Mitogenic effects of beryllium and zirconium salts on mouse splenocytes in vitro

Toxicology

Letters,

30 (1986) 89-95

89

Elsevier

TOXLett.

1530

MITOGENIC EFFECTS OF BERYLLIUM MOUSE SPLENOCYTES IN VITRO

AND ZIRCONIUM

(Beryllium; zirconium; mouse splenocytes; lymphocytes; ROGER MRC

J. PRICE Toxicology

Surrey,

and DAVID

SALTS ON

mitogenesis)

N. SKILLETER

Unit, Medical

Research

Council

Laboratories,

Woodmansterne

Road,

Carshalton,

SM.5 4EF (U.K.)

(Received

October

8th,

(Revision

received

and accepted

1985) November

28th,

1985)

(Zr) salts,

BeSO

SUMMARY The beryllium dependent incorporation 100-200

into lymphocyte

to assist

results indicate augment

of mouse

pM) was greater

low concentrations found

(Be) and zirconium

stimulation

tion of delayed

DNA, although

subsequent that at defined

(concanavalin

concentrations

responsiveness

hypersensitivity

as measured response

each exerted by an increase induced

range of Zr(SO&

concentrations

A; ConA)-mediated

a concentrationin [3H]thymidine

by Zr(SO&

(2-3 fold at l-5 FM). Preincubation

(< 1 PM) or a broad lectin

and Zr(SO&,

the maximal

than that by BeSO

of BeSO

the functional

spleen cell proliferation

and production

lymphocyte

proliferation.

granulomas

The

mitogens

cells, which may help explain the characteristic of immunological

with

(2-100 PM) was also

Be and Zr salts can both act as lymphocyte

of immune

(4-5 fold at

of splenocytes

by these metals

and

inducin viva.

INTRODUCTION

The metals Be and Zr have important applications in the nuclear, aerospace and metal alloy industries as well as other specialised uses [1,2]. A characteristic of human exposure to these metals and some of their compounds is the development in many individuals of cell (T-lymphocyte)-mediated delayed skin hypersensitivity followed by the appearance of immunological granulomas [3,4]. These granulomas are described by the presence of giant and epithelioid cells of mononuclear phagocyte origin associated with lymphocyte infiltration and contrast with the granulomas produced by metals such as aluminium which contain mainly phagocytosing macrophages and have been described as non-immunological [5]. Abbreviations:

0378-4274/86/$

ConA,

concanavalin

03.50 0 Elsevier

A; IL, interleukin;

Science

Publishers

TCA,

trichloroacetic

B.V. (Biomedical

acid.

Division)

90

Although the in vivo immunotoxicology of Be and Zr has been extensively studied in experimental animals [5,6], relatively little is known about the effects these metals have on immune cells in vitro. Cells isolated from spleen are known to comprise both lymphocyte and accessory (adherent macrophage type) populations and thus provide a useful in vitro system to monitor certain immune cell properties particularly mitogenic responses 171. In the present study, using the incorporation of [3H]thymidine into cultured mouse spleen lymphocyte DNA as a measure of cell proliferation, we have examined both the direct mitogenic effects of Be and Zr salts and the consequences of treatment of splenocytes with the metal salts on their subsequent response to lectin mitogens. The results indicate that Be and Zr salts stimulate lymphocyte proliferation and augment subsequent lectin-mediated mitogenesis, but that these reponses are strongly concentration-dependent. MATERIALS

AND METHODS

Materials Metal salts of analytical grade were obtained from Aldrich Chemical Co. Ltd., Gillingham, Dorset (U.K.) and aqueous solutions (1 mM) were freshly prepared before use and maintained at pH 5.0 to avoid formation of colloidal precipitates. All metal ion solutions and ConA (Sigma Chemical Co., Poole, Dorset, U.K.) were filtered through a 0.22 FM MiIlipore filter prior to addition to the culture media. The lymphocyte culture medium (RPM1 1640) was purchased from Gibco-Europe, Paisley, Scotland; foetal calf serum from Sera Lab., Crawley Down, Sussex; phosphate-buffered saline (Dulbecco A) from Oxoid Ltd., Basingstoke, Hampshire, and flat-bottomed 3.5ml capacity Linbro multi-well culture plates from Flow Laboratories, Irvine, Scotland. [3H]Thymidine was obtained from Amersham International, Bucks (U.K.). Preparation of spleen cells Spleens from COz-killed adult male MFI mice (20-25 g), were gently handhomogenized in 10 ml RPM1 1640 and filtered through a 250~PM mesh nylon gauze. The filtrate was centrifuged (200 x g, 1 min) to remove debris and the supernatant mixed with 2 ~01s. 0.87% NH&l to lyse red blood cells. The splenocytes were centrifuged (350 x g, 10 min) and washed in RPM1 1640, and finally filtered through a lOO-PM nylon gauze. The cell yield was approx. 45 x lo6 lymphocytes per mouse spleen and contained approx. O. l-0.2% adherent cells. Cell viability was measured throughout the study by trypan blue dye exclusion and estimated to be 85-90% in freshly isolated cells. Lymphocyte incubations Splenocytes (2.5 x lo6 cells) were incubated in the presence or absence of metal

