In vitro immunostimulatory properties of Abrus lectins derived peptides in tumor bearing mice.

Abstract

In vitro immunostimulatory effect of Abrus lectins derived peptide fractions (AGP and ABP) was investigated in DL bearing mice. Both AGP and ABP were found to activate splenocytes and induced production of cytokines like IL-2, IFN-[gamma] and TNF-[alpha] indicating a Thl type of immune response. Analysis of in vitro treated splenocytes by flow cytometry revealed an increase in percentage of T and B cell with high expression of activation markers ([CD25.sup.+] and [CD71.sup.+]). At the same time, expression of co-stimulatory markers was significantly high compared to tumor control. The tumor associated macrophages were able to stimulate NO production, IL-1 secretion, increased phagocytosis and decreased expression of mannose receptor. It was also observed that NK. cell was activated by AGP and ABP. These results suggest that both AGP and ABP act as immunostimulants in vitro in DL bearing mice.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Abrus; Agglutinin; Abrin; Peptide; Dalton's lymphoma (DL); Immimostimulation

Introduction

Abrin and agglutinin, lectins from the seeds of Abrus precatorius are type II ribosome-inactivating proteins that inhibit protein synthesis in eukaryotic cells. Abrin is extremely toxic to eukaryotic cells ([IC.sub.50] 0.05 nM) and Abrus agglutinin is of comparatively low toxicity (3.5 nM). Abrin is a heterodimeric glycoprotein, whereas agglutinin is a heterotetrameric glycoprotein (Hegde et al. 1991). Both lectins showed a potent antitumor and immunostimulatory properties in murine model (Ram-nath et al. 2006; Tripathi and Maiti 2005; Ghosh and Maiti 2007). Additionally, tryptic digest of Abrus agglutinin (TDA) stimulates macrophage to increase the phagocytic and bactericidal activity as well as hydrogen peroxide production. TDA also proliferates splenocytes leading to Thl response and NK cell activation (Tripathi et al. 2004).

Recent investigation has showed that Abrus agglutinin and abrin derived peptide fraction (AGP and ABP) induced mitochondrial apoptosis in human cervical cancer cells (Bhutia et al. 2008a, b). Further, in vivo therapeutic effectiveness of these peptide fractions were deciphered in Dalton's lymphoma (DL) mice model which found to mediate through apoptosis (Bhutia et al. 2008c, d). The present study was performed to demonstrate in vitro immunostimulatory properties of AGP and ABP in DL bearing mice. The effect of peptide fractions on splenocytes, its subpopulation and cytokine profile was investigated by flow cytometry. We have also analyzed the involvement of activation of NK cells and TAM by the peptide fractions.

Material and methods

Abrus lectins derived peptides

The Abrus lectins derived peptide fractions (agglutinin derived peptide, AGP; and abrin derived peptide, ABP) were isolated and characterized as previously reported (Bhutia et al. 2008a). Briefly, Trypsin (10 x [10.sup.3] BAEE unit/mg of protein) was added at a ratio of 1:50 to lectins solution (1.0 mg protein/ml in 0.01 M phosphate buffer saline [PBS]). Trypsinization was carried out at a temperature of 37[degrees]C for overnight. AGP and ABP was isolated using l0 kDa molecular weight cut off membrane (Amicon Ultra, Millipore). The peptide fractions were lyophilized and concentration was quantified using Fluorescamine with glycine as standard (Udenfriend et al. 1972). The peptide fractions (AGP and ABP) in this study were tested for endotoxin contamination using a Limulus amebocyte lysate (LAL) assay kit (Endosafe, Charles River Laboratories, Wilmington), and all fractions gave a negative response.

Reagents

MTT (3-[4,5-dimethylthiazol-2-yl]-2.5-diphenyltetra-zolium), trypsin, fluorescamine, dimethyl sulfoxide (DMSO), FITC-mannosylated BSA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Amicon Ultra (30 and l0 kDa) was purchased from Millipore. Cytotoxic assay kit from Promega, fetal bovine serum (FBS) from Hyclone, RPMI-1640 media, from Invitrogen, India was purchased.

