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-Globulin Fraction Proteins and Their Metal Complexes with Copper Cations in Induction of IL-8 ProductionM. A. Apresova, I. E. Efremova, A. A. Babayants, and S. B. Cheknev

Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 156, No. 12, pp. 791-794, December, 2013Original article submitted March 12, 2012

Plasma -globulin fraction proteins, copper cations, and metal complexes formed by copper cations with human serum -globulin induce the production of up to 4.0 ng/ml IL-8 by human blood cells. Protein modifi ed by copper cations is 1.3-1.7-fold (p<0.001-0.01) more potent than control -globulin and 1.3-fold (p<0.001) more potent than copper cations alone. Analysis of the time course of IL-8 production demonstrated that IL-8 is produced as a prolonged or delayed response cytokine under conditions of this induction.

Key Words: -globulin; metal complexes; induction; interleukin-8

Laboratory of Cell–Cell Interactions, N. F. Gamaleya Research Insti-

tute of Epidemiology and Microbiology, Ministry of Health of Rus-

sian Federation, Moscow, Russia. Address for correspondence: cheknev@gamaleya.org. S. B. Cheknev

The production of IL-8, one of the most potent che-mokines attracting the neutrophils to the foci of acute infl ammation [14,15], is associated with metal cation transport and metabolism in the cell microenviron-ment [1,5].

A special role in these processes is played by -globulin fraction proteins chelating metal from the periglobular space and undergoing conformation changes that initially involve spatial packing of pro-tein molecule in its Fc region and modulating the time course of antibody interactions with cellular Fc recep-tors (FcR) [3,13].

This rearrangement of -globulins modifi es the fl ow of intracellular signals induced via FcR activa-tion, while the pool of cytokines produced during the response differs by composition from the pool induced by proteins from the native conformation of Fc region [2,4].

This pool includes, among other cytokines, TNF-α [2] and IL-6 [4]. TNF-α induces the production of IL-8 [6] and is generated in parallel with IL-8 under con-ditions of IL-18 induction [11]. Interlekine-6 is also produced in parallel with IL-8 – in response to IL-1

[10] that is synthesized in the presence of IL-18 [11]. Induction of IL-18, like that of IL-1, is analogous to TNF-α and IL-6, is focused on the cell FcR, and is defi nitely associated with -globulin fraction proteins chelating metal cations from the microenvironment. IL-18 production during the development of the in-fl ammatory response can serve as a factor directly causing IL-8 induction [11,12].

We studied the production of IL-8 by human pe-ripheral blood cells (PBC) in the presence of -globu-lin fraction proteins, their metal complexes with cop-per, and isolated copper cations.

MATERIALS AND METHODS

Induction of IL-8 in cell suspensions, prepared by di-lution of human whole peripheral venous blood (106 cell/ml), was realized in complete nutrient medium based on double Eagle’s medium (M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitis) with 2% donor plasma, L-glutamine (form the set attached to a fl ask of medium), gentamicin (20 U/ml), and hep-arin (up to 5.0 U/ml) over 24, 48, and 72 h at 37oC in a humid atmosphere with 5% CO2 in plastic fl at-bottom 24-well plates (Costar).

Specimens of copper cation-modifi ed human se-rum -globulin (initial preparation from ICN) were used in a fi nal concentration of 0.5 μg/ml. The ef-

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fects of control -globulin preparations and solution of copper hydrosulfate (Merk) with cation content cor-responding to the quantity of metal bound to protein at the stage of experimental sample preparation were evaluated in parallel.

-Globulin from the same protein portion as the respective experimental (modifi ed) sample but con-taining no Cu salt served as copper control. The vol-ume of control protein solution was brought to the needed volume with 0.15 M NaCl.

Phytohemagglutinin P (1 μg/ml; Difco) and New-castle disease virus (10 CPD/cell) served as control inductors of IL-8 production.

The content of IL-8 in the supernatants of induced PBC, used after 1:10 and 1:200 dilution of the initial material, was measured by ELISA using ELISA Pro-cessor II (Boehring). ELISA kits (Vektor-Best) were used according to the instruction with additional tech-nological controls.

