Effect of sulphite on the oxidative metabolism of human neutrophils: Studies with lucigenin- and luminol-dependent chemiluminescence

J BlOLUMlN CHEMILUMIN 1995; 10: 9-19

Effect of Sulphite on the Oxidative Metabolism of Human Neutrophils: Studies with Lucigenin- and Luminol-Dependent Chemiluminescence

Anil Mishra," Niru Dayal and Ingrid Beck-Speier GSF-Forschungszentrum fur Urnwelt und Gesundheit, Projekt Inhalation, Neuherberg, D-85758 Oberschleissheirn, Germany

To assess the effect of sulphite on the oxidative metabolism of human neutrophils, chemiluminescence (CL) measurements were performed using lucigenin and luminol as chemiluminigenic probes. Lucigenin-dependent CL was used for measuring superoxide anion (0;) production, and luminol-dependent CL was used for determination of myeloperoxidase (MP0)-connected processes. With sulphite concentrations of 0.01 to 1 mmol/L, resting neutrophils showed an up t o sixfold increase of lucigenin-dependent CL, but only a 1.9-fold increase of luminol-dependent CL. Subsequent stimulation of sulphite-treated neutrophils wi th phorbol myristate acetate (PMA) (soluble stimulant) or zymosan (particulate stimulant) resulted in an additional significant increase of lucigenin-dependent CL compared to stimulated control cells, whereas luminol-dependent CL increased slightly by 0.01 mmol/L sulphite and decreased then continuously. Sulphite concentrations above 1 mmol/L decreased both lucigenin- and luminol-dependent CL of resting and PMA- or zymosan- stimulated neutrophils. Lucigenin-dependent CL of sulphite-treated and subsequently stimulated neutrophils was strongly inhibited by extracellularly added superoxide dismutase, whereas luminol-dependent CL was markedly reduced by the M PO inhibitor azide. The intracellular activity of MPO in neutrophils stimulated with PMA in the presence of sulphite (Smmol/L) was reduced by 55%. Sulphite (0.1 mmol/L) also inhibited strongly the activity of MPO in a cell-free system. These results indicate that micromolar concentrations of sulphite exert a stimulating effect on the 0; production of neutrophils extracellularly, but have an inhibitory effect on MPO-catalysed reactions intracellularly.

Keywords: chemiluminescence; lucigenin; luminol; sulphite; human neutrophils

I NTR 0 D U CTI 0 N

* Present address: Northern Ohio Universities, College of Medicine, Department of Physiology, Rootstown, OH, USA. Abbreviations: CL, chemiluminescence; PBS, phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate; PMN, polymorphonuclear neutrophils; TMB, 3,3',5,5'-tetramethylbenzidine; MPO, myeloperoxidase; SOD, superoxide dismutase; XOD, xanthine oxidase.

Sulphur dioxide (SOz) and sulphite are well-known air pollutants, and sulphite is often used as a food and drug preservative (1). There is evidence that some lung diseases, including asthma and chronic bronchitis, are aggravated by exposure to SO2 (2-7). Several animal and human studies show

CCC 0884-3996/95/010009-11 0 1995 by John Wiley & Sons, Ltd.

Received 26 May 1993 Revised 15 December 1993

10 A. MISHRA, N. DAYAL AND I. BECK-SPEIER

that short-term exposure to SO2 as well as long- term exposure to high (200ppm) or low (0.22 ppm) concentrations of SO2 induce inflammatory reactions in the lungs including an influx of neutrophils (PMN) (8- 10). Although exposure to SO2 causes an infiltration of PMN into the lungs, little is known about direct effects of SO2/ sulphite on PMN functions. Our in vitro studies showed that sulphite can adversely affect energy metabolism and that target organ sensitivity is related to the activity of sulphite oxidase which detoxifies sulphite by oxidation to sulphate (1 1). PMN, together with other phagocytic cells, are endowed with very low activity of sulphite oxidase, and therefore have a relatively low protective capacity against sulphite (1 1). Recently we have shown that PMN are stimulated by sulphite to produce superoxide anions (07) due to an activation of NADPH oxidase (12).

