Experimental immunology
The influence of methisoprinol on the spleen phagocyte and blood lymphocyte activity in rats - in vitro study

Introduction
The immune system is a complex interaction of checks and balances that allows regulation by oppositing stimulatory and inhibitory influences. The viral infections appear to be an important practical problem in human and animals, due to endemic spreading of the infections, high costs and generally low efficacy of the therapy. Therefore, a strategy of elimination of endemic infections should a battery prevention tasks aimed both to the elimination of the infective agents and the elevation of the non-specific anti-viral resistance of the organism. Immunomodulators comprise a group of biological and synthetic compounds and play an important role in modern alternative prevention and therapy of infectious diseases. The general opinion about the use of non-specific immunomodulators in prophylaxis of endemic infections is positive [1, 2]. A reasonable possibility for effective, non-toxic immunomodulation which should be considered for prevention of endemic infections in animals appears to be the application of potent activators of the macrophage-monocyte and lymphocyte systems [2-6]. Actually exist a large number of macrophage and lymphocyte activators of natural origin from bacteria, fungi, plants and other biological and non-biological materials [7-13]. These substances were tested in experimental and clinical investigations for the treatment of neoplastic diseases, as well as for the prophylaxis and treatment of infections in human and animals [1, 3, 8, 14, 15].
Methisoprinol (inosiplex) is a synthetic compound formed from the p-acetamidobenzoate salt of N-N dimethylamino-2-propanol and inosine in a 3 : 1 molar ratio. It exerts antitumour and antiviral activities in vivo, which are secondary to an immunomodulating influence on the non-specific and specific defence mechanisms in human and mouse [6, 18, 21, 22]. The immunomodulating effects of methisoprinol in vitro and in vivo has been assessed in many diseases including Herpes infections, the acquired immunodeficiency syndrome (AIDS) and autoimmune diseases in human [16, 21, 22].
The aim of the present investigation was to assess the in vitro influence of methisoprinol on the spleen macrophage activity and blood lymphocyte proliferation in rats.

Material and Methods
Animals and experimental design
For this study 10 adult male and 10 adult female Wistar rats, aged 12 weeks, were used. Animal experiments were carried out in conformance with the Animal Protection Law and the recommendations of the Animal Ethics Committee of the University of Warmia and Mazury in Olsztyn. Peripheral blood was obtained by venous puncture (Vacutainer set - Vacuette Greiner Labortechnik) and spleens were separated after dissection with procedure used in laboratory animals.
For each experiment performed under identical conditions, collected peripheral blood were diluted with one part of RMPI 1640 culture medium (RPMI 1640, Sigma) supplemented with sodium pyruvate and 1% antibiotics (penicilin/streptomycin, Sigma) and lymphocytes were separated on Percoll gradient (Pharmacia, Sweden). The number of isolated cells was quantified microscopically after the cells were stained with trypan blue, using a modified Neubauer chamber. The spleen of each rat was removed aseptically and single-cell suspensions were obtained by teasing the tissues in medium through a steel mesh and isolating spleen phagocytes using either a Gradisol (Polfa, Poland)
The methisoprinol (Polfa Grodzisk, No: 330300, Poland) was used in the present studies. The isolated rats blood lymphocytes were stimulated by concanavaline A (ConA, Sigma Chemical) or lipopolysaccharide (LPS Sigma Chemical).

