Experimental immunology
Influence of polysaccharide fraction C isolated from Caltha palustris L. on T and B lymphocyte subsets in mice

Introduction

Natural products have been shown to be an excellent and reliable source for the development of new drugs [1]. A large number of plant extracts used in traditional medicine have been shown to possess nonspecific immunomodulating activities [2]. Thus, they can be an alternative source of bioactive agents of medical significance. Remedies derived from Echinacea sp. might serve as an example. They have been widely used as commercial formulations, but their immunomodulatory properties are still under investigation [3].

Caltha palustris L. (Ranunculaceae) is a plant widely known and distributed in Europe, Asia and North America. The extracts obtained from Caltha palustris have been used in traditional medicine: it has been used to treat arthritis [4] leprosy, rheumatism, gonorrhea [5], and a variety of skin lesions [6]. Some studies demonstrated that water extract of Caltha palustris presents slight oncostatic activity against some mice tumors (Sarcoma S-180, Ehrlich carcinoma) [7]. Isolation of the polysaccharide fractions of tested plant and their chemical and physical properties were described [8].Also, some of their biological effects were studied in vitro [9]. It was demonstrated that fraction C stimulated both lymphocyte and the granulocyte activation, whereas it did not exert the effect on the proliferation of human leukemic cell cultures. Fraction C from Caltha palustris L. extract enhanced in vitro proliferative activity of PHA-induced human lymphocyte, and elevated the granulocytes H₂O2 production and release in the cultures without the addition of PHA.

This study examined the Caltha palustris polysaccharides fraction C in in vivo tests. Specifically, the im­munomodulatory effect on T cell subsets in thymus and T and B lymphocytes in the spleen and mesenteric lymph nodes were examined and characterized.

Material and methods

Plant material and preparation of polysaccharide fraction



The plant material was collected near Trzebnica (West Poland). The dried, powdered material (1000 g) was extracted with methanol (5L) for 48 h and afterwards, the solution was discarded. After evaporation of the dissolvent, the residual herb was extracted with distilled water to obtain water extract (A) and residue. Then, the residue was extracted with 0.1 M NaOH to obtain extract B and another residue which was extracted with 5% CH3COOH to obtain extract C. Extract C was reduced in vacuo to 2L without heating. Next, it was centrifuged and the supernatant was treated with TCA and then with acetone. Fraction C, obtained from 5% CH3COOH extract tested positive for saccharides in reaction with phenol reagent [10] and was found to contain polysaccharides of molecular weight 3,6 × 104 Da and 2.5 ×104 Da (Biogel-P10 – gel permeation chromatography). Total sugar content (39%) was determined as anhydroglucose after Dubois et al. [10], uronic acids (4.8%) by the Blumenkrantz method [11]. Investigated fraction also contained a significant quantity of ash –24%.

A stock solution of the plant extract fraction was prepared ex tempore by dissolving 2 mg of fraction C in phosphate-buffered saline solution (PBS, Institute of Immunology and Experimental Therapy, Wrocław, Poland).



Animals



The studies were conducted on male and female Balb/c mice (8-10 weeks of age), each weighing 20-22 g. The mice were obtained from a breeding laboratory at the Medical University, Wrocław, Poland. The animals were fed a commercial granulated food and water ad libidum and kept under the conventional conditions. The Local Ethics Committee approved the studies (No. 22/2008).



Drugs and treatment



The solution of fraction C from Caltha palustris extract at doses of 10, 1, and 0.1 mg/kg, was administered intraperitoneally, once or five times at 24 h intervals. The volume of each dose was 0.2 ml/mouse. The trials in control group were conducted in parallel. The mice in the control group were treated with phosphate buffered saline – PBS

(0.2 ml/mouse). Each control and experimental group consisted of seven mice.



Measurements



The following measurements were taken:

• the total number of thymocytes, splenocytes and lymphocytes of the mesenteric lymph nodes,

• the weight ratio of the thymus, spleen, mesenteric lymph nodes calculated according to the following formula: weight of organ (g)/body weight of mouse (g) × 100,

• the percentage and count of CD subsets (CD4–CD8–, CD4+ CD8+, CD4+, CD8+ in thymus, CD19+, CD3+, CD4+, CD8+ in spleen and mesenteric lymph nodes).