A major function of PMN is to kill pathogenic microorganisms by production of reactive oxygen species and release of granular enzymes, both extracellularly and, perhaps more importantly, intracellularly into the phagolysosome (1 3). During stimulation of PMN by various stimuli, the primary oxygen metabolite generated is OF which is converted to hydrogen peroxide (H202) by catalytic or non-catalytic dismutation. H 2 0 2 is used in the intracellular bactericidal system of PMN consisting of myeloperoxidase (MPO), H 2 0 2 and chloride to form the highly reactive oxidants hypochlorous acid (HOC1) and singlet oxygen (14-16). MPO is contained within primary granules and released into the phagolysosome or extracellular space upon stimulation of the cells. The HOCl generated reacts with endo- genous amines to yield N-chloramines. The MPO-generated oxidants HOCl and N - chloramines are considered as the active agents in bactericidal killing and cytotoxicity mediated by PMN (17).

The generation of oxygen metabolites by activated PMN is accompanied by light emission which is an energy product of phagocyte oxygenation activity (18,19). This native chemiluminescence (CL) of phagocytes has a low quantum yield but can be intensified by the use of chemiluminigenic probes such as luminol and lucigenin which are susceptible to oxygenation reactions producing products with high quantum yield (20-23). Luminol- and lucigenin-dependent CL are generated via different oxidative pathways and are use- ful tools to differentiate the redox activity of

phagocytic cells (22,24). Luminol-dependent CL is generated by dioxygenation of luminol and is considered as a measurement of intracellularly generated oxidants and MPO activity (20,22-28). Lucigenin-dependent CL is generated by reductive dioxygenation of lucigenin by 0; under physiolo- gical conditions and determines 0; production (21,22,24,29).

In this study, we investigated the effect of sulphite on the oxidative metabolism in populations of resting, phorbol myristate acetate (PMA)- stimulated or phagocytosis-associated human PMN by using CL measurements with luminol and lucigenin as chemiluminigenic probes to determine different oxygenation activities of the cells. Particularly, the effect of sulphite on the intracellular activity of MPO was determined by luminol-dependent CL and oxidation of tetramethylbenzidine (TMB) as enzymatic assay system, and compared with the effect of sulphite on the extracellular release of 0; determined by lucigenin-dependent CL.

MATERIALS AND METHODS

Materials

Lucigenin (1 0,l O’-dimethyl-9,9’-biacridinium dini- trate), luminol (5-amino-2,3-dihydro-1,4- phthalazinedione), phorbol 12-myristate 13-acetate (PMA), zymosan, and 3,3’-5,5’-tetramethylbenzidine (TMB) were purchased from Sigma (Deisenhofen, Germany), phosphate buffered saline (PBS-buffer) from Biochrome (Berlin. Germany), human MPO from Calbiochem (Frankfurt, Germany), catalase, superoxide dismutase (SOD) and xanthine oxidase (XOD) from Boehringer (Mannheim, Germany), and Poly- morphprep from Nycomed (Oslo, Norway). All other chemicals (analytical grade) were from Merck (Darmstadt, Germany).

Isolation of P M N

Human PMN were isolated from citrate- anticoagulated freshly drawn venous blood with Polymorphprep (30). The purified cells were resuspended in PBS buffer (without Ca2+ and Mg2+) containing 0.1 YO glucose, and viability was always over 95% by trypan blue exclusion.

SULPHITE AND OXIDATIVE METABOLISM 11

Preparation of sulphite solutions

Sodium sulphite was dissolved in PBS buffer, pH 7, containing 0.1% glucose, and the pH value was readjusted to pH 7 with HCl. Sulphite solutions were always freshly prepared.

