ISSN 10683674, Russian Agricultural Sciences, 2016, Vol. 42, No. 2, pp. 184–187. © Allerton Press, Inc., 2016. Original Russian Text © O.I. Mamykova, 2016, published in Rossiiskaya Sel’skokhozyaistvennaya Nauka, 2016, No. 1, pp. 53–57.

VETERINARY

Experimental Assessment of Immunobiological Activity of the Roncoleukine Immunomodulatory Agent O. I. Mamykova Skryabin AllRussia Research Institute of Fundamental and Applied Parasitology of Animals and Plants, Moscow, 117218 Russia email: [email protected] Received September 22, 2014

Abstract—We have found that roncoleukine (recombinant interleukin 2, rIL2) when double administered in the dose of 5000 IU/kg of body weight or 10000 IU/kg of body weight enhanced functional activity of the effector cells of the delayed type hypersensitivity. It was demonstrated that the selective action of rIL2 in the dose of 10000 IU/kg of body weight induced protective effect (more than 50%) in the mice experimentally infected with T. spiralis. Keywords: immunomodulating agent, immunobiological activity, protective effect, antibodies, cellmediated and humoral immunity DOI: 10.3103/S1068367416020087

INTRODUCTION The impairment of the immunomodulatory func tion of T lymphocytes in parasitic diseases and unde sirable side effects of anthelmintics generate a need to use immunoactive drug products together with the etiotropic ones. Inefficiency of the immunomodula tors selection methods is an essential drawback of the combined therapy. The choice of immunomodulator should be based on its immunobiological activity inso far as targeted action on the immunity factors that need to be pharmacologically controlled in order to increase the efficiency of the therapy is required, as well as on the protective effects experimentally detected in the parasitic diseases models [1, 2]. The aim of the current work was to study immuno biological properties of the roncoleukine immuno modulating agent in order to find new therapeutic means of treating helminthoses. MATERIALS AND METHODS Drug substance of roncoleukine is recombinant interleukin 2 (IL2), a 15.4 kDa polypeptide biotech nologically produced by the producer cells of the baker’s yeast Saccharomyces cerevisiae recombinant strain. In the work, we used injectable roncoleukine (rIL2), 1 cm3, which contained 0.05 mg, or 50000 IU of the recombinant interleukin 2. According to the drug product label, exogenous interleukin 2 directly influ ences the growth, differentiation, and activation of T and B lymphocytes, monocytes, and macrophages.

The drug product was double administered with 24 h or 48 h intervals subcutaneously to each individual mouse into the neck skin fold in the doses of 2000 IU/kg of body weight, 5000 IU/kg of body weight, and 10000 IU/kg of body weight. To the control group mice, no drug was administered. Experimental assessment of the immunobiological effects of roncoleukine on the cellular and humoral reactions was performed in two replication using 90 white outbred mice males with the body weight of 18– 20 g provided by the Stolbovaya farm of the Scientific Center for Biomedical Technologies of the Russian Academy of Medical Sciences. Experimental groups were formed containing 8–10 mice each in order to obtain statistically significant results. All animals were kept under similar conditions in the vivarium and were withdrawn from the experiment by cervical disloca tion or decapitation depending on the experimental purposes. The state of the cellmediated immunity upon the administration of roncoleukine was estimated by the ability of mice to develop the delayed type hypersensi tivity response, which was simulated according to the method of Kitamura [3]. Production of cellbased immunity mediators by the effector cells of the delayed hypersensitivity was stimulated by sheep erythrocytes (thymusdependent antigen). To simulate the delayed type hypersensitivity response, mice from the experi mental groups were primed by the intraperitoneal injection of 0.5 mL of sheep erythrocytes in the form of 3% suspension in sterile physiological solution. To develop sensibilization, the booster antigen dose was injected on the fifth day after injection into the right

