Experimental immunology
Modulation of murine T and B lymphocyte subsets by polysaccharide fraction B isolated from Caltha palustris L.

Introduction

Natural plant extracts can be an alternative and reliable source for the development of new drugs [1–3]. Various plant extracts used in traditional medicine for millennia have been shown to possess nonspecific immunomodulating properties and natural remedies derived from Aloe vera might serve as an example [4]. There are many commercial formulations on the basis of Aloe vera plant but their immunomodulatory activities are still under investigation [5]. Caltha palustris L. (Ranunculaceae) is a plant that is widely known and affluently 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 [6] leprosy, rheumatism, gonorrhea [7], and a variety of skin lesions [8]. Sokoloff’s studies demonstrated that a water extract of Caltha palustris shows slight oncostatic activity against some mice tumors (Sarcoma S-180, Ehrlich carcinoma) [9]. Isolation of the polysaccharide fractions of the investigated plant, their chemical and physical properties and some of their biological effects, which were studied in vitro were described. It was presented that fraction B suppressed both lymphocyte and granulocyte activation and diminished the proliferation of human leukemic cell cultures. Fraction B from Caltha palustris L. extract inhibited in vitro proliferative activity of PHA-induced human lymphocyte, and decreased the granulocytes H₂O2 production and release in the cultures without the addition of PMA [10, 11].

In this study we examined and characterized the immunomodulatory effect of polysaccharide fraction B of Caltha palustris extract on T cell subsets in thymus and T and B lymphocytes in the spleen and mesenteric lymph nodes. Our previous experiments focused on fraction C of Caltha palustris extract [12].

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. Extract B was reduced in vacuo to 2L without heating. Next, it was centrifuged and the supernatant was treated with TCA and then with acetone. Fraction B, obtained from 0.1 M NaOH extract tested positive for saccharides in reaction with phenol reagent [13] and was found to contain polysaccharides of molecular weight 3.4 × 104 Da and 3.2 × 104 Da (Biogel-P10 – gel permeation chromatography). Total sugar content (22%) was determined as anhydroglucose after Dubois et al. [13], uronic acids (2.8%) by the Blumenkrantz method [14]. The investigated fraction also contained a significant quantity of ash – 47%. A stock solution of the plant extract fraction was prepared ex tempore by dissolving 2 mg of fraction B in phosphate-buffered saline solution (PBS, Institute of Immunology and Experimental Therapy, Wrocław, Poland).



Animals



The studies were conducted on Balb/c mice (8-10 weeks of age; male and female), each weighing 20-22 g. The mice were obtained from the Breeding Center of Laboratory Animals at the Institute of Occupational Medicine, Łódź, Poland. The animals were kept under the conventional conditions. The mice were fed commercial granulated food and water ad libitum. The study protocol was approved by the Local Ethics Committee (No. 22/2008).



Drugs and treatment



The solution of fraction B from Caltha palustris extract at doses of 10, 1, and 0.1 mg/kg, was administered intraperitoneally (i.p.), once or five times at 24 h intervals. The volume of each dose was 0.2 ml per animal. The trials in the control group were conducted simultaneously. The mice in the control group received phosphate buffered saline – PBS (0.2 ml/mouse). Each control and experimental group consisted of seven mice.



Measurements



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

The measurements included: (a) the total number of thymocytes, splenocytes and lymphocytes of the mesenteric lymph nodes, (b) 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 and (c) 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).



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 h and 72 h after the last administration of fraction B 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°C) on a layer of Ficoll 400 (Pharmacia, Fiene Chemicals AB, Sweden)/Uropolinum 75% (diatrizoate sodium and meglumine diatrizoate; Polpharma, Poland) at a 1 : 3 ratio and density of 1.071. After centrifugation, the cells were collected from the interphase and washed twice (375 × g, 8 min, 4°C) with PBS supplemented with 1% bovine serum albumin (BSA, Sigma, USA) at 4°C. 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 of 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 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 instructions. Cells were incubated at 4°C for 30 min and then washed 3 times with ice-cold PBS. Fluorescence was analyzed using a flow cytometer (FACS Calibur; Becton Dickinson, Germany). Data acquisition and analysis were done using the Cell Quest 3.1f software.



