Int. J. Exp. Path. (I990) 71, 537-544

Measurement of reactive oxygen metabolites produced by human monocyte-derived macrophages exposed to mineral dusts Peter Nyberg and Matti Klockars Institute of Occupational Health, Helsinki, Finland

Received for publication 25 September I989 Accepted for publication I 2 March I990

Summary. The aim of the present work was to develop an in-vitro model for studying mineral dust-induced production of reactive oxygen metabolites by human macrophages. Monocytes isolated from human buffy coats were cultured in vitro for I-6 days. Quartz particles induced both luminol- and lucigenin-dependent chemiluminescence (CL) by the adherent cells. However, the luminol response decreased from day to day, obviously due to a decrease in the myeloperoxidase (MPO) activity of the cells, whereas the lucigenin response showed no such MPO dependence. The luminol response was inhibited by superoxide dismutase (SOD), catalase, and the MPO-inhibitor azide, while the lucigenin response was inhibited by SOD and catalase but stimulated by azide. There was a positive correlation between the lucigenin responses and the results obtained with the established cytochrome c assay for superoxide, when opsonized zymosan was used as a stimulant. The effects of quartz, titanium dioxide, chrysotile asbestos, and wollastonite particles were investigated with the lucigenin assay. Quartz and chrysotile caused prominent light emission by 6-day-old macrophages, whereas titanium dioxide and wollastonite caused weak responses. We conclude that mineral dusts induce production of reactive oxygen metabolites by human monocyte-derived macrophages, and that the quantitative responses depend on both physical and physicochemical dust properties, the nature of which are still to be defined.

Keywords: oxygen radicals, lucigenin, luminol, chemiluminescence, monocyte-derived macrophages, mineral dusts Both polymorphonuclear and mononuclear human phagocytes contain the membranebound enzyme NAPDH oxidase which, following activation by a variety of stimuli, catalyses the generation of superoxide radicals (G 2) (Rossi et al. I986). Hydrogen peroxide (H202) and hydroxyl radicals (OH*) may be formed from superoxide (Babior

1978). These highly reactive oxygen metabolites are used in the defence against microorganisms (Babior I978) and malignant cells (Weiss & LoBuglioi982), but a growing body of data indicates that they may also have a pathogenic role in a number of inflammatory diseases (Halliwell & Grootveld I987).

Correspondence: Matti Klockars, Institute of Occupational Health, Topeliuksenkatu 4 I a A, SF-oo2 50 Helsinki, Finland. 537

P. Nyberg & M. Klockars All macrophages develop from blood Vacc, Sweden; pooled, sterilized human monocytes (Van Furth & Cohn I968). Under AB + serum from the Finnish Red Cross appropriate in-vitro conditions monocytes Blood Transfusion Service; superoxide diswill differentiate to macrophages, and such mutase (5000 U/mg; from bovine erythrocells have been shown to produce reactive cytes) and cytochrome c (from horse heart) oxygen metabolites when stimulated with from Boehringer Mannheim, FRG; catalase standard NADPH oxidase stimulants (Naka- (2800 U/mg; from bovine liver), HEPES gawara et al. I98I; Seim I983; D'Onofrio & buffer, and zymosan yeast from Sigma, USA; Lohmann-Matthes I984; Jungi & Peterhans sodium azide from Merck, FRG; and DAPI (W', I988). The generation of these metabolites 6-diamidino-2-phenylindol) from Serva can be measured for instance by using the Feinbiochemica, FRG. chemoluminogenic (light-producing) probes, Purified human myeloperoxidase was a luminol and lucigenin (Van Dyke I985). generous gift from J6rgen Malmqvist, Malmo Luminol (5-amino-2, 3-dihydro-I,4-phthal- General Hospital, Sweden. The following azindione) is thought to react with hypochl- mineral dusts were used: quartz (Fyleverken orous acid, which is formed in the myeloper- Ltd, Sweden), titanium dioxide (Baker Comoxidase (MPO) catalysed reaction of H202 pany, Netherlands), chrysotile A asbestos with halide (Seim I983; DeChatelet et al. (IUAC, International Union Against Cancer, i982), whereas lucigenin (io, io'-dimethyl- standard sample), and wollastonite FW 325 9,9'-biacridinium dinitrate) is thought to (Partek Co, Finland). react directly with 0 2 (Allen I 98 I; Aasen et al. I 987; Corbisier et al. I 987; Gyllenham- Monocyte isolation mar i987). Mineral dusts have earlier been shown to Buffy coats from healthy blood donors were cause luminol-dependent CL from human obtained from the Finnish Red Cross Blood PMNL (Hedenborg & Klockars i987, i989) Transfusion Service. The mononuclear cells and they induce 0 2 production from PMNL were isolated by Ficoll density centrifugation as measured by cytochrome c reduction according to B0yum (i968). The cells were (Hedenborg & Klockars i989). It has been washed twice in a culture medium consisting suggested that generation ofoxygen metabo- of RPMI-i64O (the standard medium, not lites by phagocytes following contact with containing a-tocopherol, ascorbate or ironmineral dusts might have a role in the cysteine), I0 mIl/l 2-9% L-glutamine, IO% pathogenesis of pneumoconioses (Doll et al. AB+ serum, I00 IU/ml penicillin and I00 i983; Vallyathan et al. i988). The purpose Mg/ml streptomycin. The washed cells were of this work was to develop an in-vitro model suspended in 24 ml culture medium, and the for studying the effect of mineral dusts on the cell suspension was divided onto three sterile production of reactive oxygen metabolites by Petri dishes of diameter go mm (Primex human macrophages. Plast, Denmark). After I h of incubation at 3 70C the dishes were washed three times with PBS and the remaining adherent cells Materials and methods were gently removed with a Teflon policeman. The cells were counted in a Coulter Reagents Counter, and the cell density was adjusted to The lucigenin and luminol were purchased 0.5 X IO6 cells/ml. The monocyte purity at from LKB-Wallac, Finland; phosphate-buf- this stage was 83.3 ±I.8% (mean fered saline (PBS) Dulbecco, RPMI-I64o, value±s.d.) as measured from alpha naphand L-glutamine from Orion Diagnostica, thyl acetate esterase (ANAE) stained culFinland; Ficoll-Paque from Pharmacia, Swe- tures. The ANAE staining was performed as den; penicillin and streptomycin from Nord- described by Mueller et a]. (I975). 538