Mice and tumor system

Swiss albino mice (20[+ or -]2g, 6-8 weeks old) were used for immunomodulation. Mice were housed in open top cages and maintained on food and water ad labium. Room temperature was maintained 22 [+ or -]2[degrees]C with light and dark cycle was 14/10 h. All animal experiments were performed according to the rules of "Committee for the purpose of control and supervision of experiments on animals (CPCSEA), Ministry of Environment and Forests, Government of India" and Institutional Animal Ethics Committee, Indian Institute of Technology, Kharagpur, PIN-721302. DL (1 x [10.sup.6]/mouse) was maintained in ascetic form by serial transplantation in Swiss albino mice or in an in vitro cell culture system by serial passage (Bhutia et al. 2008c, d).

Cell lines

YAC-1 cell line was obtained from National Centre for Cell Science (NCCS) Pune, India and was maintained in RPMI-1640 media supplemented with 10% fetal bovine serum.

Lymphocyte proliferation assay of DL-bearing mice

A single cell suspension of spleen and thymus was prepared from normal and tumor bearing mice under aseptic conditions by homogenization in Hank's balanced salt solution (HBSS). The contaminating RBC was removed by hemolytic Gey's solution. Cell concentration was adjusted to 1 x [10.sup.6] cells/ml and the cells (180 [mu]l) were plated in 96 well flat bottom tissue culture plates and incubated with 20 [mu]l of various concentrations of AGP and ABP. All cultures were set up in triplicate for 72 h at 37[degrees]C in a humidified atmosphere of 5% [CO.sub.2]. Proliferation of splenocytes and thymocytes were checked by MTT assay method (Mosmamr 1983).

Immunophenotyping by flow cytometry

The splenocytes were collected after 72 h incubation with AGP and ABP. The splenocytes were washed with PBS containing 0.1% BSA and 0.01% sodium azide and [CD3.sup.+], [CD4.sup.+], [CD8.sup.+], [CD19.sup.+], [CD25.sup.+], [CD71.sup.+], [CD80.sup.+] and [CD86.sup.+]-cells were measured using anti-CD3-FITC, anti-CD4-PE, anti-CD8-PE, anti-CD19-FITC, anti-CD25-PE, anti-CD71-PE, anti-CD80-PE and anti-CD86-PE (BD, San Diego, CA) respectively. Surface staining was performed with proper combinations of antibodies for 30 min on ice followed by washing in PBS containing 0.1 % BSA and 0.01% sodium azide before data acquisition. Flow cytometry data was analyzed cellQuest pro software on a FACSCalibur Becton-Dickinson flow cytometer (Caruso et al. 1997).

Estimation of cytokines by Flex CBA kit

The culture supernatants used for cytokine assay were collected after 72 h incubation of splenocytes with AGP and ABP. Cytometric bead array (CBA) from BD (San Diego, CA) for mouse IFN-[gamma], IL-2, IL-4, IL-5, IL-10, TNF-[alpha] were performed by flow cytometer. The quantification and analysis of CBA raw data for mouse cytokines were done by Flow Cytometric Analysis Program (FACP) software (Young et al. 2008).

NK-cell cytotoxicity assay

The splenocytes were plated onto plastic tissue culture flasks at 37 [degrees]C in a [CO.sub.2] incubator for 2 h. Non-adherent cells was taken as a source of NK (effector) cells. Effector cells and target cells (YAC-1) at the ratio of 10:1, 5:1 and 1:1 were added to wells of a round bottom 96 well cell culture plate with different concentration of peptides. After 4 h cytotoxicity was performed using CytoTox96[R]Non-Radioactive cytotoxicity assay kit (Promega, MD, USA) using following formula.

% Cytotoxicity = [Experimental - Effector Spontenous - Target Spontenous/Target Maximum - Target Sponteneous]x100

Experimental: effector and target cells in ratio (10:1, 5:1 and 1:1); effector spontaneous: only effector cells; target spontaneous: only target cells; target maximum: target cell complete lysis.

Isolation of tumor-associated macrophages (TAM)

Mice with DL, were killed by cervical dislocation and tumor associated macrophages were isolated as described previously (Singh et al. 2004).