Statistical analysis



Student’s t-test for independent samples at a significance level of 5% was used to compare the efficacy of treatment. In each case, the differences between the mean values of the control group versus treated group (fraction B at a dose of 1 × 0.1 mg/kg and 1 × 1 mg/kg and 1 × 10 mg/kg, and at a dose of 5 × 0.1 mg/kg and 5 × 1 mg/kg and 5 × 10 mg/kg, respectively, after 24 h and 72 h) were studied. The calculations were made using the Statistica 9.1 StatSoft package.

Results

As Table 2A shows, a single administration of fraction B from Caltha palustris extract at doses 1 and 0.1 mg/kg temporarily but significantly increased the absolute count of mature CD8⁺ thymic cells, measured on day 1. In turn,

3 days after a single administration of the tested fraction, the significant decrease in the absolute count of CD8⁺ cells in thymus at all three doses (0.1 mg/kg, 1 mg/kg, 10 mg/kg) was found. Further, it was followed by the decrease in the absolute count of CD4⁺CD8⁺ thymocytes (double-positive cells) and accompanied by the decrease in the total number of thymocytes, at all investigated doses. The increase in the absolute count of CD4⁺ thymic cells was observed at all three doses (0.1 mg/kg, 1 mg/kg, 10 mg/kg), only 24 h after a single injection. However, a temporarily increase in the absolute count of immature CD4^–CD8^– (double-negative cells) thymocytes 24 h after a single administration was noticed. Five times (Table 2B) administration of fraction B (0.1 mg/kg) from Caltha palustris extract significantly decreased the absolute count of CD8⁺ cells on both days. Moreover, the decrease in the absolute count of CD4⁺CD8⁺ cells was observed after the multiple administration of polysaccharide fraction B at a dose of 0.1 mg/kg measured on days 1 and 3 and 10 mg/kg on day 1. Additionally, the decrease in the percentage and absolute count was noticed after five exposures to the tested agent (0.1 mg/kg) measured on both days. After five injections of fraction B, the decrease in the total number of thymocytes was observed at a dose of 0.1 mg/kg on both days and at a dose of

1 mg/kg on day 1. As Table 3A shows, a single administration of fraction B at a dose of 0.1 mg/kg increased the percentage of CD3+ (Pan-T cells) (day 1 – dose 1 mg/kg; day 3 – all 3 tested doses), which is followed by the increase in the percentage of CD4⁺ splenocytes. Multiple administration of fraction B (Table 3B) at a dose of 0.1 mg/kg increased the percentage of CD19⁺ (B lymphocytes) in the spleen on both days, whereas a single administration exerted the opposite effect, but only on day 3. However, five exposures to fraction B at a dose of 1 mg/kg and 0.1 mg/kg decreased the percentage of CD3+ cells accompanied by the decrease in the percentage CD8⁺ splenocytes on both days.

A single administration of investigated fraction B (0.1 mg/kg, 1 mg/kg, 10 mg/kg) caused the decrease in the percentage of B lymphocytes (CD19⁺) from mesenteric lymph nodes determined on day 1 and the decrease in their absolute count after administration at doses of 0.1 mg/kg, 1 mg/kg, measured on day 3 (Table 4A). Moreover, a single injection of fraction B resulted in the enhanced percentage of CD3+ cells (0.1 mg/kg, 1 mg/kg, 10 mg/kg) on day 1, accompanied by the increase in the percentage of CD4⁺ mesenteric lymph nodes lymphocytes population on day 1 (10 mg/kg, 1 mg/kg). Five exposures to fraction B (Table 4B) decreased temporarily the absolute count of CD19⁺ cells on day 1

(0.1 mg/kg, 1 mg/kg). Multiple injections of fraction B resulted in the decrease in the percentage and absolute count (day 3) of CD3+ cells and only the percentage (day 1) at all doses applied. Furthermore, the decrease in the absolute count of CD8⁺ cells measured on both days was observed, at all 3 tested doses.