The signifi cance of differences in the means dur-ing mathematical processing of the results was evalu-ated by Student’s t test.

RESULTS

The pool of early (24-h induction) IL-8, produced in the presence of control and copper cation-modifi ed -globulin and Cu cations alone, contained 761±10.69 to 1523.75±81.50 pg/ml IL-8 (Fig. 1). Spontaneous pro-duction of the cytokine by PBC was 1105±25 pg/ml.

Protein modified by copper binding 1.3 times more actively induced IL-8 (p<0.001) than control -globulin, but was 1.5-fold less potent (p<0.001) than copper cations alone (Fig. 1).

During the next 24 h of incubation, the content of IL-8 in supernatants of cell cultures, induced by control and experimental proteins, increased 1.6-2.1 times (p<0.001).

As a result, IL-8 content in the pool of cytoki nes induced to production during 48 h reached 1256.25± 90.24 to 2100.9±59.8 pg/ml (Fig. 2). Spontaneous produc tion of the cytokine by PBC was 2222.5±91.3 pg/ml. -Globulin transformed by copper cation bind-ing was 1.7 and 1.3 times (p<0.001) more active than the control protein and copper cations alone, respec-tively (Fig. 2).

During the next 24 h (72-h incubation of cells), the production of IL-8 in the presence of all control and experimental samples increased 1.9-2.2 times (p<0.001).

The production of IL-8 after 72-h induction in our experimental system reached 2805.00±121.52 to 3902.50±214.66 pg/ml (Fig. 3) vs. spontaneous pro-duction of the cytokine by PBC (1132.5±12.5 pg/ml). The production of the cytokine in the presence of the

copper metal complex reproduced the regularities of 48-h induction with the peak activity of copper-mod-ifi ed protein (Fig. 3).

Hence, activities of all studied samples increased with prolongation of cell incubation from 24 to 48 and further on to 72 h of incubation (Figs. 1-3). The production of IL-8 in the early pool was higher than its spontaneous production by PBC only in response to copper cations (1.4 times; p<0.002), while later in-duced production of IL-8 was 2.5-3.4 times (p<0.001) higher than the spontaneous one.

In response to phytohemagglutinin, human PBC produced up to 5.5 ng/ml IL-8 with the maximum after 72-h induction. Newcastle disease virus induced the production of up to 13.5 ng/ml cytokine with the peak after 48-h incubation of cells.

Wer previously found that activity of -globu-lin complex with copper, inducing the production of

Fig. 2. Production of IL-8 by human PBC after 48-h induction with

-globulin fraction proteins and their metal complexes with copper

(n=8). p<0.05 in comparison with 1; **p<0.001 in comparison with

1 and 3.

Fig. 1. Production of IL-8 by human PBC after 24-h induction with

-globulin fraction proteins and their metal complexes with copper

(n=8). Here and in Figs. 2 and 3: 1) control -globulin (0.5 μg/ml);

2) copper-modified -globulin (0.5 μg/ml); 3) copper (1.0 ng/ml).

p<0.001 in comparison with: *1 and 3, +1. n: number of observations

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TNF-α by human PBC, reached the peak during the fi rst 24 h of cell incubation and persisted throughout all periods of observation [2]. For IL-6 production this effect was detected after 48 h of incubation and persisted till 72 h of cell incubation [4]. For IL-18 it was detected only in the late pool of cytokines and was much lower than for TNF-α and IL-6.

Hence, the production of IL-8 under conditions of induction with metal complexes of -globulin fraction proteins with copper cations was more likely a result of active production of TNF-α and(or) IL-6, but not of IL-18 synthesis. It was therefore a natural manifesta-tion of stimulation of the entire macrophage-monocyte component [3,4,6], realization of intracellular signal mechanisms of congenital immunity [7,10,11], and a result of transduction focused on high-affi nity stimula-tory FcRI [6,7].

