Journal of Clinical Laboratory Analysis 6:47-53 (1992)

Microtiter Plate Assay for Phagocyte-Derived Taurine-Chloramines Veronique Witko, Anh Thu Nguyen, and Beatrice Descamps-Latscha INSERM U25, Hdpital Necker, Paris, France Despitetheir potential importance,the role of phagocyte-derived chloramines (‘‘longlived oxidants”) has not yet been investigated in inflammatory or infectious diseases. We have developed a sensitivespectrophotometric microtiter plate assay for chloramines based on their capacity to oxidize potassium iodide (KI). Consistant levels of endogenous chloramines were detected in normal human polymorphonuclear neutrophil (PMN) supernatants after stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Exogenous taurine strongly enhanced chloramine secretion and was used to quantify the chlorinating potential of PMN. Taurine-chloramines were also detectable in rnonocyte supernatants, although in smaller amounts. The specKey words:

INTRODUCTION

ificity of the KI assay was assessed both in terms of effect of compounds inhibiting (KBr) or interacting with (sodium azide and catalase) chloramine formation and by showing that PMN from patients with chronic granulomatous disease, due to a hereditary lack of oxidative response capacity, were unable to produce chlorarnines. Taurine-chlorarnine levels secreted by PMA (but not zymosan)stimulated PMN were closely related to the cellular luminol-amplifiedchemiluminescence (CL) responses although the CL assay failed to detect chloramines in PMN supernatants. We consider that this KI assay should be of use in studying the role of long-lived phagocyte-derived oxidants in clinical medicine.

phagocytes, oxidative burst, chemiluminescence, myeloperoxidase, oxyradicals, inflammation, SDectrophotometrv

for chloramines, little if any information is available concerning their possible role in inflammatory and/or immunologiIt is now recognized that reactive oxygen species (ROS) cal disorders. produced during the respiratory burst of phagocytic cells play We propose a simple spectrophotometric microtiter plate a major role in host defenses against bacterial infections (1,2), assay to determine chloramine levels in phagocytic cell incutumors (3), and parasites (4) and mediate numerous tissue bation supernatants, based on the capacity of these molecules processes associated with inflammation (5) and ischemic injury to oxidize potassium iodide (KI) (13). In the model used, ( 6 ) .Among these ROS, superoxide anion (O2-)-first generchloramine production results from chlorination of endogeated after NADPH oxidase activation-and hydrogen peroxnous amines released from phagocytes undergoing activation ide (H202)-arisen from O2- dismutation-can be measured of oxidative metabolism. Incubation of polymorphonuclear simply in spectrophotometric (7,8) and chemiluminescence neutrophils (PMN) with an exogenous amine such as taurine, (CL) assays (9,10), which is not the case for the hydroxyl a p-amino acid plentiful in leukocytes and an HOCl scavenradical (OH.) and singlet oxygen (lo2). Phagocytes can also ger, can permit the phagocyte-chlorinating potential to be generate hypochlorous acid (HOC1) via the reaction of H202 determined (14,1.5). with chloride catalyzed by myeloperoxidase (MPO) (2). Because of its high reactivity, HOCl does not accumulate in METHODS biological systems and disappears almost instantaneously in multiple reactions with available substrates. In this process, Cell Preparations it participates in the generation of a derivative group of oxiNormal PMN were obtained from heparinized (Liquemine dants known as the chloramines (1 1,12). Chloramines are Roche, 10 U/ml) venous blood of volunteer donors recruited termed long-lived oxidants; there is evidence that they can oxidize proteins and they are thought to be involved in the regulation of the inflammatory response (1 1). Chloramines, which are more stable than ROS, could thus play a promi- Received August 23, 1991; accepted August 28, 1991. nent role in the propagation of oxy-radical-mediated tissue Address reprint requests to Beatrice Descamps-Latscha MD, DSci, INSERM damage. However, given the lack of a simple, reliable assay U25, HBpital Necker, 161 rue de Skres, 75743 Paris CEDEX 15. 0 1992 Wiley-Liss, Inc.

