Molec. Aspects Med. Vol. 12, pp. 87-92, 1991 Printed in Great Britain. All dghts reserved.
0098-2997/91 $0.00 + .50 ~)1991 Pergamon Press plc.
OXYGEN RADICAL PRODUCTION BY TRANSFORMED B LYMPHOCYTES Owen T.G. Jones, John T. Hancock and Lydia M. Henderson Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
Introduction Superoxide (O~) is produced by phagocytic leukocytes (neutrophils, monocytes, macrophages and eosinophils) and contributes to the microbicidal and tumouricidal activities of these cells (Badwey and Karnovsky, 1980). It is becoming apparent that O~ can be produced by cells other than professional phagocytic cells, albeit at lower rates, where it may have functions yet to be determined. Volkman et al. (1984) found that EBV transformed B lymphocyte cell lines produced O~ when stimulated with phorbol myristate acetate (PMA), a potent activator of phagocyte O~ formation, although the rates of about 0.3nmol/min/107 cells were only 1/20 of those given by activated neutrophils (about 20 nmol/min/107 cells. This work with cell lines has been repeated (Melinn and McLaughlin, 1987; Maly et al., 1988; Hancock et al., 1989) and significantly higher activities of O1 generation found (see Fig. 1). Even in the absence of added PMA there is significant release of O~ by the lymphocytes. There is evidence that this activity is present even in normal, non-transformed B lymphocytes isolated from tonsils (May et al., 1989). Some properties of the B lymphocyte superoxide generating oxidase are described below.
Composition and Localisation of the B Lymphocyte Oxidase In disrupted cell preparations, oxidase activity was dependent upon the addition of NADPH or NADPH. The Km for NADPH was lower than for NADH in the three cell lines studied (Hancock etal., 1989) but varied between different cell lines. This may arise from small differences in composition of the oxidase or its orientation in the membrane. However, it is likely that the oxidase is an NADPH oxidase, as found in phagocytes. B cell lines which were active in producing O~ contained (Maly et al., 1988) low potential cytochrome b (cytochrome b-245 also called b558), a component of NADPH oxidase of neutrophils (Cross et al., 1981), and expressed mRNA for the Abbreviations: PMA, phorbol myristate acetate; DPI, diphenylene iodonium; EBV, Epstein-Barr, virus; HAGG, heat-aggregated immunoglobulin; 1L, interleukin, TNF, tumour necrosis factor.
87
88
O . T . G . Jones et al.
13chain of the cytochrome b (Maly et al., 1989). Use of an antibody specific for cytochrome b-245 indicated that the cytochrome was located on the plasma membrane of the B lymphocytes, (Maly et al., 1989), where the haem group could transfer electrons directly to 0 2 (Cross et al., 1982; Cross et al., 1985) releasing O2 to the outer face of the cell. Patients with chronic granulomatous disease have neutrophils which are incapable of generating 0 2 , when stimulated with PMA (Volkman etal., 1984) due to lack of cytochrome b-245 (Segal et al., 1983). EBV B lymphocytes from such patients also fail to produce O;~ when stimulated with PMA.
~
4--
m
~2
I
I
I
I
I
I
0
10
20
30
40
50
~1
60
Time (min) Fig. 1. Rate of superoxide production by EBV-B lymphocytes in response to PMA
NADPH oxidase of neutrophils is specifically inhibited by diphenylene iodonium (DPI) which covalently attaches to a protein with Mr of 45 kD (Cross and Jones, 1986; Cross, 1987) believed to be a flavoprotein component of the oxidase (Yea et al., 1990). The oxidase of EBV B lymphocytes is similarly inhibited by DPI, which also labels a 45 kD protein (Maly et al., 1988). No binding of DPI to 45 kD protein was found in non EBV-transformed B lymphocytes, which lacked the O~ generating oxidase. The similarity of components and reactivities of EBV B lymphocyte oxidase and of the neutrophil oxidase suggests that in lymphocytes the oxidase is located in the plasma membrane and has the following sequence, as proposed for the neutrophil oxidase by Cross et al. (1985): eeeNADPH --4 FAD ~ cytochrome b-245 ~ 0 2 --~ O~ protein (45kD)
Stimuli for Superoxide Generation by EBV B Lymphocytes The variety of activators O~ production is listed in Table 1. The cytokines, TNFt~, I1-18 and 116 are good activators of the oxidase; their usual role is to regulate proliferation and maturation of B lymphocytes. Tumour necrosis factor is a potent activator of NADPH oxidase of adherent neutrophils (Nathan 1987). Each of these cytokines mediates its action after binding to a specific receptor on B lymphocyte plasma membranes (De Franco, 1987).
