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FUNCTIONAL CHARACTERIZATION OF MURINE IMMUNOCOMPETENT CELLS AND MORPHOLOGY OF LYMPHOID ORGANS WITH THE INTRODUCTION OF PLANT IMMUNOMODULATOR

Olga V. Lebedinskaya1*, Elena A. Lebedinskaya1, Anatoliy P. Godovalov2, Ilona D. Makarenkova3 and Nelly K. Akhmatova3
  • 1 Acad. E.A. Wagner Perm State Medical University, hystology, cytology and embriology, Russia
  • 2 Acad. E.A. Wagner Perm State Medical University, immunology, Russia
  • 3 I.I. Mechnikov Research Institute of Vaccines and Sera, Russia

A huge number of plant-based drugs possessing the immunomodulating effects are currently used in medicine for the purpose of promoting the defense mechanisms of the body [4, 6]. Plant immunomodulators combined with the conventional therapy favor the recovery and reduce the rate of relapses [4]. Agonists of Toll-like receptors (TLRs) seem promising plant immunomodulators that are used in immunotherapy of infectious and autoimmune diseases, as well as malignancies [1]. One of these immunomodulators is fucoidan japonica, a brown sea algae-based drug. It possesses the immunomodulating activity against various immunocompetent cells such as lymphocytes, dendritic cells, natural killers and neutrophils; plays an essential role in anti-tumor defense; and provides the mobilization of hematopoietic stem cells [2, 3, 5]. However there is still scarce volume of data on the fucoidan effect on the morphological peculiarities of lymphoid organs and functional properties of immunocompetent cells. The aim of the investigation was to analyze the fucoidan effect on the murine immunophenotype and function characteristics of splenic mononuclear leukocytes (ML), as well as on structure and cell composition of lymphoid organs. Materials and Methods. Purified fucoidan from sea algae Laminaria japonica Areshoug and Kjellmaniella gyrate Miyabe was used. Experiments were fulfilled with CBA murine strains divided in two groups. First (control) group comprised intact animals, the second (test) group included mice i.p. injected with fucoidan in a dose of 200 mg/mouse. Isolation of mononuclear leukocytes was implemented according to the conventional procedure in Ficoll-Urografin density gradient (Pharmacia, USA; density 1,077 g/cm3), that formed the inter-phase ring, then these were collected with pipette and cell concentration was adjusted to a volume of 1x10^6 cell/mL. The evaluation of lymphocyte subset structure was realized via flow cytometry using monoclonal antibodies (MCA) (Catlag Laboratories, USA) against cell antigens (CD3, NK1.1., CD3/NK1.1., CD4, CD25, CD4/CD25, CD8a, I-AK, CD19, CD5.2, CD40, CD5.2/CD40). In addition, the ML immunophenotype assay was carried out with the fucoidan japonica introduction (10 mg/mL) into the splenocyte culture. The serum cytokine concentration was determined with immunoenzyme method using test-systems (Bender MedSystems, USA) according to the manufacturer instructions. The cytotoxic activity of splenic ML was revealed on NK-dependent line of erythroblast leucosis K-562 cells in 3-(4,5-dimethylthiazol-2-il)-2,5-diphenyltetrazoilum bromide reduction test (MTT-test). Paraffin sections of hematopoietic and lymphoid organs (red bone marrow, thymus, spleen and lymph nodes) taken 4 h and 24 h after the drug injection were stained with hematoxylin and eosin (by Van Hyson); methyl green and pyronin (by Brashe), and Schiff-iodine acid (by McManus). Statistical data processing was made using Student’s t-test, with standard package of statistical software Windows 2003 (StatSoft 6.0). Results. Following the fucoidan introduction into murine splenic ML cultures, the number of NK-cells, NKT-cells, activated T-helpers, B-lymphocytes statistically significantly elevated in culture suspension. It should be indicated the pronounced cell surface Toll-like receptor (TLRs) expression. Immunophenotype assay showed that i.p. fucoidan injection to animals provoked the 1,5-2-fold increase of splenic natural killers (NK1.1+) and natural killer T cells (NKT – CD3+/NK1.1+); by 2-6-fold the amount of activated T- and B-lymphocytes (CD25+). Results obtained indicate possible fucoidan effect on the activation of cells involved in antibody formation including antigen-presenting cells and T-helpers, as well as on the B-lymphocyte differentiation into mature antibody producers. As a whole, this is manifested in humoral immune response augmentation. Fucoidan is characterized by dose-dependent activation of the cytotoxicity when it is introduced in mononuclear leukocyte culture. Maximum ML cytotoxic activity (39,0±0,6%) was observed at fucoidan concentration in a dose of 10 mg/mL in a culture fluid. The analysis of the cytotoxic ability of murine splenic mononuclear leukocytes relatively to NK-sensitive tumor line of K-562 cells following the i.p. fucoidan introduction demonstrated that after a slight lowering of the index of ML cytotoxic activity four hours since the drug introduction there was subsequently observed the elevation of this parameter approaching maximum values 24 h after the fucoidan injection. While analyzing the murine serum cytokine level with i.p. injection of fucoidan it was revealed that the IL-6, IL-10 and to greater extent TNF-a expression (by 40-fold) increased only 96 h following the immunomodulator injection. The morphological investigation demonstrated that under the fucoidan effect the activation of both lymphoid and myeloid lineages occurred. This is evidenced by the indication of active hematopoiesis in red bone marrow; proliferation in lymphoid tissue and primary (thymus) and secondary (spleen, lymph nodes) organs of immunogenesis. Meanwhile, the lymphoid cell proliferation was equally observed in T- and B-dependent areas of secondary organs and thymus lobule cortex. It should be indicated the pronounced macrophage reaction in spleen and lymph nodes following the intraperitoneal injection of the drug. In conclusion, the implemented experiments demonstrated that the plant immunomodulator fucoidan japonica modulates the effector activity of innate and adaptive immunity and results in lymphoid cell proliferation in primary and secondary lymphoid organs. The prospect for this immunomodulator use in biotherapy is also determined by its ability to promote the expression of Toll-like receptors on the surface of mononuclear leukocytes.

