Toxicology, 50 (1988) 247-256 Elsevier Scientific Publishers Ireland Ltd.

EFFECT OF MERCURIC CHLORIDE ON MICROBICIDAL ACTIVITIES OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES

BRIGITTE BAGINSKI

Institute of Hygiene, University of D~sseldorf, Au f m Hennekamp 50, D-~O00 D~sseldorf lF.R. G.)

(Received August 3rd, 1987) (Accepted November 15th, 1987)

SUMMARY

We investigated the effects of mercuric chloride on phagocytic capacity, formation of toxic oxygen species and release of lysosomal enzymes of human polymorphonuclear leukocytes (PMNL). Our results show that HgCl 2 may alter these microbicidal functions of human PMNL without remarkable damage of cell viability. The phagocytic capacity was markedly depressed in a concentration-dependent manner. The formation of toxic oxygen species was also diminished by mercuric chloride when induced by phagocytosis. It was furthermore reduced when the PMNL were activated without phagocytosis by binding of IgG to Fc-receptors or by binding of phorbol myristate acetate to the membrane. In contrast, the release of the lysosomal enzyme lysozyme was enhanced in the presence of mercuric chloride, but not the release of fJ-glucuronidase. These effects may lead to impaired defense against infections and possibly to inflammatory reactions in adjacent tissues induced by released lysosomal enzymes.

K e y words: Human PMNL; Mercury; Chemiluminescence; Phagocytosis; Lysosomal enzymes; Cytotoxicity

INTRODUCTION

The unspecific polymorphonuclear leukocytes (PMNL) are phagocytes, which are very quickly accumulated on sites of infections [1]. Their

Abbreviations: PMNL, polymorphonuclear leukocytes; DMEM, Dulbecco's minimum essential medium; LDH, lactate dehydrogensae; PMA, phorbol myristate acetate; DMSO, dimethyl sulfoxide; IgG, immunogiobulin G; Hepes, N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (biological buffer).

0300-483X/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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microbicidal functions are essentially involved in the defence against bacteria. Following phagocytosis or contact with soluble stimuli PMNL undergo an increase in non-mitochondrial oxidative metabolism leading to the formation of toxic oxygen species such as superoxide anion, hydrogen peroxide and the hydroxyl radical [2,3]. It is a well known fact that the functions of human PMNL may be altered, that is mainly decreased, by several drugs such as anti-phlogistics or cortisone-derivates [4--6]. It was observed in several studies that heavy metals such as lead or cadmium impaired the defence against infections in animals [7--9]. Also the functions of isolated phagocytes of rodents were mainly impaired due to exposure to lead and cadmium in vitro [10-14]. In human PMNL we have previously demonstrated a reduction of phagocytosis due to lead and cadmium [15]. The oxidative metabolism, however, was influenced in a different manner by cadmium; when induced by phagocytosis it was reduced and when induced by specific binding of aggregated IgG to Fc-receptors it was enhanced [16].

Mercury recently becomes toxicologically important for humans because of its wide industrial use. One effect on phagocytes is already reported: Mercuric chloride (HgC12) reduced in vitro the formation of toxic oxygen products in human PMNL and rat alveolar macrophages when the oxidative metabolism was induced by phagocytosis [13,17].

It is not yet clarified if this effect is due to an influence of HgC12 on the phagocytic capacity or on the oxidative metabolism itself. In the present study we therefore investigated in isolated human PMNL the effect of HgCle on phagocytic capacity, the oxidative metabolism when it was induced by phagocytosis of zymosan particles and when it was induced without phagocytosis by binding of heat-aggregated IgG to Fc-receptors or by unspecific binding of phorbol myris tate acetate to the cell membrane. Furthermore, we measured the release of lysosomal enzymes in presence of HgC12.

MATERIALS AND METHODS

HgC12, analytical grade, was obtained from Merck, Darmstadt, F.R.G. A stock solution (10 -2 M) in double distilled water was kept frozen and was freshly diluted for each experiment in Dulbecco's Minimum Essential Medium (DMEM, Boehringer Mannheim, F.R.G.).