Assay procedure
The proliferative ability of the blood lymphocytes stimulated by mitogens were determined by MTT assay, first described by Mosmann [23] and used in animals by Siwicki et al. [9] and Wagner et al. [24]. MTT [3-(4,5-Dimethyl thiazol-2-yl0-2,5-diphenyl) tetrazolium bromide] (Sigma) was dissolved in PBS at concentration of 5 mg MTT/ml and sterilized by filtration. This stock solution was used for the MTT assay. One hundred ml of isolated lymphocytes were distributed in 96-well culture plates (Costar, USA) at concentration 1 × 106 cells/ml in RPMI 1640 medium containing 2 mM L-glutamine, 0.02 mM 2-mercaptoethanol, 1% Hepes buffer, penicillin/streptomycin (100 U/100 mg/ml) and 10% fetal calf serum (FCS) with or without methisoprinol at different concentrations. In each experiment, the concentrations of methisoprinol used in the RPMI 1640 medium (Sigma) were 0, 0.5, 1, 5, 10, 25 and 50 mg methisorinol/ml of medium. The mitogens: ConA at concentration 5 mg/ml or LPS at concentration 20 mg/ml were then added (100 ml/well) to each well. All samples were tested in triplicate. The mixture containing cells in medium with different concentration of methisoprinol and mitogen were incubated for 72 h at 37°C, with 5% carbon dioxide atmosphere (Asab incubator, Sweden). After incubation, 50 ml of MTT (5 mg/ml PBS) solution were added to each well and incubated 4 h in 37°C. The microtiter plates were centrifuged (1400 × g, 15°C, 5 min) and the supernatant was removed carefully by an Eppendorf pipette. To each well 100 ml of a DMSO (Sigma) were added and after 10-15 min. incubation, the solubilized reduced MTT was evaluated in an ELISA microreader (Alab Plate Reader, USA) at 620 nm wavelength.
The activity of spleen phagocytes was analysed using respiratory burst activity (RBA) assay and potential killing activity (PKA) assay.
Respiratory burst activity was determined in spleen phagocytes after stimulation with PMA (Phormol Myristate Acetate, Sigma), as described by Chung and Secombes [25] and adapted for animals by Siwicki et al. [9]. One hundred ?l of isolated phagocytes were distributed in 96-well culture plates (Costar, USA) at concentration 1 × 106 cells/ml in RPMI 1640 medium containing 10% fetal calf serum (FCS) with or without methisoprinol at different concentrations. In each experiment, the concentrations of methisoprinol used in the RPMI 1640 medium (Sigma) were 0, 0.5, 1, 5, 10, 25 and
50 mg methisorinol/ml of medium. After 2 of incubation, cells was mixed with 100 ml 0.2% nitro blue tetrazolium (NBT, Sigma) and 1 ml of PMA at concentration of 1 mg/ml in ethanol. After 30 min. of incubation at 37°C, the supernatant was removed from each well. The cell pellet was washed with absolute ethanol and also three times with 70% ethanol. The amount of extracted reduced NBT after incubation with 2M KOH and DMSO (dimethylsulfoxide, Sigma) was measured colorimetrically at optical density (OD) 620 nm in microplate reader (Tecan, Sunrise).
The potential killing activity (PKA) of spleen phagocytes was determined after stimulation cells with killed Staphylococcus aureus strain 209P, according to the method presented by Rook et al. [26] adapted for animals by Siwicki et al. [9]. One hundred ml of isolated phagocytes were distributed in 96-well culture plates (Costar, USA) at concentration 1 × 106 cells/ml in RPMI 1640 medium containing 10% foetal calf serum (FCS) with or without methisoprinol at different concentrations. In each experiment, the concentrations of methisoprinol used in the RPMI 1640 medium (Sigma) were 0, 0.5, 1, 5, 10, 25 and 50 mg methisoprinol/ml of medium. After 2 h incubation cells were mixed with 100 ml of 0.2 % NBT and 10 ml killed bacteria (containing 106 bacteria). The mixture was incubated for 1 h at 37°C and the supernatant was removed. The cell pellet was washed with ethanol and it was dried at room temperature. This was followed by the addition of 2M KOH and DMSO to each well. The amount of extracted reduced NBT was measured colorimetrically at optical density (OD) 620 nm in microplate reader (Tecan, Sunrise).

Statistical analysis
The results were analyzed using the Student’s t-test. Means and standard deviations for all values were calculated and presented in the figures. Differences between the treatment means were considered statistically significant if p < 0.05 with use of GraphPad Prism 5 software.

