Germany) at 7.5 [micro]g/ml was used as a positive control and medium only as a negative control. The effect of the polysaccharides on the rat spleen cells was determined by measuring cell proliferation and cytokine secretion.

Stimulation of rat peritoneal macrophages

Peritoneal rat macrophages were collected by infusing the peritoneal cavity with ice-cold PBS. The cells were washed twice with PBS-FCS. The cells were then suspended in culture medium at a concentration of 2 X [10.sup.6] cells/ml. The cells were cultured in the presence of the polysaccharides Pc-1, Pc-2, Pc-3 and Pc-4 and for comparison thamnolan from Thamnolia vermicularis var. subuliformis (Olafsdottir et al., 1999b) (concentration range between 11 and 100 [micro]g/ml) in 0.5 ml of culture medium in 48 well plates (Nunc) and incubated for 3 days at 37[degrees]C, 5% [CO.sub.2] and 100% humidity. Lipopolysaccharide (LPS; Sigma) at 10 [micro]g/ml was used as a positive control and medium only as a negative control. 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. [.sup.3]H-thymidine (Amersham Biosciences) was added at 0.5 [micro]Ci per well and the cells incubated for further 16 h. The cells were harvested on glass fibre filters using Filtermate cell harvester (Packard, USA) and the radioactivity bound to the filters was determined using a [beta]-scintillation counter (TopCount, Packard), with data expressed as counts per minute (cpm). The results represent means of four repeat experiments and are expressed as stimulation index, which was calculated by dividing cpm of stimulated cells with cpm of unstimulated cells.

Cytokine ELISA

The supernatant from the cell cultures 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 method using reagents from R & D Systems (Abingdon, UK). Maxisorp plates (Nunc) were coated with purified mouse anti-rat IL-10 or anti-rat TNF-[alpha] and incubated at room temperature overnight. The wells were blocked with 1% BSA, 5% sucrose and 0.05% Na[N.sub.3] in PBS for 1 h at room temperature. Tissue culture supernatant, diluted 1:2, was added to both plates as well as recombinant rat IL-10 (0.25-16 ng/ml) or recombinant rat TNF-[alpha] (31.2-2000 pg/ml) for making the standard curve and the plates were then incubated overnight at 4[degrees]C. Following four washes with PBS the plates were incubated with biotinylated goat anti-rat IL-10 or biotinylated goat-anti-rat TNF-[alpha] for 2h at room temperature. The plates were washed as before and then incubated with HRP-conjugated streptavidin for 20 min at room temperature, followed by four washes. Finally the plates were incubated with TMB substrate (Kirkeagaard and Perry Laboratories, MD, USA) and the reaction stopped by addition of 0.18 M sulphuric acid. Absorbance was measured at 450 nm and the amount of IL-10 and TNF-[alpha] production by the rat peritoneal macrophages and IL-10 secretion by rat spleen cells was read from standard curves which were constructed using the concentration of the recombinant IL-10 or TNF-[alpha] on x-axis and the absorbance on y-axis. Each experiment was run in duplicate and the results represent means of four repeat experiments.

Statistical analysis

The data were represented as mean [+ or -] standard deviation (sd) of four experiments. Statistical evaluation was performed using Student's t-test for unpaired observations between Pc-1, Pc-2, Pc-3 or Pc-4 and nigeran for rat spleen cell proliferation assay and IL-10 secretion of spleen cells, and thamnolan for the TNF-[alpha] secretion measurements. Nigeran and thamnolan did not induce any response.

Results and discussion

Isolation and structure elucidation

The cold water soluble polysaccharide fractions III-a and III-b and fraction V from the alkali extraction from P. canina L. were purified with gel filtration to give the homogenic heteroglycans Pc-1, Pc-2, Pc-3 and the galactoglucomannan Pc-4. The monosaccharide composition and mean [M.sub.r] of the four heteroglycans is shown in Table 1.

No glucans could be detected in this lichen and the galactoglucomannan Pc-4 (yield 1.6%) from the alkali extract is the most abundant polysaccharide of P. canina. The protein content of Pc-1, Pc-2, Pc-3 and Pc-4 was determined to be less than 1.0% (Bradford, 1976) and [.sup.1]H-NMR spectra of the four glycans showed no signs of proteins or lipopolysaccharides.