91

ion additions in 1 ml RPM1 1640 medium containing 5% heat-inactivated foetal calf serum maintained in culture plates at 37°C pH 7.3 in a humidified atmosphere of 95% air 5% CO2. When used ConA was included in the medium at an optimal concentration of 2 pg/ml, cell proliferation was measured after the indicated treatments by a 2-h pulse-labelled [3H]thymidine (0.1 &i) incorporation into lymphocyte DNA. The aspirated well contents were sequentially centrifuged (300 x g, 10 min) and washed with 1 ml RPM1 1640 and 3 ml phosphate-buffered saline, followed by precipitation overnight at 0°C in 5% TCA. The centrifuged (2500 x g, 10 min) tissue pellet was dissolved in 0.5 ml 1 M NaOH overnight mixed with 0.5 ml distilled water, 100 ~1glacial acetic acid and 10 ml Beckman MP Scintillant prior to measurement of 3H-radioactivity in a Packard Tri-Carb Model 460 scintillation counter. RESULTS AND DISCUSSION

Fig. 1 shows that after an optimum incubation period of 3 days Zr(SO& was a more effective mitogen to mouse splenocytes than BeSOd. Furthermore, stimulation of [3H]thymidine incorporation into lymphocyte DNA was produced by only a narrow range of BeSO concentrations (l-5 PM) whereas maximal effects of Zr(SO& were observed over a much broader and higher concentration range (20-200 PM). 10-

0

I 20

5 Metal

I I I loo 200 300 400

salt concentration

(@II

Fig. 1. Comparison of the stimulation of mouse spleen lymphocyte DNA synthesis by beryllium and zirconium. Mouse splenocytes (2.5 x lo6 cells) were incubated in the presence of various concentrations (abscissa) of either BeSO (0), Zr(SO)d (0) or Ah(S04)3 (A) for 3 days prior to measurement of pulselabelled

[3H]thymidine

5 determinations.

incorporation

(2 h) into lymphocyte

DNA. Values (ordinate)

are means

+ SD of

92

No incorporation of radiolabel was detected in the presence of 10 mM hydroxyurea, demonstrating that the metal salt stimulation was due to replicative DNA synthesis and not unscheduled DNA repair [8,9]. The metal ions Hg’ + and Zn’ ’ have been shown to induce lymphocyte proliferation in vitro with optima at approx. 10 PM and 100 pM, respectively, possibly by direct action of the metal ions on T-cells HO-121 and we have demonstrated previously Be2 + binding to lymphocytes [22]. Both Be and Zr salts readily form hydroxides at physiological pH and thus differences in the dose-response of lymphocyte replication to these metal salts may simply be a reflection of the proportions and availablility of free metal ions. In this respect it would be of value to know the differential hydroxide precipitation characteristics of BeS04 and Zr(SO& in the culture medium but the detailed analyses required for this estimation have yet to be carried out. The decrease in [3H]thymidine incorporation induced by metal salt concentrations above 5 pM BeSO or 200 PM Zr(S04)2 may have been associated with some cytotoxic effects since the presence of 50-100 FM BeSO or 200-400 pM Zr(SO& was shown to cause a 25% and 15% loss of cell viability respectively. However, a more probable explanation might be that of an opposing effect of the inhibition of cell division since it is known that, at least in non lymphoid cells, intracellular access of a number of metal ions including Be’+ can block DNA synthesis [24,25]. Facilitation of T-lymphocyte activation by lectin mitogens requires the presence of accessory cells [ 13,141 which were known to be present in the splenocyte preparations. Pretreatment of splenocytes with compounds followed by lectin stimulation can thus be used to monitor affects of such materials on the normal mitogenic response of lymphoid cells [7]. We have used this approach to examine the action of optimal and suboptimal mitogenic concentrations of BeSO and Zr(SO& on the subsequent response of splenocytes to ConA. A characteristic of our in vitro test system was that a 20-h preincubation of cells in the absence of lectin resulted in an 80% decrease in the ConA-stimulated [3H]thymidine incorporation into lymphocyte DNA. Our earlier studies had established that this was due to loss of the accessory cells or their activation factors from the culture since ConA responsiveness could be restored by subsequent addition of macrophages [23]. However, Table I shows that pretreatment with appropriate concentrations of BeSO or Zr(SO& for 20 h substantially maintained the proliferative response. This was most marked in the case of Zr(SO4)z for which a 40-60% retention of lymphocyte responsiveness to ConA was achieved over a broad range of concentrations (20-100 yM) and thus the effect appeared to be independent of the Zr(SO& concentrations already shown to cause direct mitogenesis (cf. Fig. 1). In contrast only pretreatment of splenocytes with a normally nonmitogenic concentration of BeSO (viz. 0.2 FM) resulted in a significant retention of the response to ConA, concentrations above 1 pM BeSO being strongly inhibitory (Table I). A similar marked loss of lymphocyte responsiveness to mitogens in vitro has been