48

Witko et al.

at the Centre de Transfusion de l’H6pital Necker (16). Briefly, PMN were separated from erythrocytes by means of Plasmagel (Roger Bellon Laboratoire, Paris, France) sedimentation followed by Ficoll-Hypaque (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) density centrifugation. Residual erythrocytes were lyzed by treating the cell pellet with a lysis buffer containing ammonium chloride. The purified PMN were then resuspended in phenol-red-free Hank’s balanced salt solution (HBSS) pH 7.4 (Eurobio, Paris, France) at the desired concentration. In some experiments, monocytes were also purified by using the classical adherence procedure. Briefly, the mononuclear cell layer of the Ficoll-Hypaque gradient was pipetted, washed twice, adjusted to a monocyte concentration of 106/ml, plated in 24-well Falcon 3047 tissue culture dishes (Falcon Labware, Becton Dickinson and Co., Oxnard, CA), and incubated for 60 min at 37°C. The dishes were then washed 5 times with HBSS to remove non-adherent cells (mainly lymphocytes). Oxidative burst-unresponsivePMN from two children with X-linked chronic granulomatous disease (CGD) were kindly provided by Professor C. Griscelli (Unit6 d’HCmatologie Pediatrique, HGpital Necker Enfants Malades) with informed parental consent.

Chloramine Production and PMN Supernatant Preparation Neutrophils resuspended in HBSS at various concentrations were incubated for the indicated periods at 37°C under constant gentle shaking in the absence (controls) or presence of the following stimulating agents: phorbol myristate acetate (PMA; 4P-phorbol,12P-myristate, 13a-acetate, Sigma) at a final concentration of 1 pg/ml and opsonized zymosan particles (Zymosan A from Saccharomyces cerevisiae, Sigma), prepared as previously described (16), at 2 mgiml. At the end of the incubation period, the samples were centrifuged at 4°C (1,200g for 10 min) and the supernatants were collected. Adherent monocytes were stimulated directly in the culture dishes and supernatants were prepared following the same protocol as for PMN. In some experiments aimed at amplifying or inhibiting PMN chloramine formation, taurine and KBr, respectively, were added to the cell preparations immediately before the stimulus of chloramine secretion. In experiments to investigate the effect of oxy-radical scavengers (catalase and superoxide dismutase) or an MPO inhibitor (sodium azide) the compounds were added 15 min before the stimulus. These chemicals were purchased from Sigma. All cell supernatants were stored at - 20°C and tested the following day.

Chloramine Determination in PMN Supernatants Chloramines were determined by colorimetric measurement of the triiodide ion formed by the oxidation of potassium

iodide in solution, according to the method of Kimura et al. [ 131. Chloramine-T (N-chloro-p-toluene-sulfonamide sodium salt, Sigma, St. Louis, MO) a standard commercial product, was used to calibrate the assay. A solution of chloramine-T (100 mM) in distilled water stored at 4°C was diluted daily in HBSS to appropriate concentrations (0- 100 nmol/ml). Two-hundred microliters of test samples (supernatants or the chloramine standard solution) was distributed in 96-well Falcon microtiter plates. Ten microliters of l . 16 M KI was then added, followed by 20 p1 of acetic acid (Prolabo, Paris, France) 2 min later. The absorbance of the reaction mixture was immediately read at 340 nm in a microplate reader (model MR 5000, Dynatech, France) against a blank containing 200 p1 HBSS, 10 pl KI, and 20 p1 acetic acid. Within the range of 0 to 100 nmol/ml, absorbance at 340 nm followed Beer’s law, assuming an extinction coefficient of 26 mM- ‘.cm I .

+

~

ChemiluminescenceAssay The PMN oxidative response was measured at the same time as chloramine in terms of cellular luminol-amplified CL [16]. Briefly, 100 pl of PMN suspension (lo6 cells/ml) was placed in polystyrene tubes containing 50 pl of luminol solution (Sigma, 0.2 mg/ml) and 10 pl of HBSS (resting CL) or stimulating agents, e.g., PMA (1 pg/ml) or opsonized zymosan (2 mg/ml). The tubes were incubated at 37°C under constant agitation (120 rpm) for 15 min. CL intensity was measured by mean of photometry (Biolumat Model LB 9500, Berthold, Wildbad, FRG) and expressed as counts per second (cps).