The measurements were determined at two time points: 24 (day 1) and 72 h (day 3) after last administration of fraction C from Caltha palustris extract.



Assay of thymocyte, splenocyte and lymphocyte of mesenteric lymph node subpopulations



The mice were anesthetized with halothane, (Narcotan, Zentiva, Prague, Czech Republic), and killed by cervical dislocation 24 and 72 h after last administration of fraction C from Caltha palustris extract. Thymus, spleens and mesenteric lymph nodes were removed and placed in disposable Petri dishes containing sterile, ice-cold PBS.

The suspended cells were removed from the lymphatic organs by gentle passage through a nylon mesh and then centrifuged (2250 × g, 15 min, 4^oC) on a layer of Ficoll

400 (Pharmacia, Fiene Chemicals AB, Sweden)/Uro­polinum 75% (diatrizoate sodium and meglumine diatrizoate; Polpharma, Poland) in a 1 : 3 ratio at a density of 1.071. After centrifugation, the cells were collected from the interphase and washed twice (375 × g, 8 min, 4^oC) with PBS supplemented with 1% bovine serum albumin (BSA, Sigma, USA) at 4^oC. After the second wash, the cells were resuspended in PBS with 1% BSA at 1 × 107 cells/ml. The viability of each cell suspension, as determined by trypan blue dye exclusion, was 90-95%. The cells were resuspended in 100 µl PBS solution containing 1% BSA. The thymocytes, splenocytes and lymphocytes of mesenteric lymph nodes were stained with the monoclonal antibodies: Rat Anti Mouse CD4 : FITC/CD8 : RPE dual color reagent (Serotec, Kidlington UK), at the dilutions recommended by the manufacturer. The splenocytes and the lymphocytes of the mesenteric lymph nodes were also stained with the antibodies: Rat Anti Mouse CD19 : FITC/CD3 : RPE dual color reagent (Serotec, Kidlington. UK), according to the manufacturer’s instruction.

Cells were incubated at 4^oC for 30 min and then washed 3 times with ice-cold PBS. Fluorescence was analyzed using a flow cytometer (FACS Calibur; Becton Dickinson, Germany). Lymphocyte marker distribution was analyzed using the Cell Quest 3.1f software.



Statistical analysis



The data obtained in the study were analyzed statistically using a t-test. The differences were considered significant at p < 0.05.

Results

As can be seen in Table 1, a single administration of fraction C from Caltha palustris extract at all three doses (0.1 mg/kg, 1 mg/kg, 10 mg/kg), on day 1 after injection, temporarily but significantly decreased the percentage and the absolute count of mature CD8+ thymic cells. Whereas 3 days after a single administration, the significant increase of the percentage of the CD8+ cells in thymus at the doses of 10 and 0.1 mg/kg was found (Table 1). Further, 1 day after the administration of a single dose of 10 mg/kg, the decrease of the total number of thymocytes was observed. It was followed by the decrease of absolute count of immature CD4+ CD8+ thymocytes (double-positive cells).

As can be seen in Table 1, five times administration of fraction C from Caltha palustris extract increased significantly the absolute count and the percentage of CD4+ thymic cells, irrespective of the dose applied. Additionally, the increase of total number of thymocytes was observed on day 1 after the last administration of five injections at doses 1 and 0.1 mg/kg of fraction C.

No changes in the absolute count and the percentage of immature CD4– CD8– (double-negative cells) thymocytes were observed.

Single administration of fraction C from Caltha palustris extract at a dose of 10 mg/kg increased the percentage and absolute count of CD3+ (Pan-T cells), which corresponded with a decreased percentage of the CD8+ lymphocytes in the spleen (Table 2).

However, five exposures to fraction C (1 and 0.1 mg/kg) increased percentage of CD4+ splenocytes, as can be seen in Table 2. Multiple administration of examined fraction C at the doses of 0.1 and 10 mg/kg increased the percentage of CD19+ (B lymphocytes) in the spleen but a single administration did not exert such influence.