Results
In presented study we examined the in vitro influence of different concentrations of synthetic immunomodulator methisoprinol on the proliferative response of blood lymphocytes T and B as measured by the MTT assay. The influences of different concentrations of methisoprinol on the proliferative response of blood lymphocytes T stimulated by ConA are presented in the Table 1. The results showed that the methisoprinol at concentration between 5 and 50 mg/ml significantly increased the proliferative response of T lymphocytes, compared to the control presented by cells incubated without methisoprinol (only ConA). The highest proliferative responses of lymphocytes were observed at concentrations between 10 to 50 mg/ml, but we have not observed statistically significant difference between male and female animals..
The influences of different concentrations of methisoprinol on the proliferative response of blood lymphocytes B stimulated by LPS are presented in the Table 1. The analysis of the results showed that the methisoprinol also increased the proliferative response of B-cells at concentrations between 5 and 50 mg/ml, compared to the control presented by cells incubated without methisoprinol (only LPS). The highest proliferative responses of B-cells were observed at concentrations between 10 to 50 mg/ml. The present study shows that the concentrations between 5 to 50 mg/ml of methisoprinol increase the mitogens-induced proliferation rate of rats blood lymphocytes T and B, without difference between male and female rats.
The statistically significantly (p < 0.05) higher values of the phagocytic ability and potential killing activity of spleen phagocytes were observed at concentrations of methisoprinol between 5 to 50 mg/ml, compared to the control presented by cells incubated without methisoprinol. The influence of different concentrations of methisoprinol on the respiratory burst activity and potential killing activities are presented in the Table 2. The analysis of the results showed that the methisoprinol increased the metabolic ability (RBA) and potential killing activity (PKA) of spleen phagocytes at concentrations between 5 and 50 µg/ml, compared to the control. The highest RBA and PKA values were observed at concentrations between 10 to 50 µg/ml and statistically significant difference between male and female animals was not observed.

Discussion
Several publications have compiled a large amount of data on the immunomodulating activity of methisoprinol [17, 21]. This product potentiates or augments immunological events by triggering agents such as mitogens, antigens or lymphokines. Mitogen or antigen-stimulated T-lymphocyte differentiation and proliferation are accelerated by concurrent exposure to methisoprinol [15]. The methisoprinol strongly stimulated differentiation of cells of the T-lymphocyte lineage [20]. The methisoprinol-induced increase in the concanavalin A-stimulated blastogenic response was more apparent in cell cultures showing an initially low blastogenic response [19]. Similarly, in vitro methisoprinol markedly enhanced concanavalin A-stimulated T-lymphocyte cell proliferation in cells from patients with severely impaired cell-mediated responsiveness, including patients with AIDS or persistent generalised lymphadenopathy [5]. The pokeweed mitogen-stimulated T-cell-dependent B-cell proliferative responses in cells from patients with AIDS or AIDS-related complex have also been reported to be enhanced by methisoprinol [14]. By direct interaction in vitro, methisoprinol has been shown to stimulate various parameters of metabolic activity of macrophages and monocytes [18, 21]. Incubation with methisoprinol in vitro stimulated the production of interleukin-1 in human monocytes and macrophages, and increased the levels of lisosomal enzymes in the latter [21].
In our preliminary study the strong stimulating influence of different concentrations of methisoprinol on the blood proliferative response of T and B-lymphocytes were observed. Also methisoprinol at different concentration activated metabolic ability and potential killing activity of spleen PMN and MN phagocytes. This is very important for restoration or modulation of immunity after suppression induced by different viruses or xenobiotics.