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hind foot pad (experimental) in the volume of 25 μL of the 15% sheep erythrocyte suspension, while the same volume of the 0.83% NaCl solution was injected into the left foot pad (control). The response level was assessed by the increase in the weigh of the feet 24 h after the injection. The intensity of the delayed type hypersensitivity cellular response was estimated by the response index (RI) shift value, indicated as a percent age. Response index was calculated according to the following formula: RI = (Me – Mc)/Mc × 100%, where Me is experimental foot weight and Mc is con trol foot weight. The state of the T cellmediated humoral immu nity was estimated by testing the ability of the mouse’s organism to produce antibodies against the immuniz ing agent, the thymusdependent antigen (sheep erythrocytes), upon roncoleukine administration. To stimulate the production of haemagglutinins, all mice were immunized by the intraperitoneal injection of the optimal dose of the test antigen, 0.5 mL of 3% sheep erythrocyte suspension. Antibody titer in the serum of each mouse was measured at the peak of the primary immune response using the microtechnique variant of the direct haemagglutination test [4]. Antibody titer was expressed as log1/2. To compare immune responses in the experiment and the control, the drug compound action index was calculated which is the ratio of the antigen titer in the experiment to the antigen titer in the control. An action index value equal to 0.7 or lower indicates the humoral immune response suppression under the action of the drug compound under study, while the index value equal to 1.3 or higher indicates the stimulatory action of the drug compound. In a series of trials using the mice experimental tri chinosis model, we studied the protective properties of roncoleukine (rIL2) as a criterion of activation of the animal organism’s resistance to helminthic infections and of the immunomodulatory action of the drug compound under study. In each trial, we used 10–12 white outbred mice males with the body weight of 18– 20 g infested by Trichinella spiralis invasion larvae. Experimental infection was performed by the peroral introduction of T. spiralis larvae using an esophageal probe. The invasion larvae dose per mouse varied and was 140 larvae in the I and II trials and 20 larvae in the III trial. Injectable roncoleukine was administered in the dose of 10000 IU of the recombinant interleukin 2 (20000 IU/kg of body weight in total). Subcutaneous injections were performed two times at different time intervals after the infection (intestine phase and mus cle phase of the T. spiralis life cycle). Infested animals to which drug compound was not administered served as the control. Roncoleukine protective effect was estimated 40– 45 days after the infection by the number of T. spiralis larvae in a gram of muscle tissue using the digestion in RUSSIAN AGRICULTURAL SCIENCES

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artificial gastric juice technique. Artificial gastric juice was prepared by dissolving 2 g of pepsin and 10 mL of concentrated HCl in a liter of tap water. After com plete digestion, larvae were washed and their number was calculated under a binocular microscope with 16× magnification. The sections of diaphragmal and mas seter muscles obtained from each mouse under study were tested for the presence of encapsulated T. spiralis larvae using the compression trichineloscopy tech nique. The protective effect was estimated by the values of the invasion retardation index (R), which was calcu lated according to V.A. Chernov’s formula [5]: R = (C – E)/C × 100, where C and E is the number of T. spiralis larvae detected in a gram of muscle in the animals from the control group and experimental group, respectively. Statistical processing of the experimental data was performed using STUDENT 200 software. The statis tical significance of the differences for the features under study was assessed by the Student’s ttest, and the significance level was 0.05. RESULTS AND DISCUSSION The intensity of the delayed type hypersensitivity cellmediated response illustrates the ability of phar maceutical compounds to affect the production of the cellbased immunity mediators with diverse effects by the primed lymphocytes that engage the cells of the mononuclear phagocytes system in the immune response. The efficient delayed hypersensitivity response is initiated by the repeated contact of the primed lymphocytes with the corresponding antigen in the presence of macrophages. For this reason, the intensity of the cellmediated response indirectly rep resents the rate of the functional activity of the mac rophages. Those pharmacological compounds or bio logically active substances which cause the delayed hypersensitivity response index shift have effects on the cellmediated immunity [3]. The results of testing the delayed hypersensitivity effector cells are presented in Fig. 1. Subcutaneous injections of roncoleukine (rIL2) in the tested doses caused cellmediated reactions of varying intensity. When the drug compound was administered in the dose of 2000 IU/kg of body weight in two successive injections with a 24 h interval, it caused statistically insignificant enhancement of the delayed hypersensi tivity cellmediated response, namely, a 22% increase over the control level (6.58 ± 0.5%). The delayed hypersensitivity response index value was 8.03 ± 1.12% (p > 0.05). The increase in the drug compound dose resulted in a more profound index shift. When roncoleu kine was administered in the doses of 5000 IU/kg of body weight and 10000 IU/kg of body weight, also with 24 h intervals, the delayed hypersensitivity response indices were 12.11 ± 2.11% and 9.29 ± 0.70%, respectively, 2016

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Delayed hypersensitivity response index, % 12 10 8 6 4 2 0 I II III Control Fig. 1. Intensity of the delayed hypersensitivity cellmedi ated response upon the double subcutaneous rIL2 injection. I, II, and III—roncoleukine in the doses of 2000 IU/kg of body weight, 5000 IU/kg of body weight, and 10000 IU/kg of body weight, respectively.