Preparation of human PMNL Human PMNL were isolated from veneous heparinized blood of healthy

adults by separation on Ficoll-Paque (Pharmacia, Freiburg, F.R.G.) [18]. The PMNL were collected from the pellet. Remaining erythrocytes were lysed hypotonically with cold double distilled water for 20 s and the same volume of 1.8% NaCl was added subsequently. The PMNL were suspended in Dulbecco's Minimal Essential Medium with Hepes (Boehringer Mannheim, F.R.G.) and adjusted to the cell number needed for the respective experiments. Morphologically more than 95% of the cells were PMNL.

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Each of the following experiments was performed in triplicate, it was repeated at least 4 times on different days.

Cell viability Cell viability was tes ted using dye exclusion test with trypan-blue. One

hundred microliters cell suspension (1 × 107 cells/ml) in siliconized glass vials were mixed with 50/zl HgC12 (final conc. 10 -4-10 .7 M). Following 20 h of incubation at 37°C in a 5% CO 2, 950/0 air atmosphere aliquots of the incubated cells were added to an equal volume of 0.5% trypan-blue solution (Flow Laboratories, Meckenheim, F.R.G.) for 90 s, and the number of stained cells/200 PMNL was counted. The activity of lactate dehydrogenase (LDH) was determined photometrically using a commercial test-kit (LDH-Monotest- Opt, Boehringer Mannheim, F.R.G.). PMNL were incubated with HgC12 as described above. The tes t was performed with aliquots of 100 pl cell supernatant prepared by 5 min centrifugation of the cell suspension with 100 g.

Phagocytosis The phagocytic capacity was assessed by counting of ingested yeast cells

(Saecharomyces cerevisiae) according to the method of Schmid and Brune [19]. Yeast cells were opsonized with pooled human serum for 30 min at 37 °C and washed twice in DMEM. PMNL suspension (1.5 ml, 2.5 × 105 cells/ml) were placed into multiwells (diameter 2 cm). After 30 min of incubation for adherence at 37°C in a 5% CO 2, 95% air atmosphere the medium was replaced by fresh medium with 10-5-10 -7 M HgCI 2 and PMNL were incubated for further 30 or 60 min. Then the medium was replaced by medium containing yeast cells. The ratio yeast celI/PMNL was 10:1. After 30 min of incubation the number of ingested yeast cells/200 PMNL was counted.

Chemiluminescence Lucigenin, zymosan and phorbol myristate acetate (PMA) were purchased

from Sigma Chemical Co., St. Louis, U.S.A. Human standard immunoglobulin was obtained from Behringwerke, Marburg, F.R.G. Chemiluminescence was induced by 3 different stimuli:

(1) Zymosan particles, a preparation of yeast cell walls, in a concentration of 10 ~g/ml. A stock solution was kept frozen in 0.9% NaC1.

(2) Heat aggregated IgG (human Standard Immunoglobulin was shaken for 30 min in a waterbath at 63 °C [20]) in a concentration of 60 ~g/ml.

(3) Phorbol myris ta te acetate (PMA) in a concentration of 10 ng/ml (stock solution with 10 mg/ml in DMSO).

The substances were freshly diluted in DMEM for each experiment. In preliminary tests non-aggregated IgG did not stimulate the oxidative metabolism of PMNL up to a concentration of 1500 ~g/ml.

PMNL suspension (50 pl) were filled into minivials (1.5 ml, 2.5 × 105

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PMNL/vial) and 10 rain incubated for adherence at 37°C in a 5% CO 2, 95% air atmosphere. Jus t before chemiluminescence measurements HgC12 or control medium, the respective stimulus and lucigenin (0.1 mM) were added. The final volume was 200 ~l. Measurements were performed for 10 s in a Biolumat 9500 (Berthold, Bad Wildbad, F.R.G.) at 37 °C every 10 min over a period of 90 min.

Chemiluminescence curves in presence of HgC12 were only compared to the daily control curves. To assess the effect of HgC12 the counts of exposed cells were expressed in percent of the controls at the same time point (maximum of the control curve).

Measurement of lysomal enzymes The amount of lysozyme was estimated by lysis of Micrococcus luteus

suspended in phosphate buffer (0.05 M, pH 7.0). The activity of ~- glucuronidase was assessed using 4-nitrophenylglucuronide as substrate. Lysozyme (EC 3.2.1.17), Micrococcus luteus, /~-glucuronidase (EC 3.2.1.31) and 4-nitrophenylglucuronide were obtained from Boehringer Mannheim, F.R.G. The methods of measurements were modified according to a recommendation of Boehringer Mannheim, F.R.G.

