Induction of metal-binding proteins by zinc is accompanied by reduction of other proteins including antibodies

0306~4492/85 $3.00 + 0.00 <‘, 1985 Pergamon Press Ltd

INDUCTION OF METAL-BINDING PROTEINS BY ZINC IS ACCOMPANIED BY REDUCTION OF OTHER PROTEINS INCLUDING ANTIBODIES Department

of Zcology.

tlniversity College of Wales, Aberystwyth Telephone: (0970) 3 I I I

SY23 3DA, LJK.

Abstract-l. Exposure of carp lymphoid cells to zinc causes formation of metal-binding proteins (metailothioneins). 2. Metallothionein induction is accompamed by a suppression of the production of other proteins. 3. Antibodies are among the proteins which are decreased by zinc exposure. 4. Similar results from 01 t%,cl cxoeriments indicate that zinc might affect even fish living in water still regarded

Exposure other

as unpolluted.

of a variety heavy

metals

of organisms induces

to zinc

formation

as well as of

metal-

binding

proteins known as metallothioneins (MT) (Klgi and Nordberg. 1979; Cherian and Nordberg. 1983). They are of low molecular weight, contain 23.--35% cysteinyl residues and lack histidinyl and aromatic amino acid residues. Their main role seems to be the intracellular binding of metals for transport or compartdetoxification, storage, mentalization (Foulkes, 1982). The induced synthesis of MT by low doses of toxic metals and protection against subsequent higher doses are analogous to the synthesis of immunoglobl~li~~s by an antigen in immune responses. Hovvever, little is known about the effect of the synthesis of MT on the immune system in either mamInals or fish, although zinc is known to be essential for its functioning (Scholen c’f N/.. 1979: Bach, 1981; Hansen rt al., 1981). Zinc is a common pollutant in the aquatic environment (Forstner and Wittman, 1981), is known to induce MT in fish (Marafante, 1976; Overnell et al.. 1977: Noel-Lambot et al., 1978; Kito et ul.. 1982~ Ley ct trl., 1983) and to accumulate in their lyln~hoid organs (Chipl~~~n it al., 1958; Joyner et ul., 1964: Badsha and Goldspink, 1982; Kito et cd., 1982a). Previous studies irl rim have shown that it inhibits DNA synthesis of carp lymphoid cells (Cenini and Turner, 1983). The present work investigates the induction of MT by zinc in carp immunocompetent cells and its effect on the synthesis of other proteins. with particular- reference to antibodies. In Co experiments were carried out to investigate the validity of the it? vitro findings for the living fish.

Anaesthetized carp (tricaine methanesulphonate 0.1 g/l) were bled immediately before sacrifice in order to minimize blood content in the lymphoid tissue. Cell suspensions were obtained from anterior kidney, middle kidney and spleen.

Disaggregated lymphoid cells were incubated in microtitration test plates (200 pI!well; 250,000 lymphocytes/well) for 24 hr at 22 C in the absence or presence of ZnCl,. The culture medium was Eagle’s M.E.M. supplemented with 100 i.u.:ml benzylpenicillin, 100 p&/ml streptomycin, 20 i.u.:ml heparin and 300~~gg/ml L-glutamine. No serum supplement was necessary. Viabilities were determined with the fluorescein diace~dte~ethidium bromide test (Takasugi, 197 I ). Unless otherwise specified, zinc was used at a concentration of IO-‘M. This is the concentration previously found to induce maximum inhibition of DNA synthesis without affecting cell viability (Cenini and Turner, 1983).

Cultured cells or whole lymphoid organs, both from IO iish. were washed and homogenized in 0.01 M Tris-HCI buffer. pH 7.4. The homogenates were ultracentrifuged at 105.OOOfi for 1 hr at 4 C to remove nuclear and mitochondrial fractions. The particle-free fraction was applied to a Sephadex G-75 column (1.8 x 45 cm). Equal volumes of control and experimental samples were used; all control samples contained the same quantity of proteins estimated by the method of Lowry et al. (1951) (8 mg). Fractions of 5 ml were collected at a Row rate of 25 ml/hr. Calibration of the Sephadex column for molecular weight determination was achieved with insulin, cytochrome c, tryspin and pepsin (Andrew. 1970). Metal determination was by atomic absorption s~ectr(~ph~)tometry; absorbance of the eluted fractions was measured at 280 nm. In consideration of previous tin&n&s by Kito et al. (1982a) in carp, the zinc peak in fraction 14-16. corresponding to the position of cytochrome c, was regarded as the MT fraction.