References
1. Nolte I (1992): The therapy of chronic diseases of the respiratory tract in dogs and cats. Tierarztl Praxis 20: 301-305.
2. Van Kampen KR (1997): Immunotherapy and cytokines. Semin. Vet Med Surg (Small Animals) 12: 186-192.
3. Blecha F (1988): Immunomodulation: a mean of disease prevention in stressed livestock. J Animal Sciences 66: 2084-2090.
4. Murayama TS, Matsuume-Sasakai B, Furukawa T, Rinaldo CR (1989): Biological response modifiers enhance the activity of natural killer cells against human cytomegalovirus-infected cells. J Med Virol 29: 102-108.
5. Campo M, Chiavaro I, Canfarotta F (1982): Effect of levamisole and methisoprinol on in vitro lymphocyte reactivity in chronically irradiated subjects and patients affected by neoplasias. J Immunopharmacol 4: 127-137.
6. Fudenberg HH, Whitten HD (1984): Immunostimulation: synthetic and biological modulators of immunity. Annual Rev Pharmacol Toxicol 24: 147-174.
7. Gibka J, Skopińska-Różewska E, Siwicki A et al. (2008): Stimulation of humoral immunity in mice by undecan-2-one, undecan-2-ol and their derivatives. Centr Eur J Immunol 33: 47-49.
8. Kormosh N, Laktinov K, Antoshechkina M (2006): Effect of a combination of extract from several plants on cell-mediated and humoral immunity of patients withadvanced ovarian cancer. Phytother Res 20: 424-425.
9. Siwicki AK, Krzyżanowski J, Bartoszcze M et al. (1998): Adjuvant properties of killed Propionibacterium avidum KP-40 in vaccination of dogs against canine parvovirosis. Dtsch Tierarztl Wschr 105: 186-190.
10. Siwicki A, Skopińska-Różewska E, Hartwich M et al. (2007): The influence of Rhadiola rosea extracts on non-specific and specific cellular immunity in pigs, rats and mice. Centr Eur
J Immunol 32: 57-62.
11. Skopińska-Różewska E, Strzelecka H, Wasiutyński A et al. (2008): Aqueous and hydro-alcoholic extracts of Echinacea purpurea (L) Moench as traditional herbal remedies with immunotropic activity. Centr Eur J Immunol 33: 78-82.
12. Skopińska-Różewska E, Wójcik R, Siwicki A (2008): The effect of Rhadiola quadrifida extracts on cellular immunity in mice and rats. Pol J Vet Sci 11: 97-104.
13. Wójcik R, Siwicki A, Skopińska-Różewska E et al. (2008): The in vitro influence of Rhadiola quadrifida extract on non-specific cellular immunity in pigs. Centr Eur J Immunol 33: 193-196.
14. Tsang KY, Fudenberg HH, Galbraith GM et al. (1985): Partial restoration of impaired interleukin-2 production and Tac antigen (putative interleukin-2 receptor) expression in patients with acquired immune deficiency syndrome by isoprinosine treatment in vitro. J Clinl Investig 75: 1538-1544.
15. Nakamura T, Miyasaka N, Pope RM et al. (1983): Immunomodulation by isoprinosine: effects on in vitro immune functions of lymphocytes from humans with autoimmune diseases. Clin Exper Immunol 52: 67-74.
16. Campoli-Richards DM, Sorkin EM, Heel RC (1986): Inosine pranobex - a preliminary review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy. Drugs 32: 383-424.
17. Ginsberg T, Hadden JW. Immunopharmacology of methisoprinol. In: Fudenberg HH, Whitten HD (eds.). Immunomodulation: New Frontiers and Advances. Plenum Press, New York 1984; 331-348.
18. Hadden JW, Wybran J (1981): Immunopotentiators: Isoprinosine, NPT 15392 and azimexone: modulators of lymphocyte and macrophage development and function. Adv Immunopharmacol 1: 457-468.
19. Portoles A, Ojeda G, Rojo JM, Pilar M (1983): Modulatory effect of isoprinosine on lymphocyte proliferative response under immunodepressive conditions. J Immunopharmacol 5: 245-256.
20. Touraine JL, Gay-Ferret G, Sanadji K et al. Isoprinosine: synergistic effects with NPT 15392 in vitro and activity on suppressor T-lymphocytes in auto-immune mice in vivo. In: Serrou B et al. (eds.). Current Concept in Human Immunology and Cancer Immunomodulation. Elsevier Biomedical Press B.V. 1982; 491-499.
21. Wybran J, Appelboom T. Isoprinosine (inosiplex): immunological and clinical effect. In: Fudenberg HH, Whitten HD (eds.). Immunomodulation: New Frontiers and Advances. Plenum Press, New York 1984; 363-374.
22. Lam KC, Lin HS, Lai CL et al. (1988): Isoprinosine in classical acute viral hepatitis. Digestive Diseases 23: 893-896.
23. Mosmann T (1983): Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.
24. Wagner U, Burkhardt E, Failing K (1999): Evaluation of canine lymphocyte proliferation: comparison of three different colorimetric methods with the H-thymidine incorporation assay. Vet Immunol Immunopathol 70: 151-159.
25. Chung S, Secombes SJ (1988): Analysis of events occuring within teleost macrophages during the respiratory burst. Comp Biochem Physiol 89: 539-544.
26. Rook GAW, Steele J, Umar J, Dockrel HM (1995): A simple method for the solubilization of reduced NBT and its use as
a colorimetric assay for activation of human macrophages by gamma interferon. J Immunol Meth 82: 161-167.