Lysozyme: A suspension of Micrococcus luteus was dissolved in double distilled water to an optical density of 0.75 _+ 0.05 at 450 nm measured vs. air in a Lambda 1 spectrophotometer (Perkin-Elmer, Uberlingen, F.R.G.).

A 0.1-ml suspension of PMNL (5 x 106/ml) was incubated with 25 ~1 HgC12 (10 -5 M) or DMEM respectively in controls (final vol. 0.2 ml} for 30-120 rain. Following the incubation the PMNL were centrifugated with 700 g for 5 rain at 4°C. A 0.1-ml aliquot of supernatant was added to 0.5 ml suspension of Micrococcus luteus, the optical density at 450 nm was read over a period of 2 min was AOD/2 min was calculated.

fl-Glucuronidase: 4-nitrophenylglucuronide (0.1 M) was prepared in double distilled water. Supernatants of PMNL incubated with or without HgC12 were prepared as described above, aliquots of 0.1 ml were added to 0.5 ml acetate buffer (0.1 M, pH 4.5) with 0.01 ml 4-nitrophenylglucuronide solution. The mixture was incubated for 18 h at 37°C. Then the reaction was stopped by addition of ice-cold NaOH (0.5 M) and the optical density of the mixture was read vs. acetate buffer at 405 nm.

RESULTS

Firstly, we showed that the concentrations of HgC12 used in our assays were not cytotoxic for the PMNL. The highest concentration of HgC12 we used was 10 -5 M, and within 20 h the number of PMNL stained with trypan- blue was only slightly increased by 10 -5 M HgC12, the release of the cytoplasmatic enzyme LDH was slightly enhanced. Higher concentrations of HgC12 were rather cytotoxic (Table I).

In order to exclude incorrect results because of totally lysed PMNL the

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TABLE I

EFFECT OF MERCURIC CHLORIDE ON VIABILITY OF HUMAN PMNL"

No. of trypan-blue-stained cells/100 PMNL

LDH activity (U/l)

3 h exposure 20 h exposure 20 h exposure

Control 3.3 ± 1.0 3.2 ± 1.4 44.3 ± 5.3 HgCl~ 10 -T M 3.4 ± 0.9 2.2 ± 0.8 52.2 ± 7.3 HgC12 10 -6 M 3.4 ± 1.2 4.6 ± 2.0 53.6 ± 4.5* HgCl~ 10 -6 M 6.8 ± 2.8* 7.4,'± 3.7 55.3 ± 9.5 HgCl 2 5 x 10 -5 M 21.9 ± 9.8** n.d. n.d.

"The number of dead trypan-blue-stained cells was determined in 100 PMNL following 3 h and 20 h of preincubation with HgCI 2. The activity of LDH was measured following 20 h of exposure to HgC12 in the supernatants of cell suspensions with 106 PMNL. Values are means ± S.D., n = 5. Significant differences to the control values: *P < 0.05, **P < 0.01 (Students t-test), n.d., not done.

ce l ls w e r e c o u n t e d b e f o r e and a f t e r t h e e x p o s u r e to t h e m e t a l sa l t , and cel l

n u m b e r s w e r e n e a r l y iden t i ca l .

In c o n t r a s t to t h e e f f e c t on cel l v i ab i l i t y , HgC12 r e m a r k a b l y i n h i b i t e d t h e p h a g o c y t i c c a p a c i t y of h u m a n P M N L in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r .

A l r e a d y f o l l o w i n g 30 rain of e x p o s u r e t h e p h a g o c y t i c c a p a c i t y of P M N L w a s

t o t a l l y b locked by 10 -5 M H g C l 2 and s i g n i f i c a n t l y r e d u c e d by 10 -e M. E v e n 10 -7 M HgC12 r e d u c e d t h e p h a g o c y t i c c a p a c i t y r e m a r k a b l y fo l l owing 60 min

of e x p o s u r e (Table II).