As can be seen in Table 3, five times administration of fraction C from Caltha palustris extract at all doses under investigation augmented the absolute count and the percentage of CD19+ (B lymphocytes) in the mesenteric lymph nodes. The strongest stimulating effect on B lymphocytes of mesenteric lymph nodes was observed after five injections of fraction C at a dose of 0.1 mg/kg. A decrease in

the percentage of CD4+ and CD3+ lymphocytes at all doses and in the percentage of CD8+ at the doses of 1 and

10 mg/kg was also demonstrated (Table 3). However, after 3 days from last administration of the fraction C at the dose of 1 mg/kg, the increase in the percentage and in the absolute count of CD3+ mesenteric lymphocytes, with an accompanying decrease in B lymphocytes, was found (Table 3).

Discussion

Polysaccharide compounds in other plants (such as Solanum nigrum, Opilia celtidifolia, Potentilla anserina) have been reported to have immunomodulating activities [12-14]. In the present study, we confirmed these properties also in polysaccharide fraction C of Caltha palustris.

The number and ratio of two main lymphocyte T subsets (CD4+ cells-T helpers, and CD8+ cells-T cytotoxic/suppressors) have been recognized as the most meaningful parameters for evaluating the balanced state of immunomodulation and homeostatic responses of the intrinsic immune system [15]. CD4+ T cells recognize antigens presented by the major histocompatibility complex class II (MHC II) proteins and mediate both cellular immune responses through Th1 cells and humoral immune responses through Th2 cells. The recognition of antigens presented by MHC I molecules and mediation of cellular immune responses through cytotoxic T cells are the domain of CD8+ cells.

In the present study, the increase of the CD4+ lymphocyte population (thymus, spleen) after administrating fraction C from Caltha palustris extract was reported. This effect was similar to the results reported by Wu et al. [16], who investigated the traditional Chinese medicine, Chi-Shie-Shuang-Bu-An-Shen-Tang (CST). The oral administration of sterilized CST for 3 weeks caused an increase in the population of CD4+ T cells in spleen. The authors suggest that probably polysaccharide component was responsible for this activity. According to tha data obtained in this study, the increase in CD4+ population may be potentially benefitial, especially in the states of CD4+ lymphopenia. Such conditions may result from systemic diseases, i.e. systemic lupus erythrematosus [17] or action of drugs, i.e. cyclophosphamide [18], and may increase the risk of infection.

CD4+ and CD8+ are also two subpopulations which, in peripheral blood, play a crucial role in adult host defense toward cancer [19]. Li et al. [12] demonstrated the protective effect of fraction 1a of polysaccharides, isolated from Solanum nigrum Linne (SNL-P1a), on thymus in tumor-bearing mice. They studied the profile of CD4+ and CD8+ in PBMC (peripheral blood mononuclear cell) and showed that treatment with SNL-P1a following tumor implantation caused a significant increase in the number of CD4+ T-lymphocyte and a decrease in the number of CD8+ T-lymphocyte in peripheral blood of tumor-bearing mice.

In our study, a significant decrease in the number of single-positive CD8+ thymocytes after administration of fraction C from Caltha palustris extract was found.

Our study showed that multiple administration of examined fraction C increased the CD19+ B– lymphocyte

population. Stimulating activity of other polysaccharide extracts was demonstrated in the studies on polysaccharide isolated from the radix of Platycodon grandiflorum (PG) [20]. Platycodon grandiflorum was found to markedly increase polyclonal IgM antibody production and the proliferation of B cells. Moreover, the intraperitoneal administration of PG in mice immunized by using T-dependent antigen, sheep red blood cells (SRBCs), resulted in increased IgM antibody production in B cells.

The present study demonstrated that fraction C from Caltha palustris extract can change the percentage and absolute number of T cell subsets in thymus, and T and B lymphocytes in the spleen and mesenteric lymph nodes. The effect of the examined substance depended on the number of consecutive doses applied. However, more experiments should be carried out to explain the detailed mechanism that induced the immunomodulatory activity.

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