Assay for nitrite production

Production of nitric oxide was estimated by measuring nitrite levels in cell supernatant of 24 h activated TAM with Greiss reagent (1% sulfanilamide in 2.5% phosphoric acid, 0.1 % napthylethyldiamine dihydrochloride in 2.5% phosphoric acid). Absorbance was read at 550 nm.

Assay for arginase activity

Arginase activity was measured in cell lysates of 24 h activated TAM as previously described (Corraliza et al. 1994). The results are expressed directly as the O.D/1 x [10.sup.6] cells.

Phagocytosis of latex beads

TAMs were seeded to 6 well plates at 2 x [10.sup.5] cells per well were treated with different concentration of AGP and ABP for 24 h followed by incubation with latex beads (amine-modified polystyrene, fluorescent red, l.0 [mu]m mean particle size, Sigma, USA) in fresh medium for 2 h. The TAMs were then harvested with trypsin EDTA and flow cytometry was performed using CellQuest pro software on a FACSCalibur Becton-Dickinson flow cytometer. The phagocytosis was observed as mean fluorescence intensity (Ozaki et al. 1995).

Macrophage mannose receptor (MMR) assays

TAMs were harvested after activation and incubated with saturating concentrations of FITC-mannosylated BSA (250 ng/ml ligand/5 x [10.sup.5]) for l h at 4 [degrees]C. Subsequently, cells were analyzed by using CellQuest pro software on a FACSCalibur Becton-Dickinson flow cytometer. The MMR expression was observed as mean fluorescence intensity (Stein et al. 1992).

Assay of IL-1 activity

TAM obtained from DL-bearing mice was incubated with different concentration of peptide fractions in vitro for 24 h culture supernatants were harvested and checked for IL-1 activity by a standard thymocyte proliferation assay (Singh et al. 2004). The amount of IL-1 secretion in term thymocytes proliferation from TAM was evaluated from MTT assay.

Statistical analysis

All data were given as the mean [+ or -] S.D. Experimental results were analyzed by Student's t-test. P<0.05 was considered as the level of significance for values obtained for treated compound to control.

Results

AGP and ABP enhance the proliferative capacity of immune cells

Immunosuppression prevails in tumor bearing mice and as a first step of our investigation we have deciphered the effects of AGP and ABP on immune cells proliferation using colorimetric MTT assay. The proliferation of lymphocytes increased in a dose-depended manner of peptide fractions and decreased at higher concentration. The splenocyte proliferation index (SPI) was maximum at l0 ng/ml of AGP whereas for ABP the highest SPI was observed at 1 ng/ml. Proliferation index of lymphocytes derived from normal and tumor bearing mice were not significantly different. Both AGP and ABP showed similar proliferative properties with higher index towards thymocytes as compared to splenocytes. In comparison to splenocytes and thymocytes proliferation of Abrus agglutinin and abrin (Ramnath et al. 2006; Tripathi and Maiti 2005) (data not presented), AGP and ABP were found to be less potent mitogen (Fig. 1).

[FIGURE 1 OMITTED]

AGP and ABP influence splenocyte sub-populations

We then investigated the in vitro effects of AGP and ABP on splenocytes sub-population of tumor bearing mice through flow cytometry. It was found that Abrus lectins derived peptide fractions were able to increase in number of [CD3.sup.+] cells (T cells) and [CD19.sup.+] cells (B cells) in splenocytes significantly than tumor control group. AGP found to increase [CD3.sup.+] and [CD19.sup.+] population of splenocytes in dose depend manner whereas the trend of ABP was limited at higher concentration. Among the T cell population, [CD4.sup.+] (T helper) cells increased significantly in both AGP and ABP treated groups and [CD8.sup.+] (cytotoxic T cells) almost remained unaltered. Moreover, the [CD4.sup.+] / [CD8.sup.+] ratios of treated AGP and ABP splenocytes were significantly higher than that of tumor control with approaching towards normal ratio of [CD4.sup.+] and [CD8.sup.+]. Similarly, co-stimulatory markers ([CD80.sup.+] and [CD86.sup.+]) increased significantly as compared to tumor control which was higher than the values found in normal control. The results further showed that there was significant increase in percentage of activation marker of lymphocytes ([CD25.sup.+] and [CD71.sup.+]) in splenocytes treated with AGP and ABP (Table 1).