Dust-induced production of oxygen radicals Cell cultures Round cover glasses, diameter of 9 mm (Menzel, FRG), were kept overnight in 50% ethanol and 50% ether, then washed in sterile water, and finally sterilized in an autoclave. The glasses were transferred into 48-well tissue culture clusters (well bottom areas i cm2; Costar, USA). One ml of the monocyte suspension was added to each well. The cells were incubated at 3 7TC, in 5% C02, and I00% humidity for i-6 days. The culture medium was not changed during culture. Cell quantification Preparation of standard curve. Mononuclear cells were isolated by density centrifugation from I0 ml venous blood samples from healthy volunteers. The cells were washed once in PBS and counted three times in the Coulter Counter. Using the mean value from these countings a suspension of 2 X 106 cells/ml was prepared. 50, I00, I5o, and 200 p1 of the suspension were diluted in distilled water to a final volume of i ml. The

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Fig. i. Standard curve for adherent cell counting with the DNA staining technique. 0.1-0.4 x 10' cells stained with 0.2 pg/ml DAPI in 9 mM HEPES buffer. Light emission recorded at 454 nm with the light source on 3 72 nm. Mean values ± s.e.m. from three experiments performed in triplicate.

539 cell DNA was allowed to react with i ml of a solution containing 0.2 pg/ml DAPI and 9 mM HEPES buffer in distilled water. The cells were disrupted by sonication for 3 x Is. Inmmediately after sonication fluorescence emission at 454 nm was recorded with a Perkin-Elmer 512 double-beam fluorescence spectrophotometer with the light source on 372 nm. The experiment was performed in triplicate and repeated three times with cells from different batches. The standard curve used in this work is shown in Fig. i.

Quantification of adherent cells. The cell plates were washed twice in PBS and transferred into cuvettes containing i ml of distilled water. The DNA staining was performed as described above. Fluorescence was registered from three parallel samples, and the cell number was read from the standard curve. Lysozyme assay The lysozyme content of the culture medium was measured according to Osserman and Lawlor (i9666) on days I, 3, and 6 using human lysozyme as standard. Culture medium of the same age from cell-free wells was used as blank controls.