Discussion

In the present study, we confirmed immunomodulating activities of polysaccharide fraction B from Caltha palustris extract. Many polysaccharide compounds in other plants (such as Solanum nigrum, Opilia celtidifolia, Potentilla anserina) have been reported to have immunomodulating properties [15-17]. The most meaningful parameters for evaluating the balanced state of immunomodulation and homeostatic responses of the intrinsic immune system are the number and ratio of two main lymphocyte T subsets (CD4⁺ cells – T helpers, and CD8⁺ cells – T cytotoxic/suppressors) [18]. The recognition of antigens presented by the major histocompatibility complex class II (MHC II) proteins and mediation of both cellular immune responses through Th1 cells and humoral immune responses through Th2 cells are the domain of CD4⁺ T cells. The CD8⁺ cells recognize antigens presented by MHC I molecules and mediate cellular immune responses through cytotoxic T cells. Wu et al. [19], who investigated the traditional Chinese medicine, Chi-Shie-Shuang-Bu-An-Shen-Tang (CST), reported the significant increase in the population of CD4⁺ T cells in spleen after oral administration of sterilized CST for 3 weeks. The authors suggest that probably the polysaccharide component was responsible for this activity. In the present study, the increase in the CD4⁺ lymphocyte population (thymus, spleen) after a single administration of fraction B from Caltha palustris extract was noticed, but on the other hand, the decrease in this population was observed after the multiple administration in mesenteric lymph nodes. According to the data obtained in the cited study, the increase in CD4⁺ population may be potentially beneficial, especially in potential treatment of the states of CD4⁺ lymphopenia characteristic of systemic diseases, i.e. systemic lupus erythromatosus [20] or action of drugs, i.e. cyclophosphamide [21] may increase the risk of infection. However, the decrease noticed in mesenteric lymph nodes after a longer exposure to the tested fraction might be problematic.

CD8⁺ and CD4⁺ subpopulations, which in peripheral blood, play also a crucial role in adult host defense toward cancer [22]. The fraction 1a of polysaccharides, isolated from Solanum nigrum Linne (SNL-P1a) has been demonstrated to have a protective effect on thymus in tumor-bearing mice. Li et al. [15] studied the profile of CD4⁺ and CD8⁺ in PBMC (peripheral blood mononuclear cell). The authors 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 percentage of single-positive CD8⁺ thymocytes and splenocytes after administration of fraction B from Caltha palustris extract was found.

Our study showed that a single administration of examined fraction B decreased the CD19⁺ B lymphocyte population in mesenteric lymph nodes while multiple administration decreased this parameter only temporarily. In turn, CD19⁺ B lymphocyte population of spleen has been stimulated by five times administration of the tested agent. Han et al. [23] demonstrated in their studies the stimulating activity of polysaccharide extracts isolated from the radix of Platycodon grandiflorum (PG). Platycodon grandiflorum was found to markedly increase polyclonal IgM antibody production and the proliferation of B cells. Furthermore, an increase in IgM antibody production in B cells was demonstrated after the intraperitoneal administration of PG in mice immunized using T-dependent antigen, sheep red blood cells (SRBCs). Also Duradao et al. [24] demonstrated stimulating activity on splenic B cells in in vitro and in vivo test of polysaccharide fraction E extracted from Prunus dulcis seeds. The present study showed that fraction B from Caltha palustris extract is able to change the percentage and absolute number of CD4⁺ T cell subpopulation in thymus, and CD4⁺, CD8⁺ cell subpopulations in spleen and B lymphocytes in the spleen and mesenteric lymph nodes. The effect of the investigated substance depended on the number of consecutive doses applied. Further research on the structure and relationship between the structure and immunomodulatory activities of the polysaccharides fraction B from Caltha palustris extract is required.



The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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