M.ATERIAI>S AND METHODS

Common carp. Cr~ri~rus crrv+ci L.. of both sexes. weighing 1255175 g, were collected from a local lake and kept at 19 0 in the aquarium. When studies on the 61 I+O induction of MT were carried out, fish were kept for 6 days in water with 5 !!&/I (ppm) ZnCl?. ‘I3

Cultured ceils were fixed with 2% fo~aldehyd~% glutaraldehyde. dehydrated and embedded in TAAB resin (mix C). Nominal 100 nm thick sections were analysed with an USC energy-dispersive X-ray spectrometer interfaced with a Link 290 Multichannel analyser fitted to a JEOL JEM 100-CX electron microscope. Accelerating voltage:

PIETRO CENINI

214

100 kV; counting time (live): 200 set; tilt angle, 40“. Two separate measurements were taken for each cell, and the different probe areas were chosen in a way that when combined they covered as much as possible of the cell cytoplasm. Results are expressed as Relative Mass Fraction (RMF) f SE. RMF = (P - b)/W where P is the number of characteristic counts in a peak, h is the background under the peak and W is the white radiation for the resin immediately adjacent to the specimen (measured in channels between 5.09 and 6.04 keV). Determination qf leucine, thymidine and cysteine uptake Cells were cultured in the absence or presence of ZnC1, (10-5, 10m4 and 10m3M). Cells were radiolabelled at the beginning of the culture by addition to each well of 0.5 PCi of [‘Hlleucine (specific activity 50Ci/mM), 14C cysteine (specific activity 40 mCi/mM) or [3H]thymidine (specific activity 5 Ci/mM), all in volumes of 10 ~1. Radioactivity was counted for IOmin in a liquid scintillation spectrometer. Results are expressed as Stimulation Index (mean counts per min of three experimental wells divided by control counts per min). Immunological test.7 Carp were injected intramuscularly with lo9 sheep red blood cells (SRBC). After 10-12 days lymphoid cells were cultured in the presence and absence of zinc (10m4M) as described previously. The supernatant was then used for the agglutination test, while the cells were washed in 0.15 M NaCl and incubated at 22°C for 2 hr in a Cunningham chamber (3 x lo5 white cells/ml plus IO9 SRBC/ml and 5% complement; serum from a single carp was used as a complement source). No effect of Zn ions on SRBC agglutination was observed at the experimental concentration. RESULTS AND DISCUSSION

The formation of MT following in vitro exposure of carp lymphoid cells to zinc was found to be greater in cells from the kidneys than the spleen (Fig. lA, B and C). Since techniques for separating different lymphoid cells of carp are poorly developed it was not possible using gel filtration chromatography to investigate whether MT formation varied in different cell types. Using X-ray microanalysis, however, it could be shown that the increased amount of bound sulphur (due to the numerous cysteine residues of the MT molecule) varied slightly between the morphologically different types of lymphoid cells recognized in carp (Cenini, 1985), the granulocytes being the most affected (Fig. 1D). No significant difference was found between the same cell types from different organs. In the case of the anterior kidney (where haemoglobin content due to blood contamination was minimal) protein determination in the particlefree fraction used for chromatography indicated a decreased amount of proteins with aromatic amino acid residues (and therefore non-MT) following zinc treatment (-30x), as asssessed by the method of Lowry et al. (1951). The absorption of the eluted fractions at 280nm showed that these proteins were mainly of high molecular weight. Studies on protein synthesis were therefore carried out using [‘Hlleucine. This particular amino acid, normally used for in uitro assay of protein synthesis, had the additional advantage of being absent in the carp MT-I molecule and present only in traces in the MT-II molecule (Kito et al., 1983). Figure 2A shows that the [3H]leucine uptake in carp lymphoid cells was indeed inhibited by