Chemiluminescence assay The cell plates were washed twice in PBS and transferred into the luminometer cuvettes (Clinicon; LKB-Wallac, Sweden) containing O.I mM lucigenin or luminol and in some experiments 25 Mg/ml SOD, I00 pg/ml catalase, or 0.2 mM azide (final concentrations). The reactions were started by adding mineral dusts or opsonized zymosan to the solutions. All reactions were performed at 3 70C in PBS (pH 7.4), and the samples were prewarmed to 3 70C for 20 min before the assay. The final volume of all samples was i mil. Light emission was detected at 2-min intervals with an LKB-Wallac Luminometer 1251 operated by the LKB-Wallac Phagocytosis Program I251-124 which was run in an Apple IIe computer.

P. Nyberg & 540 The results are expressed as areas under the CL emission curves (calculated by the Phagocytosis Program according to Simpson's rule (Van Dyke & Van Dyke I985)), or as representative light emission curves. In the area calculations the corresponding areas of negative controls containing cells alone in lucigenin or luminol were subtracted.

Superoxide assay The cell plates were washed twice in PBS and transferred into cuvettes containing 8o gM cytochrome c and 25 ug/ml catalase (final concentrations) in PBS. The catalase was supplemented to prevent reoxidation of reduced cytochrome c. After 6o min of incubation with opsonized zymosan at 3 70C the samples were cleared by centrifugation and the absorbance at 5 50 nm was read with a Shimadzu UV-3000 double-beam spectrophotometer. From these values the absorbance of 80o gM cytochrome c +2s5 jg/ml catalase in PBS without cells was subtracted, and the production of 0° was calculated using a millimolar extinction coefficient of 21 MMI xcm-1 (Johnston et al. 1978).

M. Klockars Table i. Characteristics of mononuclear phagocytes differentiating in vitro. Mean values ± s.e.m. from three experiments performed in triplicate

Number of cells (x io5)

Day of culture

Lysozyme in the culture ANAE-positive medium cells (%) (mg/ml)

I

i.80±0.I5

87.7±4.6

3

i.87±0.I9 2.20±0.20

97.0±o.6 100±0.0

6

0.50±0.0I 0.96±0.i9

2.67±o.65

lucigenin as probes (Fig. 2). On day i the light output from the luminol-dependent system was about sixfold that of the lucigenin-dependent system, but on day 3 the luminol responses had already decreased to the lucigenin level. On day 6 the lucigenin responses were stronger than the luminol responses. The cell number-corrected luci-

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Results Basal characteristics of the cell cultures are shown in Table i. The gain of adherent cells increased slightly with increased age of the cultures. Already on day 3 of culture nearly all cells were ANAE-positive macrophages, and on day 6 no ANAE-negative cells could be seen. The lysozyme content of the culture medium increased steadily from day i to 6. Quartz particles caused light emission from the cell cultures both with luminol and

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Fig. 2. *, Luminol- and 0, lucigenin-dependent chemiluminescence from cell cultures exposed to Ioo jg/ml quartz particles on days I, 3, and 6 of culture. Mean values±s.e.m. from three experiments performed in triplicate.

Dust-induced production of oxygen radicals

54I Table 2. Effect of 25 ug/ml SOD, IOO ug/ml catalase, and 0o2 mM azide on lucigenin- and luminoldependent chemiluminescence (CL) by cells exposed to IOO ig/ml quartz dust (final concentrations) on day i of culture. CL expressed as percentage of areas under curves for uninhibited cells. Mean values ± s.d. from one experiment performed in triplicate

Lucigenin-dependent CL Luminol-dependent CL

SOD

Catalase

Azide

6.4 ±0.2 26.5 ± 1.7

I 7.8 ± 2.7 2I.7+0.7

23I.I ± 76.o 15.1 ±4.3

genin responses increased with increasing age of the cultures. The effects of the 012 scavenger SOD, the H202 scavenger catalase, and the MPO inhibitor azide are shown in Table 2. SOD and catalase had an inhibitory effect in both CL assays, whereas azide had a stimulatory effect on lucigenin-dependent CL and an inhibitory effect on luminol-dependent CL.

When I10 Mm H202 (final concentration) was added to a solution containing I0og/ml MPO and o. i mM luminol in PBS, light emission followed. This response could be completely inhibited with ioo pg/ml catalase or 0.2 mM azide (curves not shown). Attempts to correlate the quartz-induced lucigenin-dependent CL responses with superoxide production as measured by cytochrome c reduction were not successful. However, when the strong NADPH oxidase

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Fig. 3. Correlation between lucigenin-dependent chemiluminescence and 0 2 production measured with the cytochrome c reduction assay. Cells stimulated with opsonized zymosan in the concentrations (pg/ml) mentioned in the figure on day 6 of culture. Mean values ± s.d. from one experiment performed in triplicate.