Statistical Analysis Results expressed as mean 2 SEM were compared by using Student’s two-tailed t test (paired or unpaired, as appropriate). Correlation coefficients were calculated by using simple regression analysis. Differences were considered significant when the P value was 0.05 or less.

RESULTS

Endogenous Chloramine in Normal PMN Supernatants PMN (2.5 X 106/ml)from normal subjects were incubated under various conditions to study chloramine production. As shown in Table 1, no chloramine production was detected in supernatants of resting cells. In contrast, stimulated neutrophils produced chloramines within the range of 5-22 nmollml. Mean and individual chloramine value did not differ significantly between PMA- and zymosan-stimulated cells. Figure 1 shows the kinetics of chloramine formation by PMN from normal subjects. With both PMA and zymosan, chloramine formation was detected as early as 15 min after stimulation and reached a maximum at 60 min. Thereafter, chloramine production induced by PMA fell, whereas that induced by zymosanremainedstable, respectively 12.5 ? 1.70

Measurement of Phagocyte Chloramines TABLE 1. Chloramine Production in Supernatants of Resting and Stimulated Normal PMNa Donor

Control (HBSS)

PMA

Opsonized zymosan

0 0 0 0 0 0 0 0 0 0

21.43 7.50 14 13.49 7.91 5.24 7.36 13.37 18.37 16.86

22.80 12.75 13 12.95 9.85 6.80 11.01 22.09 17.21 13.11

0

12.55 1.71

14.16 1.62

1

2 3 4 5 6 7 8 9 10 Mean SEM

0

"Each cloramine concentration was determined in triplicate and is given in nmol/ml. There was no significant difference between the two stimuli paired t test).

and 4.8 2 2.62 nmoYml (P = 0.03) at 60and 120 min with PMA and 14.2 f 1.63 and 10.7 k 3.33 nmoVml (NS) with zymosan. The specificity of the KI assay for chloramine determination was checked by adding KBr (10 mM), a halide interfering with the generation (or accumulation) of N-C1 derivatives (17), just before PMA and opsonized zymosan. As shown in Figure 2, the production of chloramines was significantly inhibited at 60 min in the presence of KBr (by 95.8 f 4.25% and 92.2 f 4.70% in the presence of PMA and opsonized zymosan, respectively vs. KBr-free control). The influence of the cell concentration on chloramine generation is shown in Figure 3. It was not possible to detect reproducible chloramine production in supernatants of preparations containing < 2.5 x lo6 cells/ml. Chloramine production increased with increasing PMN concentrations, especially in the presence of PMA. It is noteworthy that chloramine synthesis can only result from chlorination of endogenous amines released from the

. 5

without KBr

with KBr

Zymosan Stimulating agents

PMA

Fig. 2. EffectofKBron chloramineproduction. PMN (2.5 X 106/ml) were stimulated with either PMA (1pg/ml) or serum-opsonized zymosan (2 mg/ml) in the presence or absence of KBr (10 mh4) for 60 min. Data are the mean 2 SEM of five experiments with different donors.

activated cells in this model. In the case of prolonged incubation, substrates (endogenous amino-compounds) would therefore be a limiting factor for chloramine formation. Lastly, chloramine was indetectable in PMN lysates regardless of the stimulating agent (data not shown), probably due to the scavenging effect of intracellular compounds (18).

Enhancement of Chlorarnine Generation by Exogenous Taurine: Assessment of the PMN Chlorinating Potential The amount and nature of the endogenous N-Cl derivatives generated by PMN depend on the availability of amino compounds. Indeed, the addition of amines is known to enhance chloramine generation (12). PMN can form lipophilic oxidants, e.g., the monochloramine NH2-Cl, but they do not accumulate in supernatants because of their lipophilicity. Studies of the physical characteristics of endogenous N-Cl deriv-

25

-i

v

0

49

D

PMA

Zymosan

20

a rl

E

15

8

e

10

(1

8

1

5

0 0

30

60

90

120

Minutes

Fig. 1. Typical experiment showing kinetics chloramine formation by PMN from a healthy donor. PMN were stimulated in HBSS buffer with either PMA (1 pgiml) or serum-opsonized zymosan (2 mg/ml) at a final cell concentration of 2.5 X lo6 cells/ml.