and were 84.0 and 41.1% higher, than the respective control indices’ values. The effect of the delayed hype sensititvity cellmediated response enhancement was statistically significant (p < 0.05). Therefore, inject able roncoleukine (rIL2) in the tested doses upon the administration in two successive subcutaneous injec tions with the 24 h interval is able to enhance cell mediated immune response in white outbred mice. The results of the direct haemagglutination test showed that injectable rIL2 in the tested dose range had no effect on the rate of the antibody production against the test antigen (Fig. 2). When rIL2 was administered in the doses of 5000 IU/kg of body weight and 10000 IU/kg of body weight in two succes sive subcutaneous injections, the titer of haemaggluti nins in the blood serum was logS 5.85 ± 0.48 and logS 6.14 ± 0.49, respectively, which did not differ from the control titer (logS 5.57 ± 0.40). The action indices were 1.1 and 1.05, respectively, and they also indicated the nonresponsiveness of the humoral immune reac tions to the drug compound under study. Upon the rIL2 administration in the dose of 2000 IU/kg of body weight in two successive subcutaneous injections with the time intervals of 24 h or 48 h, we observed a tendency towards the enhancement of the T cell mediated humoral immune response. In the haemag glutination test, haemaglutinins in the blood serum were detected in the titers of logS 6.83 ± 0.75 and logS 6.50 ± 0.37, respectively, and were 22.6% and 16.6% higher than the respective control titers. The action index values of 1.22 and 1.16 evidenced the tendency to the enhancement of the humoral immune response to the thymusdependent antigen under the action of the drug compound in the dose of 2000 IU/kg of body weight. Increasing the time interval between the two rIL2 administrations had no effect on the antibody production rate. We have demonstrated the tolerance of immunecompetent cells, whose functioning and cooperative action provides for the antibody produc tion against thymusdependent antigens, to the tested doses of injectable roncoleukine.

log1/2 antibody titer 7 6 5 4 3 2 1 0 I Ia

II

III

Control

Fig. 2. Rate of the antibody production against the thy musdependent antigen upon the double subcutaneous rIL2 injection. I and Ia—roncoleukine in the dose of 2000 IU/kg of body weight (with the intervals of 24 h and 48 h, respectively); II—roncoleukine in the dose of 5000 IU/kg of body weight; III—roncoleukine in the dose of 10000 IU/kg of body weight.

In such a way, in our experiments, we have demon strated the selective action of injectable roncoleukin (rIL2) on the lymphoid cells. The mechanism of immunobiological action of the drug compound is determined by its predominantly stimulating effect on the cellmediated immunity and macrophages. We observed the indifference of the immunecompetent cells, whose functioning lies behind the Tcellmedi ated humoral immune response, to the immunomod ulating agent’s action. Selective mode of action of the immunoactive compounds appears to be a valuable quality, since an imbalance between the cellmediated and humoral immunity factors is usually observed in parasitic diseases. An impairment of the immuno modulating function of Tlymphocytes, which control migration, proliferation, and differentiation of stem cells, is usually noted. The depletion of the Tcells pool stimulates antibody synthesis. The chief regulator of the immune response is normally considered to be the suppressor Tcells, whose functioning results in the accumulation of the inhibitory factor that nonspe cifically influences B cell proliferation, thereby deter mining the antigen producer cells clone size [6]. Insuf ficient production of the factors regulating Blympho cytes by the Tlymphocytes in invasive diseases results in the polyclonal Blymphocyte activation [7, 8]. Tak ing into account the above stated and our own obser vations, in order to disturb the balance in the host– parasite system and to induce the protective immunity, the action of an immunomodulating agent should be directed at enhancing functional activity of the Th1 cells of the helper T cell population and at suppressing the activity of the Th2 helper T cells responsible for the development of the humoral immune response [2]. This idea served an impulse to study the protective properties of rIL2 in the dose of 10000 IU/kg of body weight, whose administration in two successive doses resulted in the pronounced selective action of the drug compound.