Immunological activity

The effect of the P. canina heteroglycans on immunological activity was analysed by culturing rat spleen cells and rat peritoneal macrophages in the presence of the polysaccharides and subsequently measuring cell proliferation and cytokine secretion. The polysaccharides showed mitogenic activity in the proliferation assay and stimulated IL-10 secretion by the spleen cells, whereas they stimulated TNF-[alpha] secretion, but not IL-10 secretion, by the macrophages (Figs. 1-3).

All the polysaccharides caused a dose-dependent proliferation of the rat spleen cells, with the highest concentration, 100 [micro]g/ml, giving the highest stimulation index (Fig. 1). The Pc-1 induced the highest SI (10.9 [+ or -] 4.5), and the Pc-4 the lowest SI (2.3 [+ or -] 0.4), which are significantly higher than the SI for nigeran, which did not cause proliferation, when analysed by student's t-test. Pc-1, Pc-2 and Pc-3 did all stimulate significant spleen cell proliferation at lower concentrations. PHA at 7.5 [micro]g/ml was used as a positive control and induced substantial proliferation, with SI being 28.6 [+ or -] 10.6 (data not shown).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The IL-10 secretion from stimulated spleen cells were measured by sandwich ELISA. All of the four polysaccharides stimulated the spleen cells to secrete IL-10 above background levels (spleen cells cultured with medium only) and the levels reach statistically significant higher levels than when cells were stimulated with nigeran in most concentrations tested (Fig. 2). With the exception of Pc-3, the highest concentration, 100 [micro]g/ml, induced the highest levels of IL-10 secretion with Pc-1 of 420 [+ or -] 178 pg/ml (p = 0.02), Pc-2 of 505 [+ or -] 89 pg/ml (p = 0.005), Pc-3 of 157 [+ or -] 88 pg/ml (p = 0.04) and Pc-4 of 176 [+ or -] 87 pg/ml (p = 0.04). At concentration of 33 [micro]g/ml, all the polysaccharides induced less IL-10 than at a concentration of 100 [micro]g/ml except Pc-3, which induced IL-10 secretion of 242 [+ or -] 78 pg/ml (p = 0.007). At a concentration of 11 [micro]g/ml, Pc-1 and Pc-2 yet induced significantly higher IL-10 secretion than nigeran (266 [+ or -] 86 pg/ml, p = 0.04 and 334 [+ or -] 78 pg/ml, p = 0.007, respectively). PHA at 7.5 [micro]g/ml was used as a positive control and stimulated IL-10 secretion of 547 [+ or -] 379 pg/ml (data not shown).

The results of the in vitro TNF-[alpha] and IL-10 secretion by peritoneal macrophages (pM0) show that the polysaccharides stimulated the macrophages to secrete TNF-[alpha] above background levels (Fig. 3) but not IL-10 (data not shown). At a concentration of 100 and 33 [micro]g/ml all the polysaccharides induced the pM0 to secrete significantly higher levels of TNF-[alpha] than when stimulated with thamnolan. The highest levels observed were for Pc-1 at 100 and 33 [micro]g/ml which stimulated 521 [+ or -] 288 pg/ml (p = 0.01) and 488 [+ or -] 210 pg/ml (p = 0.008), respectively, and for Pc-3 at 33 and 11 [micro]g/ml which stimulated 497 [+ or -] 375 pg/ml (p = 0.05) and 469 [+ or -] 395 pg/ml (p = 0.07), respectively. LPS (10 [micro]g/ml) was used as a positive control and induced TNF-[alpha] secretion of 111 [+ or -] 44 pg/ml above background levels (data not shown).

This study shows that all four polysaccharides from P. canina have an effect on the immune system by inducing proliferation and IL-10 secretion of rat spleen cells and TNF-[alpha] secretion of rat peritoneal macrophages.

The polysaccharides induced IL-10 secretion by rat spleen cells but not by peritoneal macrophages. However, the polysaccharides induced peritoneal macrophages to induce TNF-[alpha] secretion. Since the peritoneal macrophages did not secrete IL-10 following stimulation with the polysaccharides, it can be suggested that the IL-10 secretion of the spleen cells does not originate from monocytes/macrophages. It has, however, been noted that mouse macrophages from spleen and peritoneum do not secrete the same cytokine profile (D. Petursdottir and I. Hardardottir, Pers. Comm.). The cellular origin of the IL-10 within the spleen cell culture still remains to be determined.