Fig. 3. influence of cell concentrations on PMN chloramine production. PMN were incubated at concentrations of 0 to 10 X lo6 cells/ml for 60 min with HBSS, PMA (1 pg/ml), or serum-opsonized zymosan (2 mg/ml). Data represent mean 2 SEM and the number of donors is indicated in parentheses. *Significantly different from value with 2.5 X lo6cells/ml.

50

Witko et al.

atives present in the supernatants of activated neutrophils have revealed that they are hydrophilic and heterogenous (17). Amino acid analysis has shown that the major hydrophilic chloramine produced by neutrophils is taurine-chloramine. The effect of exogenous taurine on PMN chloramine production is shown in Figure 4. No taurine-chloramine was generated in the absence of stimulating agent and taurine did not interfere in the KI assay. Addition of taurine (15 mM) just before PMN stimulation with PMA or zymosan resulted in a sharp increase in chloramine generation (13- and 28-fold, respectively). The increased sensitivity of the KI assay in the presence of taurine was also shown by the facts that: 1) taurinechloramine production did not decrease after 60 min; 2) production was detected at cell concentrations as low as 0.5 X 106/ml;and 3) regardless of the incubation conditions, chloramine formation in response to opsonized zymosan was significantly greater than with PMA (Table 2).

Specificity of the KI Assay for Taurine-Chloramine Determination Incubation of PMN with KBr (10 mM), a potent inhibitor of chloramine formation, almost completely abrogated the production of taurine-chloramines, with percentages of inhibition (mean k SEM, n = 7 experiments) of, respectively, 99.1 k 0.91% in the presence of PMA and 98.9 k 0.72% in the presence of zymosan, respectively. As shown in Table 3, regardless of the stimulating agent both sodium azide (0.1 mM)-an MPO inhibitor-and catalase (10,000 U/ml)-an H202scavenger-profoundly inhibited chloramine formation. In contrast, superoxide dismutase (100 Uiml) tended to augment taurine-chloramine synthesis. Taken together, these 300

.

Without taurine With taurine

h

r( r(

B

TABLE 2. Chloramine Production by Stimulated Normal PMN in the Presence of Taurine' Stimulus PMA

Cell concentration (x lo6 PMN/ml) 0.5

1 2.5 Zymosan

0.5 1 2.5

Incubation time (min) 15

60

120

6.9t2.85 (n = 3) 20.5-r- 3.09 (n = 3) 59.0 t 7.55 (n = 3) 1 7 . 8 5 1.04 (n = 3) 40.5 3.91 (n = 3) 84.6 -C 6.35 (n = 3)

16.7 t 2.03 (n = 3 ) 42.1 2 3.16 (n = 9) 78.1 2 9.4 (n = 7) 2 7 . 9 2 1.43 (n = 3) 78.8 t 8.64 (n = 9) 138.8 t 24.28 (n = 7)

24 t 1.6 (n = 3) 47.1? 8.28 (n = 9) 79.8 12.33 (n = 7) 3 6 . 7 t 1.21 (n = 3) 1 0 4 . 9 t 13.7 (n = 9) 152.1 t 2 4 . 5 6 (n = 7)

*

*

"Each chloramine concentration was determined in triplicate and is given in nmol/ml. Results are expressed as mean t SEM. Numbers in parentheses indicate the number of donors.

results show the specificity of the KI assay for chloramine determination in our experimental conditions. We also investigated the specificity of the chloramine assay by studying PMN from two patients with X-linked chronic granulomatous disease (CGD). Due to a genetic defect in the cytochrome b558 heavy chain of the NADPH-oxidase complex, these PMN are totally devoid of oxidative response capacity (19). In the incubation conditions described above and despite the presence of taurine, no chloramine was detected in the supernatants of PMA- and zymosan-stimulated PMN from either patient. These results confirmed that chloramine formation is highly dependent on phagocyte oxidative activation pathways and showed that this KI assay could be suitable for detecting this genetic disorder.