Lucigenin- and luminol-dependent CL measurements of PMN

Lucigenin-dependent CL was determined according to Gyllenhammar (29), and luminol-dependent CL was measured according to Dahlgren (26) using a six-channel Biolumat LB 9505 (Berthold, Wildbad, Germany). For lucigenin-dependent CL measurements, PMN (3 x lo4 cells) were pre- incubated in 0.5 mL PBS buffer, pH 7, containing 0.1 YO glucose and 0.8 mmol/L lucigenin for 10min at 37°C in the Biolumat. CL measurements were started by adding sulphite in various concentrations to the cells, and registered for 20min at 37°C. CL was measured as CL-counts integrated over 20min for 3 x lo4 cells. These sulphite- treated cells were subsequently activated by PMA (l00ng) or zymosan (500pg), and CL was measured again for 20min at 37°C. For luminol- dependent CL measurements the same protocol was used, in which lucigenin was replaced by 0.02 mmol/L luminol when PMN were stimulated with PMA (26) or by 0.2mmol/L luminol when PMN were stimulated with zymosan (23). Stock solutions of lucigenin and luminol were prepared according to Allen (23), and then diluted with PBS buffer, pH 7. When used, the inhibitors for oxygen radical production SOD (1OOpg for lucigenin-dependent CL (29), 20 pg for luminol- dependent CL (26)), catalase (100 pg for lucigenin-dependent CL according to Gyllen- hammar (29), 10 pg for luminol-dependent CL according to Dahlgren (26)), or azide (0.1 mmol/L (28,31)) were added before the addition of sulphite and PMA.

XOD (1Opg) was added to 0.5mL oxygen- saturated 0.1 mmol/L potassium phosphate buffer, pH 7.6, containing 0.1 mmol/L xanthine, 0.8mmol/L lucigenin and 0.1, 1 or 10mmol/L sulphite. The reaction was started by the addition of XOD, and lucigenin-dependent CL was measured at 25°C as CL-counts of a 10-s integral after lmin reaction in a one-channel Biolumat (Berthold, Wildbad, Germany). Lucigenin- dependent CL of the XOD/xanthine system corresponded to 6.1 x lo5 cpm.

Luminol-dependent CL generated by H202 was determined in PBS buffer, pH 7, in the absence and presence of sulphite. H202 (0.25 mmol/L) was added to 0.5 mL PBS buffer, pH 7, containing 0.02mmol/L luminol and 0.1, 1 or lOmmol/L sulphite. Luminol-dependent CL was measured at 37°C as CL-counts integrated over 1Omin in a six-channel Biolumat (Berthold, Wildbad, Germany). The luminol-dependent CL of the H202 system corresponded to 1.5 x lo4 cpm.

Determination of intracellular MPO activity in sulphite-treated PMN

PMN (1 x lo6 cells/mL) were incubated in PBS buffer, pH 7, containing 0.1% glucose for 30min at 37°C in the presence of 0.5, 1 or 2mmol/L sulphite, respectively, and additionally stimulated with PMA. Stimulated control cells were incubated simultaneously. After incubation, cells were centrifuged at 400 g for 5 min at room tempera- ture. To remove sulphite, the cell pellet was washed once with PBS buffer, pH 7. The cell pellet was homogenized at 0°C by sonification (Labsonic, Braun-Melsungen, Melsungen, Germany) three times for 15s at 1OOW. After centrifugation of the homogenate at 10,OOOg for 1Omin at 4"C, activity of MPO was determined in the super- natant by the spectrophotometric method with TMB according to Suzuki et al. (32) in a con- tinuous assay at 30°C. Protein content was determined according to Lowry et al. (33).