Oxygen Radical Production
89
Protein A-bearing staphylococci (Pansorbin) activates NADPH oxidase of EBV B lymphocytes. Pansorbin is likely to act by causing a cross-linking of lymphocyte surface immunoglobulins IgG, IgM and IgE. Although antilgG induced the respiratory burst response in a B cell line, antilgM was ineffective (Maly etal., 1988). It is probable that cross-linking of surface immunoglobulins activates B cells by activating phospholiphase C, causing the release of inositol triphosphate (IP 3) from membrane phosphatidylinositol 4,5bisphosphate (PIP2), together with diacylglygerol (see review by De Franco, 1987). IP 3 causes the release of Ca 2+ from intracellular stores and Ca 2÷, with DAG, activates protein kinase C. The same mechanism appears to operate in B lymphocytes. This proposed mechanism for oxidase activation is supported by the use of inhibitors and ionophores on EBV B lymphocytes. Both A23187 and ionomycin, which make membranes permeable to Ca 2+, activate superoxide production by EBV B lymphocytes. (Our results here are in conflict with those of Volkman et al., 1984). Activation of the EBV B lymphocyte oxidase by heat aggregated immunoglobulin suggests that the B lymphocyte Fc receptor, like that of the neutrophil, is coupled to an activation system for the NADPH oxidase. In neutrophils activation of Fc receptors leads to release of IP 3 and increased intracellular Ca 2+ but the relationship is less clear in B lymphocytes (see review by De Franco, 1987). Bacterial lipopolysaccharide (LPS) is a B cell activator, causing proliferation and differentiation (Andersson et al., 1977). It is also a powerful activator of NADPH oxidase of macrophages and neutrophils and of EBV B lymphocytes. It is not clear whether LPS activates O1 production following binding to a receptor or through perturbation of the lymphocyte membrane by the lipid component of LPS. Membrane perturbing agents such as detergents and DCCD can activate neutrophil NADPH oxidase, perhaps by changing membrane fluidity. Table 1. Stimulifor O~ generationby EBV B lymphocytes (a)
Receptor-mediated TNF(x IL-113 IL-6 Protein A (Pansorbin) Heat aggregatedimmunoglobulin(HAGG) *Lipopolysaccharide(Re mutant)
(b)
Receptor-independent Phorbol myristateacetate(PMA) Fluoride (+A13+) Arachidonate Ca2+ ionophores(ionomycinor A23187)
Hancock, Hendersonand Jones, unpublished. Ineffectiveas activators were: FMLP, TNI],IL-lct, INFct,INFI3and opsonizedzymosan. The maximumrate of O~ production,given by PMA, was about 10 nmol/min/mgprotein. *Lipopolysaccharid¢may not require a receptor. We have found that arachidonic acid is a good activator of the EBV-B lymphocyte oxidase (Table 1) just as it is for neutrophils (Badwey and Karnovsky, 1986; Henderson et al., 1989). It is possible that arachidonic acid has an effect which is quite specific, acting as a second messenger within the membrane. Some support for this view is given by experiments with a variety of inhibitors of phospholipase A2, such as RO 4639, each of which inhibits O1 production by EBV B lymphocytes; the inhibition is overcome by the addition of arachidonate to the assay suspension. An involvement of a G protein in activation is suggested by the stimulatory activity of F-, together with A13+.