Table 1. Flow cytometric analysis of splenocytes sub-populations.

Group                         Normal             Tumor

[CD3.sup.+]               57.17[+ or -]4.5    31.97[+ or -]5.5
[CD19.sup.+]              34.7[+ or -]6.8     19.28[+ or -]3.7
[CD4.sup.+]               20.17[+ or -]3.8    12.15[+ or -]1.95
[CD8.sup.+]               7.71[+ or -]1.8     10.17[+ or -]2.6
[CD4.sup.+]/[CD.8.sup.+]  2.6[+ or -]0.22     1.17[+ or -]0.19
[CD25.sup.+]              15.8[+ or -]3.3     7.81[+ or -]1.53
[CD71.sup.+]              18.95[+ or -]1.6    15.88[+ or -]1.45
[CD86.sup.+]              22.13[+ or -]3.2    11.19[+ or -]1.3
[CD80.sup.+]              38.71[+ or -]2.54   32.55[+ or -]4.0

Group

                          Treated (ng/ml)

                                AGP

                                 1                  10

[CD3.sup.+]               34.54[+ or -]5.9    45.67[+ or -]4.5 *
[CD19.sup.+]              26.69[+ or -]4.4    29.8[+ or -]5.6 *
[CD4.sup.+]               14.54[+ or -]2.0    18.1[+ or -]2.2 *
[CD8.sup.+]               8.24[+ or -]1.2     9.24[+ or -]0.96
[CD4.sup.+]/[CD.8.sup.+]  1.7[+ or -]0.34 *   1.9[+ or -]0.27 *
[CD25.sup.+]              13.84[+ or -]1.7    12.89[+ or -]1.1
[CD71.sup.+]              13.84[+ or -]1.7    12.89[+ or -]1.1
[CD86.sup.+]              21.26[+ or -]2.8 *  16.99[+ or -]1.8
[CD80.sup.+]              nd                  nd

Group

                          Treated (ng/ml)

                                 AGP

                                 100

[CD3.sup.+]               52.3[+ or -]4.7 *
[CD19.sup.+]              28.8[+ or -]4.7 *
[CD4.sup.+]               20.2[+ or -]3.4 *
[CD8.sup.+]               10.89[+ or -]1.9
[CD4.sup.+]/[CD.8.sup.+]  1.85[+ or -]0.17 *
[CD25.sup.+]              17.61[+ or -]1.9 *
[CD71.sup.+]              17.61[+ or -]1.9 *
[CD86.sup.+]              15.38[+ or -]1.2
[CD80.sup.+]              nd

Group

                          Treated (ng/ml)

                                 ABP

                                 0.1                1

[CD3.sup.+]               30.56[+ or -]5.5    49.94[+ or -]7.8 *
[CD19.sup.+]              18.9[+ or -]3.95    27.53[+ or -]5.2 *
[CD4.sup.+]               15.35[+ or -]2.0    18.57[+ or -]2.3
[CD8.sup.+]               9.94[+ or -]1.2     8.23[+ or -]2.59
[CD4.sup.+]/[CD.8.sup.+]  1.54[+ or -]0.17    2.25[+ or -]0.3 *
[CD25.sup.+]              6.69[+ or -]0.56    14.3[+ or -]1.54 *
[CD71.sup.+]              6.69[+ or -]0.56    14.3[+ or -]1.54 *
[CD86.sup.+]              20.2[+ or -]2.3 *   19.37[+ or -]1.5 *
[CD80.sup.+]              42.58[+ or -]3.9 *  40.43[+ or -]4.2 *

Group

                          Treated (ng/ml)

                                 10

[CD3.sup.+]               43.6[+ or -]6.4 *
[CD19.sup.+]              24.3[+ or -]4.8
[CD4.sup.+]               16.1[+ or -]2.2
[CD8.sup.+]               9.15[+ or -]1.9
[CD4.sup.+]/[CD.8.sup.+]  1.75[+ or -]0.20 *
[CD25.sup.+]              13.25[+ or -]1.4
[CD71.sup.+]              13.25[+ or -]1.4
[CD86.sup.+]              17.45[+ or -]1.8
[CD80.sup.+]              33.55[+ or -]2.9

P values < 0.05 are considered significant compared with PBS control
(* significant compared to PBS control); nd - not determined.