Lucigenin- and luminol-dependent CL measurements of 0, and H202 Luminol-dependent CL measurements of

the system MPO-H202-chloride Lucigenin-dependent CL was generated by 0; produced by the XOD-xanthine system (29). The influ- The activity of the system MPO-H202-chloride ence of sulphite on lucigenin-dependent CL was measured by luminol-dependent CL (26,29) produced by the XOD/xanthine system was deter- in the absence and presence of sulphite. MPO mined by adding various sulphite concentrations. (42ng) was incubated in 0.5mL PBS buffer, pH

12 A. MISHRA, N. DAYAL AND I . BECK-SPEIER

7, containing 0.2 mmol/L luminol with various sulphite concentrations (0.005, 0.01, 0.05, 0.1, 0.5, 1, and 2mmol/L) at 30°C in the Biolumat. The reaction was started by addition of H202 (0.2 mmol/L), and luminol-dependent CL was measured at 30°C for 15 min.

RESULTS

Effect of sulphite on lucigenin- and luminol-dependent CL of human PMN

Human PMN were incubated with various sulphite concentrations at 37°C for 20min, either in the resting state or subsequently stimulated by PMA as soluble stimulant, or by zymosan as particulate stimulant. Control cells were treated simultaneously.

As seen in Fig. 1, resting PMN showed an increase up to sixfold in lucigenin-dependent CL by sulphite concentrations of 0.01 to 1 mmol/L. Subsequent stimulation of sulphite-treated PMN by either PMA or zymosan resulted in an additional increase of lucigenin-dependent CL with a similar concentration dependence as for resting cells. PMN, incubated with 1 mmol/L sulphite and subsequently stimulated by PMA, exhibited a

2.8-fold increase in lucigenin-dependent CL as compared to PMA-stimulated control cells. PMN, stimulated by zymosan in the presence of 0.1, 0.5 or 1 mmol/L sulphite, showed an about 1.7-fold increase in lucigenin-dependent CL compared to zymosan-stimulated control cells. Higher sulphite concentrations (2 to 10 mmol/L) diminished lucigenin-dependent CL of resting and PMA- or zymosan-stimulated PMN. When opsonized zymosan was used as stimulant for immune activation of PMN, similar results were obtained as those shown for zymosan (data not shown).

Fig. 2 shows luminol-dependent CL of resting and subsequently stimulated PMN in the presence of various sulphite concentrations. In contrast to lucigenin-dependent CL, luminol-dependent CL of resting PMN was increased only slightly (up to 1.9-fold) by sulphite concentrations of 0.01 to 1 mmol/L and decreased by higher sulphite concentrations (2 to 10 mmol/L). Subsequent stimulation of PMN by PMA or zymosan resulted in a small increase of luminol-dependent CL at 0.0 1 mmol/L sulphite (1.4-fold increase for PMA-stimulated PMN, 1.3-fold increase for zymosan-stimulated PMN). A decrease of luminol-dependent CL of PMA- or zymosan-stimulated PMN was readily apparent at sulphite concentrations of 0.5 mmol/ L and higher. Similar results were obtained for

26

24 - 22 - EB Zymoson-

m resting PMN BH781 PMA-stimuloted PMN - A Lo

h - v Y

stimuloted PMN T T 7

0 0.01 0.05 0.1 0.5 1 2 5

S u I p h it e Con cent ra t io n [m rno I / L]

Figure 1. Effect of sulphite on lucigenin-dependent CL of resting and PMA- or zymosan-stimulated PMN. Resting PMN (3 x lo4) were incubated with various sulphite concentrations for 20min at 37"C, and subsequently stimulated by PMA or zymosan. Lucigenin-dependent CL was determined as described in the section, 'Materials and methods'. Values are given as mean i SEM of absolute CL-counts integrated over 20 min for 3 x 1 O4 cells, and numbers in parentheses represent number of experiments


90

80

70

60

50

40

30

20

10

0 0 0.01 0,05 0.1 0.5 1 2 5 10

Sulphite Concentration [mmOl/L]