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Representative lucigenin light emission from cell cultures exposed to IOO pg/ml Fyle quartz (FQ), titanium dioxide (TiO2), chrysotile A asbestos (CA), and Finnish wollastonite (FW) on day 6 of culture. Shaded area, background chemiluminescence from unstimulated cells. 4.

curves

P. Nyberg & 542 stimulant opsonized zymosan was used to induce superoxide production by the cell cultures, a close correlation between the results obtained with the two assays was observed (Fig. 3). Fig. 4 shows prominent lucigenin-dependent light emission from 6-days-old cell cultures exposed to quartz particles and chrysotile asbestos fibres. Light emission induced by the inert control mineral dusts (titanium dioxide and wollastonite particles) was significantly weaker. Discussion Using the cell isolation and culture techniques described in Methods we obtained a strongly adherent, ANAE-positive, and continuously lysozyme-secreting macrophage population. Quartz stimulation of the cells caused both luminol- and lucigenin-dependent CL. The continuous decrease in the luminol response was obviously due to the loss in MPO activity described for maturing mononuclear phagocytes (Seim I983), since the lucigenin response showed no such decrease. This MPO independence of lucigenin CL is consistent with earlier reports (Seim I983; Jungi & Peterhans I988; Williams & Cole I98I). Lucigenin-dependent CL has been thought to be 0 1-specific. However, we have earlier shown that in the xanthine-xanthine oxidase system it is most likely dependent on H202, too (unpublished results). This seems to be the case also in this cellular system, since the quartz-induced lucigenin-dependent CL was strongly inhibited not only by SOD but also by catalase, and stimulated by the MPO inhibitor azide that inhibits the halide-linked metabolism of H202. The strong inhibitory effect of catalase in the present work has not been reported earlier, but several groups have observed the stimulatory effect of azide (Jungi & Peterhans I988; Gyllenhammar I987; Stevens & Hong i984). The inhibition of luminol-dependent CL by catalase and azide is in accordance with the

M. Klockars hypothesis that luminol reacts with hypochlorous acid (DeChatelet et al. I982; Dahlgren & Stendahl I 983). The inhibitory effect of SOD on luminol-dependent CL is somewhat confusing, but Hodgson and Fridovich (I973) have suggested that 0° is needed in one of the intermediate steps in the luminol reaction, and that this 01 might be produced within the reaction. This hypothesis could explain the inhibitory effect of SOD on luminol-dependent CL. The CL from the H202MPO system confirms that luminol reacts with light emission in an environment where hypochlorous acid is likely to be produced. The correlation between lucigenin-dependent CL and cytochrome c reduction is consistent with observations from cell-free xanthine-xanthine oxidase systems (Aasen et al. I987). It seems possible to use lucigenin-dependent CL as a quantitative assay for superoxide, although cautiously, since the role of hydrogen peroxide in the lucigenin assay still remains unclear. The prominent CL responses caused by quartz and chrysotile asbestos dust compared to those by titanium dioxide and wollastonite have been shown in our earlier work with human PMNL using luminoldependent CL (Hedenborg & Klockars I987; I989). These observations are clinically interesting since both quartz and chrysotile asbestos are well known pathogenic dusts whereas titanium dioxide and wollastonite asbestos are thought to be inert dusts. Thus it is possible that the pathogenicity of certain mineral dusts is partially linked to their ability to induce production of reactive oxygen metabolites by both PMNL and mononuclear phagocytes. In summary, this work shows that human peripheral blood monocytes cultured to macrophages are metabolically active and generate reactive oxygen metabolites when stimulated with NADPH oxidase stimulants. Of the CL assays for oxygen metabolites the lucigenin-dependent CL seems to be more convenient for macrophage cultures since it is not dependent on the MPO activity of the cells. The method may be used for testing

Dust-induced production of oxygen radicals oxidant production from mononuclear phagocytes in general, and it seems suited for analysing the generation of reactive oxygen metabolites from macrophages exposed to mineral dusts. Acknowledgements This study was supported by grants from Finska Uikaresillskapet. We thank the Finnish Red Cross Blood Transfusion Service for the leucocyte buffy coats, Dr D. Godelaine, ICP, Brussels for useful advice, and Ms Leena Juusela for expert technical assistance. References AASEN T.B., BOLANN B., GLErrE J., ULVIK R.J. & SCHREINER A. (I 9 8 7) Lucigenin-dependent chemiluminescence in mononuclear phagocytes.

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