JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 7 229-238 (1992)
Chemiluminescence of Human Bloodstream Monocytes and Neutrophils: an Unusual Oxidant(s) Generated by Monocytes during t h e Respiratory Burst B er na rd D a v i e s t a n d S t e v e n W. Edwards* Department of Biochemistry, University of Liverpool, PO Box 147, Liverpool L69 3BX. UK
Maximal rates of 0 ; and H,02 production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% o f those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce l o w levels of HOCl and related compounds. These combined reductions in 0; generating ability and lower myeloperoxidase levels result i n l o w levels o f luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of 0 ; and H202 than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production o f either of these t w o oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads t o photon emission. This oxidant(s) is not O;, H,O,, * OH, '02or NO, but is derived from 0; generated during the respiratory burst and is generated in greater quantities by activated monocytes compared w i t h neutrophils. Thus, lucigenin chemiluminescence is an indirect measure o f superoxide release. Keywords: chemiluminescence; neutrophils; monocytes; oxidants; respiratory burst
INTRO DUCTlO N
The production of reactive oxygen metabolites by phagocytic cells of the immune system such as neutrophils, monocytes and macrophages is recognized to be a key event in the function of these cells during infection and inflammation. In neutrophils the enzyme systems responsible for the generation of these oxidant species have been extensively inSupported by North West Cancer Research Fund and the Cancer Research Campaign. *Author for correspondence. t Present address: Department of Biological Therapy, Imperial Cancer Research Fund Laboratories, 44, Lincoln Inn Fields, London WC2A 3PX, UK. 0884-3996/92/040229-10$10.00 01992 by John Wiley & Sons, Ltd.
vestigated and partially characterized (Curnutte, 1988; Segal, 1989a; Edwards, 1991). These include a plasma membrane bound NADPH oxidase which generates 0;(Babior, 1984), and it is from this product that all other oxygen metabolites are derived. Additionally, neutrophils possess large quantities of myeloperoxidase (Klebanoff and Clark, 1978) which reacts with 0; and H 2 0 2 (derived from the dismutation of 0;) to form hypochlorous acids and related species. .OH may be formed via a metal-catalysed Haber Weiss type reaction involving 0; and H,O, (Halliwell and Gutteridge, 1989) and '0, is believed to be formed via reactions involving HOCl and H,O,. In neutrophils the importance of these reactive oxidants Received 28 November 1991 Revised 28 February 1992
230 in the killing of certain microbial pathogens is confirmed by observations of patients with chronic granulomatous disease which have defective oxidase function (Holmes et al., 1967; Segal, 1989b) and an impaired resistance to infections. Phagocytes from these patients cannot kill some types of pathogens and hence must rely on non-oxidative killing processes (Spitznagel, 1984; Lehrer et al., 1988) but these are inadequate to kill all microbial pathogens. In contrast to neutrophils, the oxidative metabolism of monocytes has not been as extensively investigated. It is known that bloodstream monocytes possess an NADPH oxidase which appears to be structurally identical to that of neutrophils and is activated by analogous mechanisms (Bromberg and Pick, 1984, 1985; Thelan and Baggiolini, 1990). It is also appreciated that, compared to neutrophils, monocytes contain lower levels of myeloperoxidase. However, the consequences of reduced levels of myeloperoxidase on the overall oxidative metabolism of monocytes has not been established. For example, it has been shown that myeloperoxidase activity regulates the duration of Oi/H,O, formation via the NADPH oxidase (Edwards and Swan, 1986) and also that utilization of H , 0 2 levels by myeloperoxidase can limit the potential to form - O H (Winterbourn, 1986). Therefore, it is likely the reactive oxidants generated from 0 ; in neutrophils will be different to those generated in monocytes, and this may have important consequences on the functions of these two cell types. The aim of the present study, therefore, was to compare the reactive oxidants generated by bloodstream monocytes with those produced by neutrophils. Monocytes generate lower levels of 0; per cell than do neutrophils and possess