zinc ions. As a further confirmation of this differential effect of zinc on the synthesis of proteins a test using radioactive cysteine as well as leucine was carried out in the same fish (the DNA synthesis was in this case also assessed). Figure 2B shows that while the synthesis of DNA and leucine-containing proteins was decreased, the uptake of cysteine [which forms 30% of carp MT (Kito et al., 1983)] was greatly increased. This selective effect of zinc is similar to the finding of Crossley et al. (1982) in Euglerm grucilis and emphasizes the role of this metal in metabolic regulation (Vallee and Falchuk, 1981). It has been proposed (Crossley et al., 1982) that zinc ions as well as protein-bound zinc could affect gene expression, either by direct interaction with nucleic acids or by modulating the binding of regulatory proteins to their particular genes. Consequently, although MT induction and inhibition of the synthesis of other proteins might be two independent phenomena, the possibility exists that Zn-MT might itself play a role in this inhibition. In this case, although binding of zinc to MT probably has the generally recognized protective role against zinc toxicity (the metal is no longer diffusible and it may be prevented from binding with various enzymes and proteins involved in biological reactions), Zn-MT might itself have a detrimental effect, even if less severe than the one exerted by the free metal. Since zinc is known to be an essential trace element for the functioning of the immune system (Scholen et al., 1979; Bach, 1981; Hansen et a/., 1982) and the organs considered in this study are the sites of the immune response (Rijkers et al., 1980; Secombes et al., 1982), investigations have been carried out to determine whether the proteins decreased by zinc exposure included antibodies. Antibody production by zinc-treated lymphoid cells was analysed using an haemolytic plaque assay and a haemagglutination test (Table 1). The former indicated that both the number of plaque-forming cells and the amount of antibodies secreted by a single cell (measured as plaque size) were diminished when cells were previously treated with zinc. The decreased antibody secretion was confirmed with the agglutination test. As far as other organisms are concerned, recent studies by Dresner et al., (1982) have shown that zinc inhibits protein synthesis in mammalian bone marrow cell suspension while MalaG et al. (1983) found that it markedly depresses the in vitro antibody response in cultured spleen cells; Zn-MT induction in such cells might be expected since this is the case in the majority of nucleated cells and their formation was indeed observed in stimulated human lymphocvtes (Alexander et al.. 1982). These results. together with &hers previously mentioned (KBgi and fiordberg, 1979; Scholen et al., 1979; Bach, 1981; Vallee and Falchuck, 1981; Hansen et al., 1982; Foulkes, 1982; Crossley et al., 1982; Cherian and Nordberg, 1983), indicate that the effects observed with fish cells may be of significance to a diverse range of organisms, including mammals. The in vitro results reported here are similar to in uiao findings of other authors; in particular, O’Neill (1981) showed that exposure of carp to zinc affects both serum protein level and antibody titre, while Kito et al. (1982a) demonstrated zinc accumulation

Zinc metallothioneins

and antibodies

215

10

05

10

5

Fractidns

numbe~O

2:

,_

0 5

10

15

20

2s

5_

Spleen

,_

1.4 _

I3 _ )_ !2 _

I_

j.1_

I_

0,

Fig. 1. MT formation in carp lymphoid cells. A, B and C: Gel chromatography of the cytosol of control (---) and Zn-treated (---) cells; D: differences in sulphur levels detected with X-ray microanalysis in control (m) and Zn-treated (0) cells. Numbers in brackets represent number of individual cells analysed; vertical lines denote SE. Ne, neutrophils; Ba, basophils; Eo, eosinophils; BI, blast cells; Ly, lymphocytes; Th, thrombocytes; PI, plasma cells; MO, monocytes; Rs, cells with rod-shaped granules.