AGP and ABP induce cytokine production by activated splenocytes

Effect of AGP and ABP on cytokines level in supernatant of splenocytes were determined by CBA flex kit through flow cytometry. The results of cytokine secretion revealed that both peptide fractions activated splenocytes to release high amount of IL-2, IL-10, IFN-[gamma] and TNF-[alpha] with moderate amount of IL-12 at respective concentrations. The IL-4 and IL-5 induction either in control or AGP and ABP stimulated splenocytes remain unchanged (Table 2),

Table 2. Cytokine analysis in supernatant of AGP and ABP stimulated
splenocytes.

Group                  Cytokines (pg/ml)

         [IFN-.sub.[gamma]]  [TNF-.sub.[alpha]]  IL-2

Normal   61.34[+ or -]7.51   51.1[+ or -]6.5     6.22[+ or -]0.54

Tumor    9.44[+ or -]2.37    25.01[+ or -]3.4    2.94[+ or -]0.32

AGP (1   14.93[+ or -]2.95   28.83[+ or -]3.12   6.47[+ or -]0.65*
ng/ml)

AGP (10  29.78[+ or -]4.23*  34.13[+ or -]4.7*   6.34[+ or -]0.45*
ng/ml)

AGP      31.73[+ or -]3.98*  55.66[+ or -]5.95*  2.82[+ or -]0.23
(100
ng/ml)

ABP      25.14[+ or -]2.57*  87.70[+ or -]8.38*  7.95[+ or -]0.54*
(0.1
ng/ml)

ABP (1   22.70[+ or -]2.32*  85.48[+ or -]7.95*  7.58[+ or -]0.42*
ng/ml)

ABP (10  37.05[+ or -]4.56*  127.03[+ or -]9.6*  8.74[+ or -]0.78*
ng.ml)

Group                 Cytokines (pg/ml)

                     IL-4              IL-5

Normal           1.05[+ or -]0.12  1.59[+ or -]0.17

Tumor            1.83[+ or -]0.23  2.00[+ or -]0.15

AGP (1 ng/ml)    1.65[+ or -]0.14  ND

AGP (10 ng/ml)   1.84[+ or -]0.20  1.23[+ or -]0.11

AGP (100 ng/ml)  1.41[+ or -]0.09  ND

ABP (0.1 ng/ml)  1.56[+ or -]0.13  1.72[+ or -]0.14

ABP (1 ng/ml)    1.43[+ or -]0.19  2.32[+ or -]0.19

ABP (10 ng.ml)   1.60[+ or -]0.12  2.10[+ or -]0.23

Group                 Cytokines (pg/ml)

                 IL-10               IL-12

Normal           5.63[+ or -]1.2     2.17[+ or -]0.19

Tumor            13.63[+ or -]2.75   1.75[+ or -]0.13

AGP (1 ng/ml)    15.67[+ or -]1.9    1.64[+ or -]0.15

AGP (10 ng/ml)   22.42[+ or -]1.75*  2.54[+ or -]0.19*

AGP (100 ng/ml)  27.63[+ or -]1.93*  1.94[+ or -]0.14

ABP (0.1 ng/ml)  42.67[+ or -]4.54*  2.04[+ or -]0.18

ABP (1 ng/ml)    45.45[+ or -]3.96*  2.54[+ or -]0.15*

ABP (10 ng.ml)   66.36[+ or -]4.75*  2.89[+ or -]0.25*

P values <0.05 are considered significant compared with PBS control
(*significant compared to PBS control); ND - not detected.

NK cell activity

Tumor cell elimination is known to be mediated in part by the cytotoxic activity of NK cells. We therefore measured the cytotoxic activity of splenocytes against NK-sensitive (YAC-1) tumor cells. Splenocytes derived NK cell activity significantly increased in dose-depended way of the sample and highest activity was observed at 1 [mu]g/ml of AGP and 100 ng/ml of ABP (Fig. 2).

