JOURNAL

OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 6 223-226 (1991)

The Effect of Two Bisphosphonates on Human Neutrophil Chemiluminescence and Myeloperoxidase Activity M. J. Kowolik' University of Leeds Dental School, Leeds, UK

P. M. Hyvonen Faculty of Dentistry, University of Kuopio. Kuopio, Finland

R. Sutherland and J. A.

Raeburn

Host Defence Laboratory, Human Genetics Unit, Western General Hospital, Edinburgh, UK

In order t o assess the importance of chlorine in a drug molecule as an influence on myeloperoxidase-mediated inflammatory cell functions, the effect of the chlorinated bisphosphonate, clodronate, on human neutrophil chemiluminescence and myeloperoxidase (MPO) activity was compared t o the non-chlorinated structural analogue, etidronate. The results suggested that the presence of chlorine may be important t o the enhancement of MPO activity. In addition both drugs manifested low toxicity and both of these observations may have relevance t o host defence. Keywords: Neutrophils; chemilurninescence; myeloperoxidase; bisphosphonates

INTR 0D UCTlO N

including the antiseptic chlorhexidine digluconate, have demonstrated an enhancement of M P O activThe oxygen-dependent microbicidal systems of hu- ity both directly and in a luminol-dependent chemiman neutrophils have been subjected to extensive luminescence (CL) system (Kowolik, 1987). investigation with respect to their function in both Geminal bisphosphonates comprise a group of protective and destructive processes relevant to compounds exerting a pronounced effect on bone homeostasis and inflammatory diseases (Weiss, resporption and are thus used in the treatment of 1989). Whole-cell chemiluminescence assays pro- Paget's disease and osteolytic tumour metastases vide a screen of this function but, under specific (Jung et al., 1981). Disodium dichloromethylene conditions, can indicate activity of specific compo- bisphosphonate (clodronate), like chlorhexidine, nents, such as the enzyme myeloperoxidase (EC contains two chlorine atoms while the structural 1.1 1.1.7) or MPO, which can also be measured analogue disodium hydroxyethylidene bisphosspec1rophotometrically. In addition, this methodol- phonate (etidronate) contains no chlorine in the ogy can be applied to monitoring the effects on molecule (Fig. 1.). The purpose of this study was to further elucineutiophils of particles (Gormley et al., 1985), date the effect of drug chlorination by challenging bactci-ia or drugs (Kowolik, 1987). Studies with chlorinated antimicrobial drugs, neutrophils in an MPO-dependent and MPO-inde-

* Autlior for correspondence OX84 :096/9iiO40223 04$05 00 i 19(JI by John W k y & Sons, Ltd.

Receicied I 9 Seprrmhcr I990 Revised 25 June 1991

224

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M. J. KOWOLIK, P. M. HYVONEN, R. SUTHERLAND AND J. A. RAEBURN

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Figure 1. Chemical structure of the t w o bisphosphonate analogues

pendent CL assay and M P O directly in a spectrophotometric assay with clodronate or etidronate.

PMA (2 pg/ml) respectively. Light output was measured at 37°C in an LKB 1251 Luminometer (LKB Wallac, Turku, Finland) and recorded as millivolts (mV). Myeloperoxidase activity: enzyme activity was assayed spectrophotometrically at 420 nm according to the method of Schindler et al. (1976), using ABTS, [2, 2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)], as substrate in a halide-free system. The effect of bisphosphonates was assayed by preincubation of enzyme aliquots extracted from 5 x lo6 neutrophils with drug at varying concentrations as for the CL assays. Preincubation was for 10 or 120 min, and enzyme activity was recorded as units/l x lo6 cells/min. The total number of experiments performed with clodronate was five, and with etidronate, three. All reagents were supplied by Sigma Chemicals, MO. USA.

MATERIALS A N D M E T H O D S

Neutrophils: 20 to 30ml of peripheral venous blood was collected from healthy adult volunteers, blood group 0. Neutrophils were harvested by routine separation techniques for whole cell CL assays (Kowolik et al., 1982), or, from the same cell batch, further processed for the extraction of M P O (Kowolik and Grant, 1983). Bisphosphonates: clodronate (Leiras Pharmaceuticals, Turku, Finland) and etidronte (Norwich Eaton, New Jersey, USA) were dissolved in calcium-free and magnesium-free phosphate buffered saline (PBS, Dulbeccos's medium, Gibco Ltd, Paisley, UK), pH 7.2, (osmolality = 278 milliosmoles/kg H,O), both drugs producing a clear solution at all concentrations. A 1 % stock solution was constituted freshly on each day of experimentation and ten-fold-dilutions made with PBS. Experiments were conducted in duplicate on cells harvested at different times. The total number of experiments performed in the presence of clodronate or etidronate were four and five respectively. Chemiluminescence: chemiluminescence generated predominantly by the MPO-mediated pathway was assayed by a luminol-augmented system and that via the superoxide anion generation by a lucigenin-augmented system (Dahlgren and Stendahl, 1983). Into the reaction vial were placed 5 x lo6 neutrophils in 500 pl PBS, 100 p1 bisphosphonate solM) or lucigenin ution, 200 pl luminol (4 x 2 x 1 0 - 4 M and 200p1 group AB serum-treated zymosan (1 mg/ml) or phorbol myristate acetate,

