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Binding studies of an arabinogalactan-protein from Echinacea purpurea to leucocytes.

Abstract

Flow cytometric investigations show binding of an isolated arabinogalactan-protein (AGP) from pressed juice of the aerial parts of Echinacea purpurea to the cell surface of human leucocytes. AGP demonstrates binding to lymphocytes, monocytes and granulocytes of different donors (n = 8). Competition assays with two antibodies, directed against CD4 and CD8, revealed no interaction of AGP with these receptors, leading to the conclusion that binding of AGP to leucocytes is mediated via other structures.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Arabinogaiactan-protein; Echinacea purpurea; Flow cytometry

Introduction

Pressed juice of Echinacea purpurea is an ingredient of herbal remedies used as non-specific immunostimulants. Arabinogalactan-protein (AGP) isolated from this pressed juice is a high molecular weight glycoprotein (1.2 x [10.sup.6] Da) with a highly branched carbohydrate moiety of >90% (w/w), mainly consisting of 3-, 6- and 3,6-linked [beta]-D-Galp residues, substituted with terminal [alpha]-L-Araf residues and terminal GlcA. The Ara/Gal ratio is 1:1.8. The protein part (7.1% w/w) is rich in Hyp, Ser, Thr, and Ala (Classen et al., 2000). Wagner et al. (1988) isolated an acidic arabinogalactan (AG) from cell cultures of E. purpurea with a different structure: this AG contains Ara:Gal in a ratio of 1:1 and in contrast to our AGP 3,5-linked Ara and 1,4-linked GalA. The AG stimulates phagocytosis and release of TNF by macrophages. Our AGP has been shown to have complement-stimulating activities in vitro (Alban et al., 2002). Pharmacological investigations of AGPs from various plants have demonstrated further immunomodulatory activities. For example they enhance the proliferation and IgM-production of mouse lymphocytes as well as nitrite and IL6-production of mouse macrophages (Classen et al., 2005b). In order to get information about possible interactions of AGPs with the immune system on a cellular level, flow cytometric investigations have been carried out with the help of antibodies. In the last years, a polyclonal antiserum against a high molecular weight polysaccharide/glycoprotein fraction from E. purpurea has been raised in rabbit (Egert and Beuscher, 1992), and a monoclonal antibody raised against rhamnogalacturonan-I recognizes an AG epitope present in an AG from cell culture of E. purpurea (Steffan et al., 1995). For our investigations we used a polyclonal antibody, generated against a purified AGP from E. purpurea. Araf residues form a key part of the antigen epitope for these polyclonal antibodies (Classen et al., 2005a).

Material and methods

FACS analysis was performed with blood from eight human healthy donors. Coagulation was anticipated with Na-EDTA. Blood (100 [micro]l) was treated with FACS-Lysing solution (Becton Dickinson, USA) in order to destroy erythrocytes. Leucocytes were separated by centrifugation and washed with phosphate buffered saline (PBS pH 7.2). Isolated AGP (100 [micro]l) (concentration 0.01-100 [micro]g/ml) from E. purpurea (Classen et al., 2000) was added before incubation at room temperature for half an hour. After addition of 1 ml PBS and centrifugation and supernatant was discarded. The primary polyclonal antibody, directed against AGP and generated in rabbit (Classen et al., 2005b) was added (10 [micro]l, diluted 1:100 with PBS) and cells were incubated for half an hour. PBS (1 ml) was added to the sample, centrifugated and the supernatant discarded. The procedure was carried out again with the secondary antibody, generated in goat, directed against rabbit IgG(H + L) and conjugated with phycoerythrin (Caltag Cat#L43004, USA). Incubation was carried out in the dark. A blank value was developed by using 100 [micro]l PBS instead of AGP. Flow cytometric investigations were done by FACSCalibur (Becton Dickinson, USA) and stopped after 20,000 measured events, analyzed by cell quest software (Becton Dickinson, USA). The dot plot histogram was analyzed and the difference between the measured fluorescence intensity of blank value and AGP values shows the level of AGP binding, declared in percentage of increase.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Investigations with CD4 and CD8 antibodies were performed each with one donor in nearly the same manner. The antihuman CD4 antibody (BD Biosciences Heidelberg, Germany) conjugated with fluorescein-iso-thiocyanate (FITC) was added (10 [micro]l, diluted 1:10 with PBS) together with AGP in rising concentrations (0.01-100 [micro]g/ml) and incubated for half an hour. The FITC labelled antihuman CD8 antibody (10 [micro]l, diluted 1:10 with PBS), supplied by BD Biosciences, Heidelberg, Germany, was used in the same manner.