[FIGURE 2 OMITTED]

Effects of AGP and ABP on tumor associated macrophages

The in vitro effects of AGP and ABP on properties of TAM were investigated. After 24 h the TAM were assayed for phagocytosis, arginase activity, expression of mannose receptor, nitrite oxide and IL-1 production. TAM produced higher amount of NO as compared to normal macrophage, and this was further augmented on treatment with peptide fractions. The NO production was increased in dose depended manner and AGP was found to be more effective than ABP. Similarly, in vitro treatment of TAM with AGP and ABP resulted in increase of phagocytotic activity. The maximum phagocytosis activity was observed with 10 ng/ml of AGP and 1 ng/ml of ABP but this activity was less as compared to phagocytosis of normal macrophage. On other hand, the expression of mannose receptor decreased in dose depended way in both AGP and ABP treated group. Activated TAM showed no alternation in arginase activity upon treatment with peptide fractions. We studied IL-1 secretion in culture supernatant of activated TAM, by standard thymocytes proliferation assay. We observed an increase in IL-1 production in term of thymocytes proliferation in presence of supernatant from TAM treated with different concentration of peptide fractions (Table 3).

Table 3. Effects of AGP and ABP on tumor associated macrophages.

Group            NO ([mu]M)         Phagocytosis (mean florescence)

Normal           18.9[+ or -]1.46   194.4[+ or -]14.65

Tumor            26.7[+ or -]2.11   33.94[+ or -]2.56

AGP (1 ng/ml)    30.5[+ or -]2.3    52.85[+ or -]4.75

AGP (10 ng/ml)   37.7[+ or -]2.95*  104.28[+ or -]9.5*

AGP (100 ng/ml)  40.6[+ or -]3.24*  85.16[+ or -]6.6*

ABP (0.1 ng/ml)  28.6[+ or -]1.95   45.86[+ or -]3.75

ABP (1 ng/ml)    34.4[+ or -]2.45*  118.89[+ or -]9.64*

ABP (10 ng/ml)   37.8[+ or -]4.28*  43.17[+ or -]3.75

Group    MMR (mean             Arginase (OD at 540  IL-1 (Thymocytes
         florescence)          nm)                  proliferation)

Normal   126.8[+ or -]9.96     0.335[+ or -]0.029   0.34[+ or -]0.028

Tumor    686.59[+ or -]45.85   0.714[+ or -]0.055   0.195[+ or -]0.014

AGP (1   446.0[+ or -]32.74*   0.893[+ or -]0.076   0.234[+ or -]0.017*
ng/ml)

AGP (10  388.12[+ or -]35.4*   0.73[+ or -]0.054    0.257[+ or -]0.022*
ng/ml)

AGP      252.23[+ or -]19.55*  0.69[+ or -]0.044    0.221[+ or -]0.024
(100
ng/ml)

ABP      408.38[+ or -]45.65*  0.75[+ or -]0.052    0.268[+ or -]0.021
(0.1
ng/ml)

ABP (1   272.85[+ or -]22.46*  0.67[+ or -]0.062    0.297[+ or -]0.023*
ng/ml)

ABP (10  250.9[+ or -]20.45*   0.87[+ or -]0.079    0.247[+ or -]0.021*
ng/ml)

P values <0.05 are considered significant compared with PBS control
(*significant compared to PBS control).

Discussion

The discovery and identification of new antitumor drugs, which can potentiate the immune function has become an important goal of research in immunophar-macology and oncotherapy. Our previous study reported that in vitro and in vivo antitumor properties of Abrus lectins derived peptide fractions in mouse model. Apart from its chemotherapeutic nature, the present study investigated in vitro immunostimulatory properties of these peptide fractions in tumor bearing mice.