Figure 2. Effect of sulphite on luminol-dependent CL of resting and PMA- or zymosan-stimulated PMN. PMN (3 x 1 04) were incubated with various sulphite concentrationsfor 20 min at 37°C. and subsequently stimulated by PMA or zymosan. Luminol- dependent CL was determined as described in the section, Materials and methods'. Values are given as mean SEM of absolute CL-counts integrated over 20min for 3 x 1 O4 cells, and numbers in parentheses represent number of experiments

sulphite-treated and PMA-stimulated PMN when luminol-dependent CL was measured with 0.2 mmol/L luminol, which is the same luminol concentration as used for zymosan-stimulated PMN (data not shown). In contrast to lucigenin- dependent CL, luminol-dependent CL of stimulated PMN was much more sensitive to the effect of sulphite, with maximal stimulation shifted to 0.01 and 0.05 mmol/L sulphite and inhibition apparent at 0.5 mmol/L sulphite. Using opsonized zymosan as stimulant for immune activation of PMN, similar results were found as those obtained for zymosan (data not shown).

Effect of inhibitors on lucigenin- and luminol-dependent CL of sulphite-treated PMN

To differentiate between the extracellular and intracellular origin of the lucigenin- and luminol- dependent CL of PMN stimulated by PMA in the presence of sulphite, several inhibitors were used, such as SOD, catalase, and azide. SOD and catalase as high-molecular-weight inhibitors are expected to reduce only extracellularly produced oxygen radicals, whereas azide gains access to intracellular sites and inhibits MPO intracellularly (26,34,35). For these experiments a sulphite concentration of 0.5mmol/L was chosen which

enhanced lucigenin-dependent CL markedly and reduced luminol-dependent CL slightly. Table 1 shows that lucigenin-dependent CL of sulphite- treated and PMA-stimulated PMN was inhibited by 94% by SOD, but not by catalase or azide, whereas luminol-dependent CL was inhibited by nearly 70% by azide, but not by catalase, and only partially by SOD. In addition to these data, similar results were found for the inhibition of lucigenin- and luminol-dependent CL, when PMN were treated with 0.5 mmol/L sulphite and subsequently stimulated by zymosan. Lucigenin- dependent CL was inhibited 94% by SOD, and luminol-dependent CL was reduced 70% by azide. Furthermore, SOD also inhibited the lucigenin-dependent CL of PMN treated with 0.5 mmol/L sulphite by 90%. These findings indicate that lucigenin-dependent CL of sulphite- treated and subsequently stimulated PMN has an extracellular origin and reflects release of O;, whereas luminol-dependent CL has an intracellular origin and is mainly due to MPO activity.

Influence of sulphite on CL of lucigenin and luminol: control studies with cell-free systems

In cell-free systems, control experiments were performed to study the influence of sulphite on the


Table 1. Effect of inhibitors on lucigenin- and luminol-dependent CL o f sulphite-treated and subsequently stimulated PMN. PMN were incubated with 0.5mmol/L sulphite and subsequently stimulated with PMA in absence and presence o f SOD, catalase and azide. Control cells were treated simultaneously. Lucigenin- and luminol-dependent CL were determined as described in the section, 'Materials and methods'. Lucigenin-dependent CL o f PMA-stimulated control PMN corresponded t o 598 f 17 x lo4 CL-counts integrated over 20min for 3 x lo4 cells (n = 10) and was set t o 100%; luminol-dependent CL of PMA-stimulated control PMN corresponded t o 2475 5 210 x lo4 CL-counts integrated over 20min for 3x104 cells (n=10) and was also set t o 100%. The other values are given as percentage o f lucigenin- or luminol-dependent CL o f PMA-stimulated control PMN and are expressed as mean f SEM w i th n = 5

Inhibitors Percentage CL of PMA-stimulated PMN

Lucigenin-dependent CL

-sulphite fsulphite

None 1 0 0 f 6 2 1 7 f 2 0 SOD 1 2 f 3 1 4 f 3 Catalase 1 1 7 1 1 8 2 2 5 f 1 4 Azide 1 1 5 f 1 0 2 2 2 f 1 6

Luminol-dependent CL

-sulphite +sulphite

1 0 0 f 7 8 0 5 7 743Z9 4 8 f 1 1

1 1 2 f l 9 5 f l l 2 5 5 4 3 2 5 3

CL of lucigenin and luminol. Lucigenin-dependent CL is generated by reductive dioxygenation of lucigenin by 0; which is produced by a system consisting of XOD and xanthine (22,29,36). Sulphite did not interfere with the lucigenin-dependent CL of the 0, producing XOD/xanthine system: 0.1, 1 and 10mmol/L sulphite changed the lucigenin- dependent CL of the system by factors of 1.16, 1.25, and 0.99, respectively.