References

1. Kuznetcova T.A., Agafonova I.G., Krohal T.S., Zvyagintceva T.N., Filimonova N.V. Gematoprotective properties of fucoidan from the brown water-plant of Fucus evanescens // Pacific medical magazine. – 2010. – № 4. – С. 32-35.
2. Choi E.M., Kim A.J., Kim Y.O., Hwang J.K. Immunomodulating activity of arabinogalactan and fucoidan in vitro. J. Med. Food. 2005. 8. 446-453.
3. Frenette P.S., Weiss L. Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms. Blood. 2000. 96. 2460-2468.
4. Hemant S., Yaseen K. Immunomodulatory Plants: A Phytopharmacological Review // Pharmacognosy Reviews. Vol 1, Issue 2, 2007, 248-260.
5. Kim E.J., Park S.Y., Lee J.-Y., Park J.H.Y. Fucoidan present in brown algae induces apoptosis of human colon cancer cells // BMC Gastroenterology 2010, 10:96.
6. Kumar S.V., Kumar S.P., Rupesh D., Nitin K. Immunomodulatory effects of some traditional medicinal plants // J. Chem. Pharm. Res., 2011, 3(1):675-684.

Keywords: plant immunomodulator, fucoidan, Immunocompetent cells, morphology of lymphoid organs, functional characterization

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Poster Presentation

Topic: Immunotherapy

Citation: Lebedinskaya OV, Lebedinskaya EA, Godovalov AP, Makarenkova ID and Akhmatova NK (2015). FUNCTIONAL CHARACTERIZATION OF MURINE IMMUNOCOMPETENT CELLS AND MORPHOLOGY OF LYMPHOID ORGANS WITH THE INTRODUCTION OF PLANT IMMUNOMODULATOR. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00074

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Received: 02 May 2015; Published Online: 14 Sep 2015.

* Correspondence: MD, PhD. Olga V Lebedinskaya, Acad. E.A. Wagner Perm State Medical University, hystology, cytology and embriology, Perm, Russia, lebedinska@mail.ru