90
O.T.G. Jones et al.
Time Course of 0~, Generation EBV lymphocytes treated with a variety of stimuli release O~ over periods of 40-50 min. The response to PMA is shown in Fig. 1. The rate of O~ secretion continues to increase for about 30 rain. and then slowly declines over the next 30 mins. This contrasts with the response of neutrophils which reaches a maximum at about 7 min. and then declines sharply. This suggests that the system of down regulation in EBV B lymphocytes is quite different. The integrated yield of O½ from activated EBV B lymphocytes is very considerable: it is similar to that from activated macrophages and could have significant biological effects.
Inhibitors of O~ Generation by EBV B Lymphocytes The effects of some potential inhibitors on O~ generation are summarised in Table 2. In addition to those compounds already mentioned, it is noteworthy that KCN at lmM was without effect in inhibiting the PMA induced respiratory burst although it was a potent inhibitor of lymphocyte respiration. This shows that the lymphocyte O1 generating oxidase is quite distinct from the mitochondrial electron transport system. Staurosporine, an inhibitor of protein kinase C (Tamaoki et al., 1986) inhibited the oxidase after activation by PMA, IL-1B, HAGG or by LPS. Forskolin, which increases intracellular cAMP did not activate the lymphocyte oxidase, indeed it appeared to diminish the effectiveness of PMA; such down regulation by cAMP has been found for the neutrophil respiratory burst (Nakagawara and Minikami, 1975). Diphenylene iodonium is (Cross and Jones, 1986) a good inhibitor of phagocyte NADPH oxidase in vivo and in vitro. Table 2. Effects of potential inhibitors on O~ generation by EBV-B lymphocytes Compound tested
Stimulus
Effect
Staurosporine
PMA LPS IL- 11] HAGG
Inhibition Inhibition Inhibition Inhibition
DPI CN" RO 4639 Forskolin PCMB
All stimuli PMA PMA PMA PMA
Inhibition No inhibition Inhibition Inhibition No inhibition
Hancock, Henderson and Jones, unpublished
It was equally as effective against the EBV B lymphocyte oxidase when added to cells activated by all the stimuli listed in Table 1. It did not inhibit the respiratory activity of the lymphocytes.
The Possible Functions of O~ Secreted by B kymphocytes The biological role of the O~ released by B lymphocytes is uncertain. There are reports that oxygen free radicals stimulate fibroblast proliferation (Murrell etal., 1989a) and antioxidants
Oxygen Radical Production
91
inhibit proliferation of T lymphocytes (Chaudri et al., 1986; Chaudri et al., 1988). Further, superoxide is produced by many cell types, including endothelial cells (Rosen and Freeman 1984; Gorog et al., 1988), fibroblasts (Meier et al., 1989; Murrell et al., 1989b), and platelets (Marcus, 1979). It is possible that superoxide radicals released by cell plasma membranes act as secondary messengers, affecting adjacent cells and perhaps initiating proliferation or transformation (see a review by Saran and Bors, 1989). They have a lifetime sufficient to act over short distances such as 55-3000 nm. In the case of B lymphocytes it is possible that radicals could also contribute to antigen processing, by oxidising susceptible amino acid residues, as well as to antigen-induced lymphocyte proliferation.
References Andersson, J. and A. Coutinho, W. Lernhardt, F. Melchers, (1977). Clonal growth and maturation to immunoglobulin section in vitro of every growth-inducible B-lymphocyte. Cell 10, 27-34. Badwey, J.A. and M.L. Kamovsky, (1986). Production of superoxide by phagocytic leukocytes: paradigm for stimulus response phenomena. Current Tooics in Cellular Regulation 28, 183-208.