RESULTS

Chemiluminescence: the results are expressed as peak mV reached for each drug concentration in relation to the control value, i.e. no drug present. The background CL emitted in the negative control experiments, i.e. no cells or no intermediates present, was consistently less than 2 mV. No appreciable effect was seen after 10 minutes preincubation, and the results illustrated in Figs 2 and 3 are following 120-min preincubation of the neutrophils with the bisphosphonate. Fig. 2 shows the effect of clodronate on luminol- and lucigenin-augmented CL. The former system consistently produced higher levels of light output and this was also seen

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Figure 2. Effect of clodronate on luminol- and lucigeninaugmented neutrophil chemiluminescence in mV with SEM for a total of four experiments. (Background consistently < 2 rnV ) C L measured after 120-rntn preincubation of neutrophils with clodronate. Drug concentrations shown on a logarithmic scale

225

B IS P H0s PH 0 NATE EFFECTS 0 N N E UTR0 P H IL C H EMILI U MIN ESC ENC E SW YPO Astlvlt no..lin. 2%

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Figure 4. Effect of clodronate and etidronate on neutrophil myeloperoxidase ( M PO) activity expressed as percentage of baseline value (no drug), after 120 min incubation Means are for total number of five experiments with clodronate and three experiments with etidronate f SEM Drug concentra tions shown on a logarithmic scale

dependent mechanisms as has been shown for the antibiotic cefaclor (Grant et al., 1983) and the antiseptic chlorhexidine (Kowolik, 1987). Structurally clodronate and etidronate are identical, with the single important exception that where the former has two chlorine atoms the latter has a CH, and OH grouping. This provided as close a comparison as would be possible among bisphosphonate derivatives (Russel and Fleisch, 1975). The other principal purpose of this study was to apply CL methodology to an investigation of the interaction between these drugs and neutrophils. According to previous applications of phagocyte CL technique (Easmon et al., 1980; Gormley et a/., 1985) this was illustrated by the peak mV produced at each drug concentration. From the results of the chemiluminescence experiments in which it was possible to distinguish between the MPO-dependent and MPO-independent pathways of neutrophil activation, it was not apparent that clodronate had selectively influenced M P O function. Having extracted the enzyme from the neutrophils, however, prolonged incubation with clodronate resulted in enhanced activity, whereas etidronate had the opposite effect. The results therefore support the original hypothesis but also indicate that there are likely to be several possible mechanisms of interaction between this type of drug and these inflammatory cells. Bisphosphonates are highly water-soluble and so are unlikely to enter the cell in concentrations sufficient to have a significant effect, although there DISCUSSION is little experimental evidence to support or refute A principal objective of this study was to further this assumption (Yakatan et al., 1982). With speinvestigate the possibility that the chlorine moiety cific reference to the possible role of chlorine, the of a drug molecule may directly influence MPO- availability of this ion to react with neutrophil

in the experiments conducted with etidronate (Fig. 3). At low concentrations neither drug markedly influenced cell activity although at 1 x lo-, pg/ml clodronate produced a small increase in CL output. Depression of CL occurred with etidronate at concentrations greater than 1 p/ml in a dosedependent manner in both systems. In the case of clodronate, a similar effect was seen but only at concentrations above 10 pg/ml. A t the highest concentration of 1 x lo3 pg/ml neither drug produced a total inhibition of light output. In all experiments a typical CL response was seen in terms of mV produced with time. That is, for clodronate the peak mV occurred within 10 minutes, although that for etidronate at concentrations above 10 pg/ml took 15 to 20 minutes. Myeloperoxidase activity: Fig. 4 illustrates the effect of two bisphosphonates on M P O activity after 120- min incubation with the enzyme. Results are expressed as percentage activity in relation to loo"/, baseline with no drug present. After 10 min incubation there was little effect produced by either drug at any concentration. At 120-min incubation, however, clodronate caused an enhancement of MPO activity at all concentrations above 1 x 10- pg/ml, reaching 243 % of the control value at 1 x lo3 pg/ml. Etidronate, by contrast, produced a gradual depression in activity reaching 12% of baseline activity at the highest concentration used.