Luminol-dependent CL is generated by dioxygenation of luminol by H202 or by a system consisting of H202 and peroxidase (22,37,38). Luminol-dependent CL of H202 was not affected by sulphite: 0.1, 1 and 10mmol/L sulphite changed the luminol-dependent CL of H202 by factors of 0.98, 1.18 and 1.14, respectively. Very similar results were obtained when the effect of sulphite on luminol-dependent CL of the H202- horseradish peroxidase system was studied (data not shown). As shown in these cell-free systems, sulphite does not interact to any appreciable

extent with the CL of either lucigenin generated by 0; or luminol generated by H202.

lntracellular MPO activity of PMA-stimulated PMN treated by various sulphite concentrations

Since our measurements with luminol-dependent CL suggest that MPO-dependent processes are inhibited in sulphite-treated cells, the intracellular activity of MPO was studied in cells stimulated by PMA in the presence of various sulphite concentrations. The MPO activity was assayed by oxidation of TMB (32). Sulphite did not affect the oxidation of TMB, because it was removed prior to the assay procedure. As shown in Table 2, the intracellular MPO activity was reduced by 40% in cells incubated with 1 mmol/L sulphite, and by 5 5 % in cells incubated with 2mmol/L sulphite. This observation agrees well with our data obtained by luminol-dependent CL (Fig. 2), and indicates that sulphite inhibits MPO activity intracellularly .

Effect of sulphite on the luminol- dependent CL of the cell-free system M PO-H202-chloride

Since the intracellular activity of MPO is reduced in sulphite-treated PMN, we studied whether

Table 2. lntracellular MPO activity of PMA- stimulated PMN treated with various sulphite concentrations. PMN (I x lo6 cells/mL) were incubated for 30min at pH 7 and 37°C in the presence of various sulphite concentrations and stimulated by PMA. PMA-stimulated control cells were incubated simultaneously. After incubation the cells were isolated by centrifugation, and the intracellular MPO activity was determined by oxidation of TMB as described in the section, 'Materials and methods'. Values o f MPO activity are given as mean 3Z SEM with n = 5

Sulphite concentration (mmol/L) MPO activity (U/mg protein)

None 1.35 f 0.06 0.5 1 .I 9 f 0.03 1 .o 0.81 f 0.03 2.0 0.61 iO.02


Table 3. Effect of sulphite on the luminol- dependent CL of the MPO-H202-chloride system. Purchased MPO was incubated with various sulphite concentrations in PBS buffer, pH 7 , containing 0.2mmol/L luminol and 0.2 mmol/L H202, and luminol-dependent CL was measured at 30°C for 15min. 100% MPO activity corresponds to 28 & 3 x 10” CL- counts/l5min/mg protein ( n = 10). The other values are given as percentage of MPO activity and are expressed as mean k SEM with n repre- senting the number of experiments

Sulphite concentration Percentage lurninol-dependent CL (rnrnol/L) of MPO

None 100 0.005 105&5(n=3) 0.01 62If5 (n=3) 0.05 25 & 2 ( n = 6) 0.1 12&2 ( n = 6 ) 0.5 1 .o 2.0