T h e o x i d a t i v e m e t a b o l i s m of P M N L q u a n t i f i e d by m e a s u r i n g

c h e m i l u m i n e s c e n c e w a s a b o l i s h e d in p r e s e n c e of 10 -5 M HgC12 i n d e p e n d e n t l y

of t h e s t i m u l u s app l i ed . In p r e s e n c e of l o w e r c o n c e n t r a t i o n s

TABLE II

EFFECT OF MERCURIC CHLORIDE ON PHAGOCYTOSIS BY HUMAN PMNL"

No. of ingested yeasts/100 PMNL

30min exposure 60 min exposure

Control 143.3 ± 14.5 135.0 ± 17.1 HgC12 10 -~ M 135.8 ± 18.6 116.6 ± 18.2 HgCl~ 10 -e M 104.1 ± 25.1"* 89.6 ± 12.6"* HgCI~ 10 -5 M 8.0 ± 5.6*** n.d.

• Following 30 and 60 rain of exposure to HgCI 2 PMNL were incubated for further 30 min with opsonized yeast cells and then the number of ingested yeast cells/100 PMNL was counted. Values are means ± S . D . , n = 6 .

Significant differences to the control values: **P < 0.01, ***P < 0.001 (Students t-testL n.d., not done.

251

80.

• control • HgCI 2 10-S M • HgCI 2 I0-6 M

HgCl 2 I0-'/ M

70.

60.

50_

~0_

30_

20_

10_

O_

x103 w

o 110_

100_

o 90_

i !

0 15 310 /,5 60 7'5 9'0 mm

Fig. 1. Effect of HgCI 2 on zymosan-induced chemiluminescence of human PMNL. HgCl 2 in various concentrations and zymosan (10 /~g/ml) were added at time 0. Chemiluminescence was measured for 10 s at t ime points indicated.

,, %

100

i . i SO

0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . .

S 6 7 I S 6 7 S 6 7 14og IgG Zymosan PMA

Fig. 2. Effect of HgCl 2 on chemiluminescence of human PMNL stimulated by zymosan (10 ~g/ml), heat aggregated IgG (60 ~g/ml) or PMA (10 ng/ml). Chemiluminescence in presence of HgC1 z is expressed in percent of chemiluminescence of controls.

252

~ ~°° t ~ ~360J~2 3 2 0 1 -~

280_

2~0_

200_

160_

120_

80.

40.

O_

0 . 6 4 .

0 . 6 .

0 . 5 6 .

0 5 2 _

0 4 8 _

0 4 4

0 . 4

0 3 6

0 . 3 2 .

0 28_

0 24

0 2

0.16

0 . 1 2 .

0 0 8

0 0 4 0

[ysozyme H control : : HgC[ z 10 -s M

17-glucuromdase 0- . . . . - o control ~- . . . . -a HgC/2 10 -5 H

'0 ' 9'o ' 3 60 120 rain

Fig. 3. Effect of HgCl 2 on the release of lysosomal enzymes of resting human PMNL. HgCI 2 was added at time 0. The activity of p-glucuronidase and the amount of lysozyme were measured in cell supernatants at time points indicated.

chemiluminescence induced by aggregated IgG or PMA was reduced, too, but reduction was more impressive, when induced by phagocytosis of zymosan particles (Figs. 1 and 2).

Only the release of lysosomal enzymes was changed in an opposite manner. Unstimulated PMNL released a remarkable amount of lysozyme into the medium (Fig. 3). Following 2 h of incubation with 10 -5 M HgC12 we measured nearly all lysozyme of the PMNL in the supernatants (total lysozyme 0.587 ___ 0.110 ~g/5 × 105 PMNL, n = 6). In contrast the release of /3-glucuronidase was not influenced by 10 -5 M HgCl 2. In comparison PMA induced the release of the same amount of lysozyme within 30 min (data not shown).

DISCUSSION

Our results show that HgCI 2 may alter the microbicidal functions of human PMNL in different ways.

The phagocytic capacity -- the precondition for intracellular destruction of microbes -- was reduced in a concentration- and time-dependent manner. It is safe to assume that this reduction results in a marked impairment of the defence against microbes in the PMNL.

A further important microbicidal activity of PMNL is the generation of highly reactive toxic oxygen species during the non-mitochondrial oxidative metabolism [2,3]. This oxidative metabolism was decreased, too, by HgC12 probably leading to diminished intracellular microbicidal capacity.