A
Badwey, J.A. and M.L. Kamovsky, (1980). Active oxygen species and the function of phagocytic leukocytes. Arm, Rev. Bi0¢hem. 49, 695-726. Chaudri, G. and N.H. Hunt, I.A. Clark, R. Ceredig, (1988). Antioxidants inhibit proliferation and cell surface expression of receptors for interleukin-2 and transferrin in T lymphocytes stimulated with phorbol myristate acetate and ionomycin. Cell Immunol. 115,204-213. Chaudri, G. and I.A. Clark, N.H. Hunt, W.BH. Cowden, R. Ceredig, (1986). Effects of antioxidant on primary alloantigen-induced T cell activation and proliferation. J. Immungl. 137, 2646-2652. Cross, A.R. (1987). The inhibitory effects of some iodinium compounds on the supemxide generating system of neutrophils and their failure to inhibit diaphorase activity. Bioqhem. Pharmaqol. 36,489-493. Cross, A.R. and O.T.G. Jones (1986). The effect of the inhibitor diphenylene iodonium on the superoxide generating system of neutrophils. Bi0fhem, L 237 111-116. Cross, A.R. and J.F. Parkinson, O.T.G. Jones, (1985). Mechanism of the superoxide producing oxidase of neutrophils; 02 is necessary for the fast reduction of cytochrome b-245 by NADPH. Biochem. J. 226, 881-884. Cross, A.R. and O.T.G. Jones, R. Garcia, A.W. Segal, (1982). The association of FAD with the cytochrome b of human neutrophils. Biochem. J. 208, 759-763 Cross, A.R. and O.T.G. Jones, A.M. Harper, Segal, A.W. (1981) Oxidation-reduction properties of the cytochrome b found in the plasma membrane fraction of human neutrophils. Bipeh¢m, J. 194,599-606. De Franco, A.L. (1987). Molecular aspects of B-lymphocyte activation. Ann. Rev. Cell Biol. 3, 143-178. Hancock, J.T. and F.E. Maly, O.T.G. Jones, (1989). Properties of the superoxide-generating oxidase of Blymphocyte cell lines. Determination of Michaelis parameters. Biochem J..2~i2, 373-375.
92
O . T . G . Jones et al.
Henderson, L.M. and J.B. Chappell, O.T.G. Jones, (1989). Superoxide generation is inhibited by phospholipase A 2 inhibitors. Role for phospholipase A 2 in the activation of the NADPH oxidase. Biochem. J. 264,249-255. Maly, F-E. and O.T.G. Jones, J-F. Gauchat, A.R. Cross, A. Urwyler, C. Walker, C.A. Dahinden, A.L. De Weck, Nakamura, M. (1989) Superoxide-dependent NBT reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines. J. Immunol. 14...~2,1260-1267. Maly, F-E. and A.R. Cross, O.T.G. Jones, G. Wolf-Vorbeck, C. Walker, C.A. Dahinden, A.L. De Weck, (1988). The superoxide generating system of B cell lines. Structural homology with the phagocytic oxidase and triggering via surface ILl. J. lmmunol. 140, 2334-2339. Meier, B. and H.H. Radeke, S. Selle, M. Youens, H. Sies. K. Resch, G.G. Habermehl. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha. Biochem. J. 263, 539-545. Melinn, M. and H. McLaughlin, (1987). Nitroblue tetrazolium reduction in lymphocytes. J. Leukocyte Biol. 4_!1,325-329. Murrell, G. A.C. and M.J.O. Francis, L. Bromley, (1989). Fibroblasts release superoxide free radicals. Biocbem. Soc. Trans 17, 483-484. Stim by PMA. See also ibid. 482-483. Murrell, G.A.C. and M.J.O. Francis, L. Bromley, (1989). Cyclo-oxygenase and oxygen free radical stimulated fibroblast proliferation. 2. Fibroblasts release superoxide free radicals. 3. Oxygen free radicals stimulate fibroblast proliferation. Bi0qh¢m. ~;oc. Trans. 17,482-484. Nakagaware, A. and S. Minikami, (1975). Generation of superoxide anions by leukocytes treated with cytochalasin B. Biochem. Biophys. Res. Commun. 64, 760-767. Nathan, C.F. (1987). Secretory products of macrophages. J. Clin. Invest. 79, 319-326. Saran, M. and W. Bors, (1989). Oxygen radicals acting as chemical messengers: A hypothesis. Frog R~i, Res. Commun. 