Chloramine Generation by Monocytes Some authors have reported that monocytes can also generate taurine-chloramines (14). As the MPO content in monocytes is significantly lower than in PMN (20), we compared their

v

200 0 d

cd 4J

al

x8 d

TABLE 3. Effect of Specific Inhibitors on PMN TaurineChloramine Productiona

100

j

Inhibitor

0

PMA

20

Stimulating agents

Fig. 4. Effect of taurine on PMN chloramine production. PMN were stimulated with either PMA (1 pg/ml) or serum-opsonized zymosan (2 mglml) in the presence or absence of taurine (15 mM) for 60 min. Results are expressed as mean k SEM of triplicate determinations in a typical experiment performed with PMN from a single healthy subject.

None (HBSS) Sodium azide (0. ImM) Catalase (10,000 Uiml) Superoxide dismutase ( 100 U/ml)

HBSS

0

PMA

55.5 t 1.57 131.9k4.51

0

2.1 t 1.42

0

( -96. 3%)b 2.0 2 0.71

0

Zymosan

(-96.5%) 60.6 t 2.76 (+8.1%)

6 . 3 t 1.50 (-95.3%) 3 . 9 k 1.12 (-97.1%) 141.4t5.61 (+5.61%)

"Each chloramine concentration was determined in triplicate and is given in nmol/ml. Results are expressed at mean k SEM. bVariation relative to inhibitor-free controls (HBSS).

Measurement of Phagocyte Chloramines

51

TABLE 4. Comparison of PMN and Adherent Monocyte (MN) Taurine-ChloramineProduction" PMA

HBSS

PMN

Donor

MN

PMN

MN

Zymosan

MN/PMN

PMN

MN

MNiPMN %

70

Case 1 Case 2 Case 3 Case 4 Case 5 Case 6

0 0 0

Mean SEM

0 0

0 0 0 0 0

0 0 0

0

62.18 56.66 66.35 44.06 50.76 34.37

38.23 23.78 8.53 13.80 17.56 6.76

61.48 41.97 12.86 31.32 34.59 19.66

124.72 147.87 126.83 150.92 176.28 150.63

46.80 23.99 19.01 11.90 36.42 4.93

37.52 16.22 14.99 7.88 20.66 3.27

0 0

52.40 4.85

18.11 4.75

33.64 7.02

146.21 7.72

23.84 6.35

16.76 4.87

"Each chloramine concentration was determined in triplicate and is given in nmoliml.

chlorinating potential (Table 4). Adherence-purified monocytes (lo6 cells/ml) and PMN from individual donors were incubated in the presence of taurine for 120 min. No chloramine was detected in supernatants of resting monocytes. Chloramine production by stimulated PMN was significantly greater than that by stimulated monocytes. Unlike PMN, there was no difference between the two stimuli (PMA and zymosan) in their capacity to induce chloramine production by monocytes. However, PMA-stimulated chloramine production by monocytes was 33.6 ? 7.02% that of PMN, but only 16.8 t 4.90%followingopsonizedzymosanstimulation.

Comparative Study of the KI Assay and Cellular Luminol-AmplifiedChemiluminescence Figure 5 shows the results obtained with PMN from 13 healthy subjects for taurine-chloramine secretion measured by the KI assay and cellular oxidative responses measured in terms of luminol-amplified CL. A close correlation was observed when PMA was used as stimulus (r = 0.75, P = 0.003 at 60 min and r = 0.80, P = 0.001 at 120 min in the KI assay). By contrast, no such correlation was found with opsonized zymosan (r - 0.23, and 0.48, respectively). 300000

-3 J

0

y=-4,57e+4 + 2664,062~ R=0,75

200000

100000

0: 20

.

, 40

I

60

80

Cuorarnine concentration ( m o l l m l )

Fig. 5. Correlation between taurine-chloramine concentration in PMN supernatants after 60 min incubation and cellular luminol-amplifiedchemiluminescence.

Since the CL assay does not permit the detection of chloramines in cell-free supernatants, the KI assay could provide a complementary approach to the investigation of phagocyte oxidative metabolism.