93

TABLE

I

EFFECT

OF PREINCUBATION

CONIUM Mouse

ON SUBSEQUENT

splenocytes

OF MOUSE RESPONSE

(2.5 x lo6 cells) were incubated

serum at 37°C for 20 h under the preincubation in the presence

of 2 a&/ml ConA

into lymphocyte Preincubation

SPLENOCYTES

TO CONCANAVALIN

DNA.

and measurement

Values are means

conditions

in RPM1

conditions

WITH

1640 medium

indicated

of pulse-labelled

BERYLLIUM

AND

ZIR-

A containing

prior to a further 13H]thymidine

5% foetal calf 3 days incubation

incorporation

(2 h)

k SD of 5 determinations.

Proliferation

response

incorporation

(dpm)

to ConA

[jH]thymidine

Control (no preincubation)a

76 286 f

No additions

14 929 + 1797

BeSO

0.2 gM

29 874 + 3988

1.0 FM

13 326 i

5.0 PM Zr(S0)4

2 CM

790 +-

8550

1244 278

35 257 + 3331

20 PM

41 966 * 5210

100 NM

47 484 r 7898

a Incubation

in the presence

of ConA

(2 pg/ml)

for 3 days without

any preincubation

treatment.

observed following pretreatment of splenocytes with silica or carbonyl iron and shown to result from adherent cell destruction [7]. Mononuclear phagocytes are known to be extremely sensitive to the cytotoxicity of several Be compounds [15-171 and consequently the loss of BeSO augmented ConA mitogenesis above 1 FM BeSO might also have been caused by elimination of the adherent cell population. We were able to substantiate this conclusion from examination of the culture dishes by light microscopy which revealed a severe loss of the adherent cells. In contrast Zr(SO& caused little loss of adherent cells which suggests that it was relatively noncytotoxic to macrophages, a conclusion supported by the evidence that this is known to be the case for Zr(OH)d [lS], a major product generated from Zr(SO& in the culture medium. At present the mechanism by which pretreatment with Zr(SO&, or low concentrations of BeS04, maintained subsequent ConA-induced mitogenesis is not clear. However, Zr and Be may enhance the functional responsiveness of immune cells through stimulation of IL1 secretion by the macrophage population with resultant lymphocyte (T-cell) proIiferation via IL2 production 1191. There is some experimental evidence that BeSO can activate IL1 release [20] and such a response might also play a role in the direct mitogenic effects of the metal salt since the release of IL1 and other lymphocyte growth factors from damaged macrophages and lymphocytes could contribute to the proliferative stimulus [19,21]. It is hoped that future studies might provide data to substantiate these suggestions. In the course of our study we also examined the effects of aluminium salts on mouse splenocytes since in vivo these compounds have been considered to produce

94

only nonimmunological granulomas [5]. We were unable to demonstrate any mitogenic properties of Al2 (SO& (Fig. 1) and pretreatment of spleen cells with the metal salt (l-50 PM) eliminated ConA-induced lymphocyte proliferation. Al salts have similar physicochemical properties to those of Be and Zr in that they too readily form hydroxides at physiological pH; furthermore, previous studies have shown that colloidal Al(OH)3 is highly cytotoxic to macrophages [18]. A probable interpretation of the present findings therefore is that the induction of delayed hypersensitivity and production of the distinctive immunological granulomas by certain Zr and Be compounds in vivo may in part be related to their dose-dependent properties of directly stimulating and, through interaction with macrophages, indirectly augmenting lymphocyte proliferative responses to other agents.

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