Complex polysaccharides from higher plants, and also a few glucans and heteroglycans from fungi, have been shown to enhance lymphocyte proliferation, and influence cytokine secretion (Yamada and Kiyohara, 1999; Bao et al. 2001, 2002; Han et al. 2001). However the structures of the P. canina heteroglycans are very different from those of the polysaccharides tested before, and in addition the assays used are different. To be able to compare the activity of natural polysaccharides on cell proliferation and cytokine secretion, the assays would need to be standardized.

Many lectins and carbohydrates are known to stimulate different cell types, such as PHA and Concanavalin A which stimulate T cells, pokeweed mitogen which stimulates B and T cells and LPS which stimulates B cells and monocytes (Janeway et al., 2001). Apart from LPS, these mitogens are all of plant origin. Therefore, it would be interesting to analyse further, which cell types in the rat spleen cell culture are involved in the enhancement of cell proliferation and IL-10 secretion.

It is of interest to observe that the polysaccharides caused rat peritoneal macrophages to secrete the proinflammatory cytokine TNF-[alpha], but not the anti-inflammatory cytokine IL-10. In contrast, the rat spleen cells produced high levels of IL-10 following stimulation with the same polysaccharides. This suggests that the polysaccharides are having opposite immunological effects at different sites within the body.

In conclusion, the lichen P. canina contains biologically active polysaccharides, which are mainly heteroglycans predominated by mannose and galactose monosaccharide structural units. More detailed structural analysis using methylation analysis and NMR spectroscopy are in progress. It might be suggested on the basis of the results described above that the polysaccharides affect the components of the innate immune system involving the peritoneal macrophages and might also affect cells of the adaptive immune system due to their stimulation of proliferation of rat spleen cells and induced IL-10 secretion of spleen cells but not on peritoneal macrophages. Further research is needed to determine the immunomodulating effects of the lichen polysaccharides of P. canina in more detail.

Acknowledgements

Supporting grants from Icelandic Council of Science and University of Iceland Research Fund, The Icelandic Research Fund for Graduate Students, as well as from the Nordic Council of Ministers, are gratefully acknowledged.

References

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S. Omarsdottir (a), J. Freysdottir (b), H. Barsett (c), B. Smestad Paulsen (c), E.S. Olafsdottir (a,*)

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

(b) Lyfjathroun Biopharmaceuticals Ltd, Vatnagordum 16-18, IS-104 Reykjavik, Iceland

(c) Institute of Pharmacy, Department of Pharmacognosy, University of Oslo, P. B. box 1068, N-0316 Oslo, Norway

Received 13 November 2003; accepted 5 March 2004

*Corresponding author. Tel.: + 354 525 5804; fax: + 354 525 4071.

E-mail address: elinsol@hi.is (E.S. Olafsdottir).
Table 1. Monosaccharide composition and ratios (%) for the four
polysaccharides determined by methanolysis followed by GC of the TMS-
derivatives. Their mean molecular weights (kDa), estimated by
calibration of dextrans of known Mr, are also shown

Monosaccharides Pc-1 Pc-2 Pc-3 Pc-4

Arabinose 1 2 1 0
Rhamnose 1 5 1 0
Fucose 1 2 1 0
Xylose 2 3 4 0
Mannose 46 49 64 67
Galactose 27 31 29 30
Glucose 22 8 0 3
Mean [M.sub.r] 340 659 12 53
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Author:Omarsdottir, S.; Freysdottir, J.; Barsett, H.; Paulsen, B. Smestad; Olafsdottir, E.S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Jun 1, 2005
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Belamcanda chinensis and the thereof purified tectorigenin have selective estrogen receptor modulator activities.
Immunomodulating polysaccharides from the lichen Thamnolia vermicularis var. subuliformis.
In vitro evaluation of antibacterial and immunomodulatory activities of Pelargonium reniforme, Pelargonium sidoides and the related herbal drug...
Differential protein expression in mouse splenic mononuclear cells treated with polysaccharides from spores of Ganoderma lucidum.
Effects of flaxseed lignan on in vitro mitogen-stimulated t cell proliferation.

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