The peptide fractions showed a characteristic mitogenic action and caused a high level of proliferation of thymocytes as compared to splenocytes in tumor bearing mice. Further, the mitogenic response of splenocytes to peptide fractions was supported by estimating expression of [CD3.sup.+] (T cell) and [CD19.sup.+] (B cell) markers on the cell surface in flow cytometric assay. Activated proliferating lymphocytes express [CD25.sup.+], [CD69.sup.+], [CD71.sup.+], and HLA-DR on their surface which are expressed minimally or even absent on resting cells and thus termed as "activation antigens". The activation markers ([CD25.sup+],[CD71.sup.+]) of splenocytes were found to increase significantly as compared to control in presence of AGP and ABP as evaluated in flow cytometric analysis (Caruso et al. 1997). This study demonstrated that the nature of mitogenic stimulus of Abrus derived peptide fractions not only influenced proliferation property but also the activation status of splenocytes. Phenotypic analysis of the lymphocytes subpopulations clearly showed in increase of [CD4.sup.+] cells without any significant alternation in [CD8.sup.+] cells on peptide treatment. Consequently, peptide fractions increased the [CD4.sup.+]/[CD8.sup.+] ratio which is a major indicator for assessing the function of T cell mediated immunity. The antigen presenting cells in splenocytes expressed [CD80.sup.+] (B7-1) and [CD86.sup.+] (B7-2), which might provide co-stimulatory signals for optimal T-cell activation through the CD28 pathway. The AGP and ABP was able to activate tumor derived murine splenocytes and release of different cytokines which had role in antitumor response. The IL-2 has many immunopotentiating effects, such as proliferation of T cells, B cells, NK cells and monocytes, augmentation of cytotoxicites of T cells and NK cells which exhibit high cytolytic activities against tumor cells. The cytokines like TNF-[alpha] and IFN-[gamma] has been recognized as an important host defense cytokine that affects tumor cells. The IL-12 exerts its biological activity in T and NK cells, inducing the production of IFN-[gamma], enhancing the generation of cytotoxic cells, and stimulating antigen-activated lymphocytes. The IL-10 shows its antitumor property by enhancement of the function of natural killer cells and macrophages (Mocellin et al. 2003). The cytokine profile of treated splenocytes had a skew towards Th 1 type of immune response with negative IL-4 and IL-5 detection which supports our previous finding (Tripathi et al. 2004). The ability of these peptide fractions to stimulate Th 1 cytokines along with IL-10 by splenocytes can be exploited as an adjuvant effect. The in vitro IL-10 production associated with increased Thl cytokines explained the immunostimulatory nature of the peptide fractions which might involve in indirect tumor regression in DL bearing mice (Bhutia et al. 2008c, d) TAM is characterized as a skewed M2 macrophage population with impaired expression of NF-kB-dependent inflammatory functions (expression of cytotoxic mediators, NO) and cytokines (TNF-[alpha], IL-1, IL-12). TAM also expresses high levels of both the scavenger receptor-A and the mannose receptor accompanied with predominant production of ornithine and polyamines by arginase pathway (Sica et al. 2006). TAMs generally are not tumoricidal, but upon activation they can exert toxicity towards tumor cells. The results revealed that activated TAM showed an enhanced phagocytosis, decreased in expression of MMR with enhanced amount of IL-1, which is a co-stimulatory signal for lymphocyte proliferation indeed due to antigen presentation of macrophage. Additionally, treatment of TAM by peptide fractions resulted in enhanced RNI production without having any effect on arginase activity which indicate tumor growth interfere might associated "switching on" of the arginase pathway. A strong positive NK cell activation was also observed by peptide fractions which explain the TFN-[gamma] and IL-10 release.

This observation suggested that the AGP and ABP were able to stimulate tumor associated macrophage, proliferate splenocytes leading to Th l response and NK cell activation in tumor bearing mice in vitro. Many small peptides have been identified from natural sources for their nonspecific immunostimulatory responses against tumor suppressed condition (Bhutia and Maiti 2008). Many of these are well studied and presently under evaluation for their therapeutic potential. This study warrants Abrus lectins derived peptide fractions are possible sources of similar small immunostimulatory peptides which might involve in tumor regression.

Acknowledgments

National Doctoral Fellowship to SKB from All council of Technical Education (AICTE), India [Award No. 1-10/FD/NDF-PG/ (IIT-KH (01)/2005-06,26th 2006] is acknowledged. SKM acknowledges Council of Scientific and Industrial Research, New Delhi, India.

References

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Sujit K. Bhutia, Sanjaya K. Mallick, Tapas K. Maiti (*)

Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India

* Corresponding author. Tel.: +913222283766; fax: +913222 255303.

E-mail address: tkmaiti@hijli.iitkgp.ernct.in (T.K. Maiti).

0944-7113/$-see front matter [C] 2009 Elsevier GmbH. All rights reserved.

doi:l0.1016/j.phymed.2009.01.006

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