DISCUSSION We propose a sensitive and reliable technique for assessing chloramine production in incubation supernatants of human blood phagocytes, with possible applications to the study of these long-lived oxidants in clinical medicine. Given the KI oxidizing capacity of chloramines, they can easily be determined in microtiter plates. Most previous studies have been based on the ability of chloramines to oxidize TNB (5-thio-2nitrobenzoic acid) to DTNB (5-5'-dithibis-2-nitrobenzoic acid), and only a few studies have used the KI assay (21). We chose KI as substrate because, contrary to the the TNB method which required reagents to be prepared immediately before use (with safety precaution), those used in the KI assay are non-toxic and can be stored. Both PMA- and opsonized zymosan-stimulated PMN generate and release endogenous chloramines via amine chlorination. This endogenous production was detected at cell concentrations of above a minimal 2.5 x 106/ml. Chloramine production was maximum after 60 min of incubation and decreased thereafter probably because of a lack of substrate. The fact that the addition of taurine to the PMN incubation medium prior to stimulation significantly increased chloramine production is in agreement with biochemical studies showing that taurine-chloramine is the major species produced by PMN (17). In addition, taurine-chloramine production remained constant for 120 min in the presence of exogenous taurine, suggesting that the previously observed lack of substrate was compensated for. Moreover, since taurine quenches HOCl(22) with the generation of taurine-chloramine, the KI assay reflects an overall maximum chlorinating potential. The precise biological role of taurine is unknown, although taurine is most abundant in situations in which ROS are generated (23). As a result, the taurine concentration is higher in leukocytes than in other cells. There is now evidence suggesting that taurine plays an important role in both detoxification

52

Witko et at.

and antioxidant defense (including membrane protection), although this latter role is controversial (24). Although incubation of PMN in the presence of taurine is not a physiological situation, it mimics inflammatory reactions, during which hydrolysed peptides are released at the inflammatory site. Regardless of the stimulus, our results showed that monocytes are capable of producing taurine-chloramines. However, the chlorinating potential of monocytes was much lower than that of PMN, confirming previously reported differences in oxygen metabolism and phagocytosing potential (16,25). Our results are in keeping with these studies showing that monocytes only release about 20% of the hydrogen peroxide generated by neutrophils during phagocytosis, but in disagreement with another study in which PMA-stimulated monocytes and neutrophils were found to generate similar amounts of chloramines (14). It is noteworthy that, in our incubation conditions, the difference between PMN and monocytes was much less marked with PMA than with a particulate stimulus, i.e., opsonized zymosan which might preferentially generate intracellular ROS. Taken together, these findings show that KI oxidation can be used to detect and quantify long-lived oxidants generated by phagocytes and that it closely reflects the oxidative response of these cells. Indeed, there was a close correlation between the results of the KI assay and those of the cellular luminol-amplified CL assay, which selectively measure the MPO-associated ROS production (26), although this correlation was only observed with a soluble stimulus (PMA). The absence of correlation with zymosan could be explained by major differences in the mechanisms involved in the generation of CL with the two stimuli (10). Our results also confirm that MPO participates in chloramine synthesis and show that the KI and cellular luminol-ampliffed CL assays are complementary approaches to the investigation of phagocyte oxidative metabolism: while CL reflects intracellular ROS generation resulting from phagocyte activation, the KI assay selectively measures the release of long-lived oxidants. The proposed KI assay, which is considerably simpler than alternative methods for chloramine determination and can readily be automated, may find numerous applications in clinical medicine and be of use in elucidating the role of these oxidants in diseases associated with inflammation and/or impaired host resistance to pathogens.

ACKNOWLEDGMENTS We wish to thank F. Tresset for excellent technical assistance and M. Kadouche for the iconography.

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Measurement of PhagocyteChloramines 24. Aruoma 01, Halliwell B, Hoey BM, Butler J: The antioxidant action of taurine, hypotaurine and their metabolic precursors. Biuchem J 256251-255, 1988. 25. Reiss M, Roos M: Differences in oxygen metabolism of phdgocytosing monocytes and neutrophils. J Clin Invest 61:480-488, 1977.

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26. Dahlgren CI, Stendahlo: Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 39:736-741, 1983.