Results and discussion

Leucocytes can be differentiated into lymphocytes, monocytes and granulocytes, which can be separately analyzed by flow cytometry analysis. Fig. 1 displays a representative dot plot resulting from such kind of analysis. The amount of fluorescence intensity (Fig. 2) indicates the binding capacity of AGP to the cells. Increasing AGP concentrations induce an increase of fluorescence intensity for all cell types, demonstrating that lymphocytes, monocytes and granulocytes significantly bind AGP on their cell surface. Table 1 shows the factor of increase in fluorescence intensity for incubation of cells with increasing concentrations of AGP. Highest factors (7.0-14.0) of increase in AGP binding are obtained between 0.01 and 0.1 [micro]g/ml AGP with all cell types. Other factors for higher AGP concentrations steps are much lower (1.1-3.0). Furthermore, the AGP was tested with regard to interaction with CD4 and CD8, two receptors expressed mainly on lymphocytes (Figs. 3 and 4). Binding of the FITC-labelled antibodies against CD4 and CD8 is not reduced by increasing concentrations of AGP, indicating lacking competition of AGP with the antibodies for the same epitopes on CD4 and CD8 antigen. Furthermore it is most probable that AGP does not interact with any other epitopes on CD4 and CD8 antigen, because an AGP-CD4 or -CD8 receptor complex would inhibit binding of CD4- or CD8-antibodies due to size of AGP with a molecular weight of 1.2 x [10.sup.6] Da. Altogether the results suggest a binding of AGP to structures or receptors, which are expressed on all three tested cell types. Binding seems to be not just only unspecific. With increasing concentrations of AGP more AGP binds to the cell surface, but without linear correlation. The factor of increase of binding of AGP is very high between 0.01 and 0.1 [micro]g/ml AGP. At higher concentrations the preferred binding sites seem to be occupied and binding of AGP to other structures of the cell surface is less specific, resulting in low factors of increase (Table 1). Possible interactions of AGP with CD4 or CD8 could not be demonstrated, so further investigations are necessary to find structures or receptors binding AGP.

[FIGURE 3 OMITTED]

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Acknowledgement

The authors thank Madaus AG, Koln, Germany for financial support of this work.

References

Alban, S., Classen, B., Brunner, G., Blaschek, W., 2002. Differentiation between the complement modulating effects of an arabinogalactan-protein from Echinacea purpurea and heparin. Planta Med. 12, 1118-1124.

Classen, B., Witthohn, K., Blaschek, W., 2000. Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the [beta]-glucosyl Yariv reagent. Carbohydr. Res. 327, 497-504.

Classen, B., Mau, S.-L., Bacic, A., 2005a. The arabinogalactan-proteins from pressed juice of Echinacea purpurea belong to the "hybrid" class of hydroxyprolinerich glycoproteins. Planta Med 71, 59-66.

Classen, B., Thude, S., Biaschek, W., Wack, M., Bodinet, C., 2005b. Immunomodulatory effects of arabinogalactan-proteins from Baptisia and Echinacea. Phytomedicine, submitted for publication.

Egert, D., Beuscher, N., 1992. Studies on antigen specifity of immunoreactive arabinogalactan proteins extracted from Baptisia tinctoria and Echinacea purpurea. Planta Med. 58, 163-165.

Steffan, W., Kovac, P., Albersheim, P., Darvill, A.G., Hahn, M.G., 1995. Characterization of a monoclonal antibody that recognizes an arabinosylated (1 [right arrow] 6)-[beta]-D-galactan epitope in plant complex carbohydrates. Carbohydr. Res. 275, 295-307.

Wagner, H., Stuppner, H., Schafer, W., Zenk, M., 1988. Immunologically active polysaccharides of Echinacea purpurea cell cultures. Phytochemistry 27, 119-126.

S. Thude (a), B. Classen (a,*), W. Blaschek (a), D. Barz (b), H. Thude (b)

(a) Department of Pharmaceutical Biology, Institute of Pharmacy, University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany

(b) Institute of Transfusion Medicine, University of Jena, Stoystr. 3, 07740 Jena, Germany

*Corresponding author. Tel.: +49 431 8801 130; fax: +49 431 8801 102.

E-mail address: bclassen@pharmazie.uni-kiel.de (B. Classen).
Table 1. Factors of increase describe the increase of fluorescence
intensity between declared concentrations

 Factor of increase
[micro]g/ml Monocytes Granulocytes Lymphocytes

 0.01-0.1 14 7.6 7.0
 0.1-1 1.8 1.9 2.5
 1-10 2.4 2.0 3.0
10-100 1.4 2.2 1.1
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Author:Thude, S.; Classen, B.; Blaschek, W.; Barz, D.; Thude, H.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Jun 1, 2006
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