226

M. J. KOWOLIK, P. M. HYVONEN, R. SUTHERLAND AND J. A. RAEBURN

components may differ among chlorinated drugs. Charge effects of the ionized drugs may be equally important. It was of interest, and possible therapeutic relevance, that both drugs produced limited inhibition of CL in this experimental system. Even at high concentrations, CL reduction was partial, and it should be borne in mind that such concentrations in serum or extracellular fluid are far in excess of those achieved during any therapeutic regime (Conrad and Lee, 1981). It is not clear whether this reflects biochemical suppression or true toxicity, but it will require further investigation. The difference in effect on MPO activity after the two incubation times used was pronounced in the case of clodronate. In order to fully ascertain the relevance in viuo it would be necessary to study this aspect of the interaction over a wider time-scale in an appropriate animal mdoel. It may be, of course, that the pronounced effect of the drug on MPOactivity, contrasted with the limited effect on CL, reflects the relative contribution of MPO to light production. This would not, however, be consistent with accepted principles of neutrophil CL generation (Dahlgren and Stendahl, 1893; Nurcombe and Edwards, 1989). Of equal importance with respect to toxicity is the use of these drugs in the treatment of malignant and debilitating diseases where inflammatory cell integrity is crucial to the prevention and control of opportunistic infection. The diminution in a specific enzyme pathway may be tolerated up to 90% before a significant clinical effect is seen, particularly with an enzyme such as MPO which is present in high concentration (Harkness, 1981). The experimental model described above provides a sensititve system with which to monitor the interaction between any drug and neutrophil granulocytes, the primary mammalian defence cell. Given the above findings, further work will be necessary to clarify the mechanisms by which bisphosphonates exert their pharmacological effects on these and other inflammatory cells. More detailed analysis of CL curves, for example, such as reaction rates and total CL produced, may reveal other significant aspects of drug action and cell response. Acknowledge m e nts We are grateful to Dr H. Hanhijarvi, Leiras Pharmaceuticals, Turku, Finland, for the gift of clodronate,

and to Norwich Eaton, New Jersey USA for donating etidronate.

REFERENCES Conrad, K. A. and Lee, S. M. (1981). Clodronate kinetics and dynamics. Clin. Pharmucol. Ther., 30, 114 120. Dahlgren, C. and Stendahl, 0. (1983). Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect. Immun., 39, 736-741. Easmon, C. S. F., Cole, P. J., Williams. A. J. and Hastings, M. (1980). The measurement of opsonic and phagocytic function by Luminol-dependent chemiluminescence. Immunol., 41,67-74. Gormley, I. P., Kowolik, M. J. and Cullen, R. T. (1985). The chemiluminescent response of human phagocytic cells to mineral dusts. Br. J . exp. Path., 66, 409-416. Grant, M., Raeburn, J. A,, Sutherland, R. Harkness, R. A,, Gormley, I. P. and Kowolik, M. J. (1983). Effect of two antibiotics on human granulocyte activities. J . Antimicroh. Chemother., 11, 543- 554. Harkness, R. A. (1981). The characteristic cell of acute inflammation, the polymorphonuclear neutrophil leucocyte, and its biochemistry. Mol. Aspects Med., 4, 191 -207. Jung, A,, van Ouwenaller, C., Chantraine, A. and Courvoisier, B. (1981). Parenteral diphosphonates for treating malignant hypercalcemia. Cancer, 48, 1922- 1925. Kowolik, M. J. (1987). The oxygen-dependent microbicidal system in human gingival crevicular neutrophils. In The Biology of Phagocytes in Health and Disease: Adwnces in the Biosciences, Mauri, C., Rizzo, S. C. and Ricevuti, G . (Eds). Pergamon Press, Oxford, pp. 175-183. Kowolik, M. J. and Grant, M. (1983) Myeloperoxidase activity in human gingival crevicular neutrophils. Archs. Oral Biol., 23, 293-295. Kowolik, M. J., Cumming, C. G. and Grant, M. (1982). Interaction between human neutrophils and group B streptococci (GBS) and group antigens, monitored by luminol-dependent chemiluminescence. J. Clin. Lab. Immunol., 8, 55--58. Nurcombe, H. L. and Edwards, S. W. (1989). Role of myeloperoxidase in intracellular chemiluminescence of neutrophils. Ann. Rheumatic Dis., 48, 56-62. Russel, R. G . G. and Fleisch, H. (1975). Pyrophosphate and diphosphonates in skeletal metabolism. Clin. Orthop. Rel. Res., 108, 241-263. Schindler, J. S., Childs, R. E. and Bardsley, W. G. (1976). Peroxidase from human cervical mucus. The isolation and characterisation. Eur. J . Biochem, 65, 325-33 1. Weiss, S. J. (1989). Tissue destruction by neutrophils. New Engl. J. Med., 320, 365-316. Yakatan, G. J., Poynor, W. J., Talbert, R. L., Floyd, B. F., Slough, C. L., Ampulski, R. S. and Benedict J. J. (1982). Clodronate kinetics and bioavailability. Clin. Pharmacol. Ther., 31,402-410. ~

The effect of two bisphosphonates on human neutrophil chemiluminescence and myeloperoxidase activity.

In order to assess the importance of chlorine in a drug molecule as an influence on myeloperoxidase-mediated inflammatory cell functions, the effect o...
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