7,213-220. Segal, A.W. and A.R. Cross, N. Borregaard, N.H. Valerius, J.F. Soothill, O.T.G. Jones (1983). Absence of cytochrome b-245 in chronic granulomatous disease. A multicentre European evaluation of its incidence and relevance New En~. J. Med. 308,245-251. Tamaoki, T. and H. iVomoto, I. Takahashi, Y. Kato, N. Morimoto, F. Tomita, (1986). Staurosporine, a potent inhibitor of phospholipid/Ca2+ dependent protein Kinase. Bioghem. Bionhys. Res. Commun. 135, 397-402. Volkman, D.J. and E.S. Buescher, J.l. Gallin, A.S. Gauci, (1984). B cell lines as models for inherited phagocytic diseases: abnormal superoxide generation in chronic granulomatous disease and giant granules in Chediak-Hihashi syndrome. J, lmmunol. 133, 3006-3009. Yea, C.M. and A.R. Cross, O.T.G. Jones, (1990). Purification and some properties of the 45kDa diphenylene, iodonium-binding flavoprotein of neutrophils. BiQchcmJ. (in press).
0098-2997/91 $0.00 + .50 ~)1991 Pergamon Press plc.
OXYGEN RADICAL PRODUCTION BY TRANSFORMED B LYMPHOCYTES Owen T.G. Jones, John T. Hancock and Lydia M. Henderson Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
Introduction Superoxide (O~) is produced by phagocytic leukocytes (neutrophils, monocytes, macrophages and eosinophils) and contributes to the microbicidal and tumouricidal activities of these cells (Badwey and Karnovsky, 1980). It is becoming apparent that O~ can be produced by cells other than professional phagocytic cells, albeit at lower rates, where it may have functions yet to be determined. Volkman et al. (1984) found that EBV transformed B lymphocyte cell lines produced O~ when stimulated with phorbol myristate acetate (PMA), a potent activator of phagocyte O~ formation, although the rates of about 0.3nmol/min/107 cells were only 1/20 of those given by activated neutrophils (about 20 nmol/min/107 cells. This work with cell lines has been repeated (Melinn and McLaughlin, 1987; Maly et al., 1988; Hancock et al., 1989) and significantly higher activities of O1 generation found (see Fig. 1). Even in the absence of added PMA there is significant release of O~ by the lymphocytes. There is evidence that this activity is present even in normal, non-transformed B lymphocytes isolated from tonsils (May et al., 1989). Some properties of the B lymphocyte superoxide generating oxidase are described below.
Composition and Localisation of the B Lymphocyte Oxidase In disrupted cell preparations, oxidase activity was dependent upon the addition of NADPH or NADPH. The Km for NADPH was lower than for NADH in the three cell lines studied (Hancock etal., 1989) but varied between different cell lines. This may arise from small differences in composition of the oxidase or its orientation in the membrane. However, it is likely that the oxidase is an NADPH oxidase, as found in phagocytes. B cell lines which were active in producing O~ contained (Maly et al., 1988) low potential cytochrome b (cytochrome b-245 also called b558), a component of NADPH oxidase of neutrophils (Cross et al., 1981), and expressed mRNA for the Abbreviations: PMA, phorbol myristate acetate; DPI, diphenylene iodonium; EBV, Epstein-Barr, virus; HAGG, heat-aggregated immunoglobulin; 1L, interleukin, TNF, tumour necrosis factor.
87
88
O . T . G . Jones et al.
13chain of the cytochrome b (Maly et al., 1989). Use of an antibody specific for cytochrome b-245 indicated that the cytochrome was located on the plasma membrane of the B lymphocytes, (Maly et al., 1989), where the haem group could transfer electrons directly to 0 2 (Cross et al., 1982; Cross et al., 1985) releasing O2 to the outer face of the cell. Patients with chronic granulomatous disease have neutrophils which are incapable of generating 0 2 , when stimulated with PMA (Volkman etal., 1984) due to lack of cytochrome b-245 (Segal et al., 1983). EBV B lymphocytes from such patients also fail to produce O;~ when stimulated with PMA.