2 f 0.4 (n = 6) 1.2 f 0.3 (n = 6) 0.8 f 0.1 (n = 6)

sulphite also exhibits an inhibitory effect on the activity of MPO in a cell-free system. For these studies MPO activity was not determined by oxidation of TMB according to Suzuki et af. (32), because sulphite strongly interfered with this assay system by reducing the oxidized form of TMB. The activity of MPO was therefore determined by luminol-dependent CL (26,29). As can be seen in Table 3, the luminol-dependent CL of MPO activity was strongly inhibited even at very low sulphite concentrations. A sulphite concentration of 0.1 mmol/L reduced MPO activity by about 90%. To confirm that luminol-dependent CL reflects MPO activity, the MPO-inhibitor azide was added to the CL-assay. As expected, 0.1 mmol/L azide inhibited luminol-dependent CL of MPO activity by 99%.

DISCUSSION

When the oxidative metabolism of PMN is stimulated, oxygen radicals are produced and granular enzymes are released. By using different chemiluminigenic probes such as lucigenin and luminol, CL is one of the most sensitive methods for studying oxygenation processes occurring during stimulation of PMN. Luminol-dependent CL is mainly dependent on MPO because it is

inhibitable by the MPO-inhibitor azide and is markedly reduced in PMN from patients with MPO-deficiency (20,25,28,3 1,39). By using oxygen radical scavengers of high and low molecular weight it has been shown that luminol-dependent CL has an extracellular and intracellular origin depending on the stimulus (26,40). When PMN are stimulated by PMA, the response of luminol- dependent CL is inhibited by the MPO-inhibitor azide and is of intracellular origin or cell- associated (23,26,28). Lucigenin-dependent CL, inhibitable by SOD, is a specific measurement for 0, production (21,22,24,29). Whereas it is feasible to differentiate between extracellular and intracellular production of oxidants using luminol (26,31,40), it is probably not possible using lucigenin. It has not been established whether lucigenin is capable of penetrating membranes due to its cationic nature. The inhibition by SOD confirms that lucigenin-dependent CL determines extracellularly produced oxidants.

In our studies on the effect of sulphite on the oxidative metabolism of PMN we used lucigenin- and luminol-dependent CL measurements to determine different oxygenation activities extracellularly and intracellularly. The oxidative metabolism of PMN was activiated by PMA, a soluble stimulant acti- vating NADPH oxidase via protein kinase C, and by zymosan as a particulate stimulant for phagocytosis (41). Regarding the lucigenin-dependent CL, our results show that sulphite in low concentration (0.01 to 1 mmol/L) significantly increased the lucigenin-dependent CL of resting and PMA- or zymosan-stimulated PMN (Fig. 1). Since the increase of lucigenin-dependent CL of sulphite- treated PMN was inhibited by extracellularly added SOD (Table l), our findings indicate that low sulphite concentrations enhance the extracellular production of 0: by resting and stimulated PMN. High sulphite concentrations (5 and 10 mmol/L) reduced the lucigenin-dependent CL of resting and stimulated PMN. Our previous studies have shown that phagocytes and lung tissue, devoid of sulphite oxidase, are susceptible to high sulphite concentrations resulting in a drastic loss of ATP (1 1). Since ATP is necessary for processes associated with oxidative metabolism, such as phosphorylation of glucose by hexokinase, it is con- ceivable that a reduced ATP level also depletes NADPH equivalents needed for the NADPH oxidase-catalysed reduction of oxygen.

Contrary to what was found for lucigenin- dependent CL, our data obtained for luminol-