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Protective effects of the roncoleukine immunomodulating agent (rIL2) in the mice with the experimental invasion of Tri chinella spiralis Variant

Number of T. spiralis larvae per mouse

Immunomodulator administration scheme, days after infection

Number of T. spiralis larvae in a gram of muscles

Invasion retardation index, %

I

140 140

20th and 21st control

451.89 ± 218.46 751.59 ± 678.33

39.87

II

140 140

sixth and seventh control

455.21 ± 471.46 975.85 ± 1231.83

53.35

III

20 20

first and second control

50.38 ± 13.27 103.59 ± 10.002

51.36

Protective effects of rIL2 upon its administration to the mice with experimental T. spiralis invasion are displayed in the table. It follows from the presented data that the administration of the immunomodulator induces an increase in the organism’s resistance to the parasite invasion, decreasing the number of encapsu lated T. spiralis larvae in a gram of muscle tissue. The immunomodulator administration during the intes tine phase of the Trichinella life cycle provides for the most advantageous conditions for the resistance enhancement to the invasion. Protective effect was observed to be higher than 50%. When animals were infected with 20 T. spiralis larvae, the statistically sig nificant suppression of the invasion was obtained. On the sections of the diaphragmal and masseter muscles, only single unencapsulated T. spiralis larvae were detected by the compression trichineloscopy. In our opinion, the impairment of the capsule formation around the larvae may also be regarded as a result of the protective response induction by the immuno modulator. The drug compound administration dur ing the muscular larvae phase (20th day after the infec tion) provided for less pronounced invasion suppres sion. Thus, it appears that the encapsulated T. spiralis larvae are less sensitive to the tested drug compound. The results of our study confirm the idea that the induction of resistance to the invasion is determined by the selective action of the immunomodulating agent and its ability to induce the immune response of the Th1 type. Th1 helper cells produce IL2 and IFNγ cytokines, which have protective capacities for para sitic diseases. The switch to the Th1 type immune response (cellmediated immunity) is followed by the enhancement of the functional activity of the suppres sor T cells, which are also capable of producing INFγ, the universal protection factor [9, 10]. Production of the cellbased immunity mediators by the delayed hypersensitivity effector cells takes place under the conditions of the adaptive antigen transfer in the pres ence of macrophages. For this reason, the contribu tion of the INFγstimulated macrophages to the development of the protective immunity by means of the nitrogen oxide production cannot be ruled out, and nitrogen oxide has a role in parasite elimination [11]. The selectivity of the immunobiological action of RUSSIAN AGRICULTURAL SCIENCES

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injectable roncoleukine and its high protective effects open possibilities to use roncoleukine (rIL2) as a co drug in the complex therapy of helminthic infections in order to enhance the efficiency of the treatment. REFERENCES 1. Mamykova, O.I., Anthelmintic activity of Panacur combinations with immunoactive drugs, Tr. Vseross. Inst. Gel’mintol., 2000, vol. 36, pp. 102–117. 2. Mamykova, O.I., Immunomodulating activity of T activin prescribed with fenbendazole in various regi mens, Russ. Agric. Sci., 2008, vol. 34, no. 3, pp. 199– 202. 3. Kitamura, K., A Footpad weight assay method to eval uate delayedtype hypersensitivity in the mouse, Immu nol. Mehtods, 1980, vol. 39, no. 3, pp. 277–283. 4. Sever, J.L., Application of a microtechnique to viral serological investigations, J. Immunol., 1962, vol. 88, no. 1, pp. 398–413. 5. Chernov, V.A., Metody eksperimental’noi khimioterapii (Methods of Experimental Chemotherapy), Moscow: Meditsina, 1972. 6. Stepanenko, R.N., Thymusdependent suppressor cells, in Itogi nauki i tekhniki. Ser. Obshchie voprosy patologii (Results of Science and Technology. Ser. Gen eral Problems of Pathology), Moscow, 1976, vol. 4, pp. 90–100. 7. Terry, R. and Hudson, K., Immunodepression in para sitic infection, Fortschr. Zool., 1982, vol. 27, pp. 125– 139. 8. Cathy, S. and Nutman, Th.B., Helminth antigens selectively differentiate insensitive CD45RA+ CD4+ human Tcells in vivo, J. Immunol., 1998, vol. 16, no. 1, pp. 351–360. 9. Heinzel, F.P., Ahmed, F., and Hujer, A.V., at al., Immunoregulation of murine leishmaniasis by inter leukin 12, Res. Immunol., 1995, vol. 146, nos. 7–8, pp. 575–582. 10. Meeusen Els, N.T., Immunology of helminth infec tion, with special reference to immunophathology, Vet. Parasitol., 1999, vol. 84, pp. 259–273. 11. Fowell, D.J., Wakil, A.T., and Locksley, R.M., Inter leukin12 in murine leishmaniasis match, flame or fuel?, Res. Immunol., 1995, vol. 146, nos. 7–8, pp. 566– 571.

Translated by E. Martynova 2016