Chemiluminescence of Human Bloodstream Monocytes and Neutrophils: an Unusual Oxidant(s) Generated by Monocytes during t h e Respiratory Burst B er na rd D a v i e s t a n d S t e v e n W. Edwards* Department of Biochemistry, University of Liverpool, PO Box 147, Liverpool L69 3BX. UK
Maximal rates of 0 ; and H,02 production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% o f those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce l o w levels of HOCl and related compounds. These combined reductions in 0; generating ability and lower myeloperoxidase levels result i n l o w levels o f luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of 0 ; and H202 than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production o f either of these t w o oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads t o photon emission. This oxidant(s) is not O;, H,O,, * OH, '02or NO, but is derived from 0; generated during the respiratory burst and is generated in greater quantities by activated monocytes compared w i t h neutrophils. Thus, lucigenin chemiluminescence is an indirect measure o f superoxide release. Keywords: chemiluminescence; neutrophils; monocytes; oxidants; respiratory burst
INTRO DUCTlO N
The production of reactive oxygen metabolites by phagocytic cells of the immune system such as neutrophils, monocytes and macrophages is recognized to be a key event in the function of these cells during infection and inflammation. In neutrophils the enzyme systems responsible for the generation of these oxidant species have been extensively inSupported by North West Cancer Research Fund and the Cancer Research Campaign. *Author for correspondence. t Present address: Department of Biological Therapy, Imperial Cancer Research Fund Laboratories, 44, Lincoln Inn Fields, London WC2A 3PX, UK. 0884-3996/92/040229-10$10.00 01992 by John Wiley & Sons, Ltd.
vestigated and partially characterized (Curnutte, 1988; Segal, 1989a; Edwards, 1991). These include a plasma membrane bound NADPH oxidase which generates 0;(Babior, 1984), and it is from this product that all other oxygen metabolites are derived. Additionally, neutrophils possess large quantities of myeloperoxidase (Klebanoff and Clark, 1978) which reacts with 0; and H 2 0 2 (derived from the dismutation of 0;) to form hypochlorous acids and related species. .OH may be formed via a metal-catalysed Haber Weiss type reaction involving 0; and H,O, (Halliwell and Gutteridge, 1989) and '0, is believed to be formed via reactions involving HOCl and H,O,. In neutrophils the importance of these reactive oxidants Received 28 November 1991 Revised 28 February 1992
230 in the killing of certain microbial pathogens is confirmed by observations of patients with chronic granulomatous disease which have defective oxidase function (Holmes et al., 1967; Segal, 1989b) and an impaired resistance to infections. Phagocytes from these patients cannot kill some types of pathogens and hence must rely on non-oxidative killing processes (Spitznagel, 1984; Lehrer et al., 1988) but these are inadequate to kill all microbial pathogens. In contrast to neutrophils, the oxidative metabolism of monocytes has not been as extensively investigated. It is known that bloodstream monocytes possess an NADPH oxidase which appears to be structurally identical to that of neutrophils and is activated by analogous mechanisms (Bromberg and Pick, 1984, 1985; Thelan and Baggiolini, 1990). It is also appreciated that, compared to neutrophils, monocytes contain lower levels of myeloperoxidase. However, the consequences of reduced levels of myeloperoxidase on the overall oxidative metabolism of monocytes has not been established. For example, it has been shown that myeloperoxidase activity regulates the duration of Oi/H,O, formation via the NADPH oxidase (Edwards and Swan, 1986) and also that utilization of H , 0 2 levels by myeloperoxidase can limit the potential to form - O H (Winterbourn, 1986). Therefore, it is likely the reactive oxidants generated from 0 ; in neutrophils will be different to those generated in monocytes, and this may have important consequences on the functions of these two cell types. The aim of the present study, therefore, was to compare the reactive oxidants generated by bloodstream monocytes with those produced by neutrophils. Monocytes generate lower levels of 0; per cell than do neutrophils and possess