and MT synthesis in carp middle kidney. Supporting studies in this laboratory have confirmed the accumulation of zinc and MT formation in middle kidney in viuo, while both spleen and anterior kidney were also found to be involved in these processes (Table 2). In agreement with in vitro experiments the anterior kidney was found to be particularly affected, showing a decrease (25%) in the amount of proteins detected both by the method of Lowry et al. (1951) and by absorption at 280nm. These similarities between in vitro and in vivo findings indicate the validity of the former for the living fish. The fact that the lymphoid organs of carp and other teleosts (Chipman et al., 1958; Joyner, 1964; Badsha and Goldspink, 1982) accumulate zinc indicates that the effects reported here may be apparent even in fish living in waters with very low concentrations of the metal which are still considered as unpolluted; the strength of an

immune reaction against a possible pathogen might then be decreased. The intensity of the effects exerted by zinc was found to be greater for the kidneys than the spleen; in adult fish the kidneys are the more important organs immunologically (Rijkers et al., 1980; Zapata, 1983). This observation provides an additional reason for concern for the immune system itself in cases of environmental pollution. This study concentrated on antibody production by lymphoid cells. However since zinc was found to affect the metabolism of all of the leucocyte types (Fig. 1D) it is likely that other parameters of the immune response besides antibody production will also be affected. In addition, the proteins affected by zinc might include other substances of biological importance which are not involved in the immune response, while other cell types might be affected in

PIETKOCENINI

216

”

I

01

104

10-5

Zinc

10-3

concentration

Anterior kidney

(M)

Middle kidney

Fig. 2. Effect of zinc on protein synthesis. A: Effect on [3H]leucine uptake. Anterior kidney (e), middle kidney (0) and spleen (0). Points represent mean values of six fish + SE. Mean counts per min of control cultures: anterior kidney, 3102; middle kidney, 3345; spleen, 810. Viabilities were >95% (10m5and 10m4 M) or < 10% (IO-’ M). B: Differential effect on [3H]leucine (m), [‘4C]cysteine (D) and [‘Hlthymidine (m) uptake in lymphoid cells from the same animal following zinc exposure (10m4 M). Mean counts per min of control culture: anterior kidney, 2967 (%), 10,754 (m), 3825 (m); middle kidney, 3288 (m), 16,167 (D), 4510 (m); spleen, 793 (8), 3410 (B), 874 (m).

Table 1. Antibody production by zinc-treated cells PFC/lO’white CantroI Anterior Middle

Agglutination

cells Z,nc-treated

Control

titre

Z,nc-treated

kidney kidney

SpkXfl

PFC = Plaque-forming cells. *Arithmetic mean i SE (N = 5). # Number of fish showing the agglutination

titre (in brackets).

Table 2. Zinc accumulation and MT formation

Anterior Middle

in t$v

kidney kidney

Spleen

MTF = Metallothionein-fraction. *Arithmetic mean i SE (N = 5).

way. Furthermore, if Zn-MT is involved in the suppression of protein production, then other heavy metals of environmental importance such as chromium and nickel which are known to induce Zn-MT in carp (Kito et a/., 1983) or cadmium, which substitutes zinc in Zn-MT (Kito et al., 1982b), may a similar

reduce zinc.

or compound

the effects described

here for

Acknowledgements-The author wishes to thank Dr Rodney J. Turner for his constructive criticism and encouragement throughout this study, which was supported by a

Zinc metaliothioneins University of Wales Studentship. Thanks are also due to Dr M. P. Ireland, Dr I. ap Gwynn, Dr M. R. L. Johnston and Sarah Cross for helpful discussion, also J. Meredith and A. Henley for technical assistance. REFERENCES

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Fiirstner U. and Wittman G. T. W. (1981) Metal Pollution in the Aquatic Environment, 2nd revised Edn, 846 pp. Springer, Berlin. Foulkes E. C. (I 982) Biological Roles of ~eta~~oth~onein, 327 pp. Elsevier~North Hohand, New York. Hansen M. A., Fernandes G. and Good R. A. (1982) Nutrition and immunity: the influence of diet on autoimmunity and role of zinc in the immune response. A. Rev. Nutr. 2, 151-177. Joyner T. (1964) Exchange of zinc with environmental solutions by the brown bullhead. Trans. Am. Fish. Sot. 90, 444448.

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