The production of IL-8 during the unfolding in-fl ammatory reaction was paralleled by rather high pro-duction of prostaglandin E2 [15], which, in turn, induced further production of IL-8, maintained and stimulated the infl ammatory component of the response [15]. The MCP-1 protein was an important factor, responsible, together with IL-8, for chronic transformation of the infl ammatory focus. This protein was synthesized in parallel with IL-8 [14]. It actively attracted to foci of infl ammation the macrophageal monocytic cells, main-taining tissue infi ltration by neutrophils, their chemo-taxis caused by the production of IL-8 [6,7].

Pro-inflammatory transformation of -globulin frac tion proteins was a result of their binding the cop-

per cations, realizing at late stages of IL-8 induction a high effector potential of their own (Fig. 3) [2,4]. Therefore, their known pro-inflammatory activity seemed to be supplemented by their capacity to in-duce IL-8. Hence, they could be regarded with good grounds as anti-infl ammatory therapy targets in deve-lopment of new pathogenetic drugs.

These data suggested that copper cations could serve as prospective targets for not only anti-infl am-matory, but also for antitumor therapy. Recent stud-ies demonstrated the key role of IL-8 in the perifocal environment of the tumor [8,9]. Interlekine-8, an au-tocrine activator of tumor cells, realizing the onco-genic activity irrespective of its pro-infl ammatory and chemotactic effects, maintained the growth of tumor cells, stimulated angiogenesis, migration, adhesion, and invasion of transformed cells to the adjacent tis-sues [8,9].

REFERENCES

1. L. V. Koval’chuk, V. L. Suslikov, L. M. Karzakova, and E. V. Sokolova, Immunologiya, 25, No. 6, 336-339 (2004).

2. S. B. Cheknev, I. E. Efremova, M. A. Apresova, and A. A. Babayants, Bull. Exp. Biol. Med., 154, No. 6, 758-761 (2012).

3. S. B. Cheknyov, I. E. Efremova, E. Yu. Denisova, and E. N. Yushkovets, Ros. Immunol. Zh., 2, No. 1, 55-62 (2008).

4. S. B. Cheknyov, I. E. Efremova, A. S. Mezdrokhina, and A. A. Babayants, Med. Immunol., 14, No. 6, 483-488 (2012).

5. B. Bao, A. S. Prasad, F. W. Beck, and M. Godmere, Am. J. Physiol. Endocrinol. Metab., 285, No. 5, E1085-E1102 (2003).

6. L. H. Faccioli, A. I. Medeiros, A. Malheiro, et al., Mediators Infl amm., 7, No. 1, 41-47 (1998).

7. W. He, Y. Zhang, J. Zhang, et al., J. Endocr., 38, No. 6, 780-785 (2012).

8. W. X. Kuai, Q. Wang, X. Z. Yang, et al., World J. Gastroen-terol., 18, No. 9, 979-985 (2012).

9. Y. S. Lee, I. Choi, Y. Ning, et al., Brit. J. Cancer, 106, No. 11, 1833-1841 (2012).

10. A. V. Orjalo, D. Bhaumik, B. K. Gengler, et al., Proc. Natl. Acad. Sci. USA, 106, No. 40, 17,031-17,036 (2009).

11. M. Sahoo, I. Ceballos-Olvera, L. del Barrio, and F. Re, Sci. World J., 11, 2037-2050 (2011).

12. D. Schuhmann, P. Godoy, C. Weiss, et al., Clin. Exp. Immunol., 163, No. 1, 65-76 (2011).

13. S. Sibéril, R. Ménez, S. Jorieux, et al., Immunol. Lett., 143, No. 1, 60-69 (2012).

14. V. Slavić, A. Stanković, and B. Kamenov, Facta Universitatis, Ser. Med. Biol., 12, No. 1, 19-22 (2005).

15. V. Srivastava, I. Dey, P. Leung, and K. Chadee, Eur. J. Im-munol., 42, No. 4, 912-923 (2012).

Fig. 3. Production of IL-8 by human PBC after 72-h induction with

-globulin fraction proteins and their metal complexes with copper

(n=8). p<0.1, **p<0.01 in comparison with 1.

M. A. Apresova, I. E. Efremova, et al.