dependent CL show that resting cells exhibit only a slight (1.9-fold) increase with sulphite concentrations up to 1 mmol/L (Fig. 2). PMA- and zymosan-stimulated PMN also showed at very low sulphite concentrations (0.01 and 0.05 mmol/ L) a slight (about 1.4-fold) increase in luminol- dependent CL, which is continuously reduced by sulphite concentrations >0.5 mmol/L (Fig. 2). Therefore luminol-dependent CL appeared to be much more sensitive to the effect of sulphite than lucigenin-dependent CL, because maximal stimulation of the cells was shifted to very low sulphite concentrations. Luminol-dependent CL of sulphite-treated and subsequently stimulated PMN was strongly reduced by azide, an inhibitor of intracellular MPO activity, and partially inhibited by SOD, but not by catalase (Table 1). The inhibiting effect of azide and not of catalase pro- vides evidence that luminol-dependent CL of sulphite-treated PMN has an intracellular origin and is associated with MPO. A partial inhibition of luminol-dependent CL of PMA-stimulated PMN by SOD was also found by Takahashi et al. (28) and explained by an involvement of 0; in the chlorination cycle of MPO for restoring MPO activity as proposed by Kettle and Winterbourn (42-44). We suggest that scavenging of 0; by SOD extracellularly may also reduce the intracellular 0, steady state level so that insufficient 0; is available for restoring MPO activity, and thus reducing the luminol-dependent CL response.

Regarding our findings obtained by luminol- dependent CL measurements, we proposed that sulphite reduces MPO activity intracellularly. This was confirmed by showing that the intracellular activity of MPO in PMN stimulated by PMA in the presence of various sulphite concentrations was significantly reduced (Table 2). The inhibiting effect of sulphite on MPO activity was also seen in a cell-free system (Table 3). A reduction of luminol-dependent CL of PMN stimulated with opsonized zymosan in the presence of sulphite (0.1 1 mmol/L) was also found by Hippeli and Elstner (45), and the possibility of an effect of sulphite on MPO was discussed. In our study presented here we show that sulphite, depending on the concentration, reduces luminol-dependent CL of PMA- or zymosan-stimulated PMN and inhibits intracellular MPO activity.

To ensure that the effect of sulphite on the oxygenation activities of PMN studied by lucigenin- and luminol-dependent CL is due to the action of sulphite on the cells and not due to interaction of

sulphite with the different CL responses, we performed control experiments in cell-free systems. In cell-free systems we generated lucigenin-dependent CL by reductive dioxygenation of lucigenin by 0; (22,29,36), and luminol-dependent CL by dioxygenation of luminol by H202 (22,38). In both cases sulphite did not affect the CL response. For the lucigenin-dependent CL we conclude that sulphite does not serve as an initial reductant in the reductive dioxygenation reaction of lucigenin (cf. 0,) (21). For the luminol-dependent CL we suggest that sulphite does not interfere in the dioxygenation reaction of luminol by H202.

We conclude from our findings that sulphite interacts with different oxygenation activities of PMN extracellularly and intracellularly. Sulphite, in low concentrations (0.01 - 1 mmol/L), enhances the production of 0; extracellularly. This sulphite-induced 0, production is due to an activation of NADPH oxidase by sulphite via a signal-transduction pathway involving protein kinase C and Ca2'/calmodulin, as we have shown recently (12). Furthermore, sulphite also affects intracellular processes by reducing significantly the activity of MPO. Recently we have found in sulphite-treated PMN that other intracellular events are also changed (e.g., degranulation, vesi- culation, intracellular concentration of H202) (46). We suggest that sulphite-treated PMN are able to produce increased amounts of 0; and H202. However, because of the inhibition of MPO by sulphite these reactive oxygen species can- not be converted to the MPO-generated oxidants HOC1, singlet oxygen and N-chloramines. There- fore PMN may be activated for oxygen radical production by micromolar amounts of sulphite, although they seem to have diminished ability to generate the very potent bactericidal and cytotoxic oxidants HOCl and N-chloramines. We have recently shown that exposure of beagle dogs to a sulphite aerosol at increased ambient levels induced inflammatory reactions in the lungs accompanied by an influx of PMN (9). A modulation of the oxidant burden derived from PMN, which accumulate in the respiratory tract during exposure to SO2 or sulphite aerosols, may be a component of this process.

Acknowledgements

We thank Drs Wolf Bors and Konrad Maier for interest- ing discussions and critical reading of the manuscript.


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Effect of sulphite on the oxidative metabolism of human neutrophils: Studies with lucigenin- and luminol-dependent chemiluminescence