253

Furthermore our results indicate that the depression of phagocytosis- induced oxidative metabolism was not only due to the reduction of phagocytosis. The reduction of oxidative metabolism was also shown when it was induced without phagocytosis by binding of aggregated IgG to Fc- receptors or by membrane stimulation with PMA. But there were different degrees of reduction of the oxidative metabolism when it was induced by the different stimuli. An explanation may be that the mechanisms to activate the oxidative metabolism are not the same for the different stimuli [21-23]. Although the mechanisms are not yet clear they may be differentiated by dependence on extracellular calcium ions [23,24] or by differently located or activated NADPH-oxidase [25,26].

The oxidative metabolism was quantified in our study by measuring chemiluminescence in presence of lucigenin indicating the generation of superoxide anion [27], the first metabolite of oxygen during the oxidative metabolism. The reduction of chemiluminescence in all cases of stimulation lead to the assumption that PMNL are inhibited to initiate the formation of active oxygen species in the presence of mercuric chloride.

There are at present no further experiments to explain this effect, but recently, it has been described that Hg 2÷ - besides other enzyme-blocking effects - may form inactive complexes with NADPH-oxidase the enzyme needed to generate superoxide anion [17]. This NADPH-oxidase is bound to the cell membrane, and following engulfment of particles the toxic oxygen species may be emitted into the phagocytic vacuoles or, when activation occurs without phagocytosis, into the surrounding area of the PMNL. In the latter case the toxic oxygen species can damage adjacent tissues leading to inflammatory reactions [28,29]. It is not possible to derive from our experiments if in vivo the HgCl2-induced reduction of phagocytosis leads to an increase of toxic oxygen species outside of the PMNL with subsequent inflammatory reactions, or if the inhibition of the oxidative metabolism is strong enough to avoid this possible promotion of inflammation.

The finding on phagocytosis-induced formation of active oxygen species is in accordance with other reports in which phagocytosis-induced chemiluminescence of rat alveolar macrophages or of human PMNL was found to be inhibited by in vitro exposure to Hg 2. [13,17].

Another important microbicidal function of PMNL is the release of lysosomal enzymes. During their maturation human PMNL form 2 kinds of granula - the specific and the azurophilic granula - containing particular lysosomal enzymes [30,31].

In our study we measured the release of lysozyme as example of lysosomal enzymes from the specific and /3-glucuronidase from the azurophilic granula. For technical reasons we could only measure the release into the surrounding medium, not the release into the phagocytic vacuoles. Mercuric chloride induced a remarkable release of lysozyme into the medium by unstimulated PMNL, whereas /~-glucuronidase was not found to be increased in the medium. In control experiments (data not shown) we did not find an inactivation of ~-glucuronidase by 10 -5 M HgCl 2. This result indicates

254

t h a t only the specif ic g r a n u l a r e l e a s e t h e i r e nz ym e s in the p r e s e n c e of HgCI 2, and th is mus t be an ac t ive deg ranu l a t i on , s ince f~-glucuronidase or LDH were not i nc reased in the medium. A s imi la r r e l e a s e of l y sozyme wi thou t r e l ea se of /3-g lucuronidase was found fol lowing in v i t ro s t imula t ion by P M A [32,33].

Bes ide the poss ib le t i s sue d a m a g e by the lysosomal enzymes the r e l e a s e f rom specif ic g r a n u l e s also p r o m i s e s fac tors which g e n e r a t e the chemotac t i c comp lemen t f r a g m e n t C5~ and by this way, th is r e l e a s e may induce f u r t h e r unspecif ic i n f l a m m a t o r y r eac t i ons [34].

The above d e s c r i b e d cell funct ions we re much more sens i t ive to the influence of m e r c u r y t han cell v iab i l i ty . W h e r e a s cell funct ions were s t r o n g l y a l t e r e d a l r e a d y wi th in t he f i r s t h of con tac t wi th mercu r i c chlor ide, cell v iab i l i ty was only s l igh t ly r educed even fol lowing 20 h of e x p o s u r e to the me ta l sal t .

In conclusion HgC12 may impa i r the microb ic ida l ac t iv i t i e s and thus the defence aga in s t i nvad ing mic robes due to a r educ t ion of phagocy tos i s and gene ra t i on of s u p e r o x i d e rad ica ls . F u r t h e r m o r e , HgC] 2 may p r o m o t e i n f l a m m a t o r y r eac t ions in s u r r o u n d i n g t i s sues due to enhanced r e l e a s e of lysosomal enzymes .

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