~
4--
m
~2
I
I
I
I
I
I
0
10
20
30
40
50
~1
60
Time (min) Fig. 1. Rate of superoxide production by EBV-B lymphocytes in response to PMA
NADPH oxidase of neutrophils is specifically inhibited by diphenylene iodonium (DPI) which covalently attaches to a protein with Mr of 45 kD (Cross and Jones, 1986; Cross, 1987) believed to be a flavoprotein component of the oxidase (Yea et al., 1990). The oxidase of EBV B lymphocytes is similarly inhibited by DPI, which also labels a 45 kD protein (Maly et al., 1988). No binding of DPI to 45 kD protein was found in non EBV-transformed B lymphocytes, which lacked the O~ generating oxidase. The similarity of components and reactivities of EBV B lymphocyte oxidase and of the neutrophil oxidase suggests that in lymphocytes the oxidase is located in the plasma membrane and has the following sequence, as proposed for the neutrophil oxidase by Cross et al. (1985): eeeNADPH --4 FAD ~ cytochrome b-245 ~ 0 2 --~ O~ protein (45kD)
Stimuli for Superoxide Generation by EBV B Lymphocytes The variety of activators O~ production is listed in Table 1. The cytokines, TNFt~, I1-18 and 116 are good activators of the oxidase; their usual role is to regulate proliferation and maturation of B lymphocytes. Tumour necrosis factor is a potent activator of NADPH oxidase of adherent neutrophils (Nathan 1987). Each of these cytokines mediates its action after binding to a specific receptor on B lymphocyte plasma membranes (De Franco, 1987).
Oxygen Radical Production
89
Protein A-bearing staphylococci (Pansorbin) activates NADPH oxidase of EBV B lymphocytes. Pansorbin is likely to act by causing a cross-linking of lymphocyte surface immunoglobulins IgG, IgM and IgE. Although antilgG induced the respiratory burst response in a B cell line, antilgM was ineffective (Maly etal., 1988). It is probable that cross-linking of surface immunoglobulins activates B cells by activating phospholiphase C, causing the release of inositol triphosphate (IP 3) from membrane phosphatidylinositol 4,5bisphosphate (PIP2), together with diacylglygerol (see review by De Franco, 1987). IP 3 causes the release of Ca 2+ from intracellular stores and Ca 2÷, with DAG, activates protein kinase C. The same mechanism appears to operate in B lymphocytes. This proposed mechanism for oxidase activation is supported by the use of inhibitors and ionophores on EBV B lymphocytes. Both A23187 and ionomycin, which make membranes permeable to Ca 2+, activate superoxide production by EBV B lymphocytes. (Our results here are in conflict with those of Volkman et al., 1984). Activation of the EBV B lymphocyte oxidase by heat aggregated immunoglobulin suggests that the B lymphocyte Fc receptor, like that of the neutrophil, is coupled to an activation system for the NADPH oxidase. In neutrophils activation of Fc receptors leads to release of IP 3 and increased intracellular Ca 2+ but the relationship is less clear in B lymphocytes (see review by De Franco, 1987). Bacterial lipopolysaccharide (LPS) is a B cell activator, causing proliferation and differentiation (Andersson et al., 1977). It is also a powerful activator of NADPH oxidase of macrophages and neutrophils and of EBV B lymphocytes. It is not clear whether LPS activates O1 production following binding to a receptor or through perturbation of the lymphocyte membrane by the lipid component of LPS. Membrane perturbing agents such as detergents and DCCD can activate neutrophil NADPH oxidase, perhaps by changing membrane fluidity. Table 1. Stimulifor O~ generationby EBV B lymphocytes (a)
Receptor-mediated TNF(x IL-113 IL-6 Protein A (Pansorbin) Heat aggregatedimmunoglobulin(HAGG) *Lipopolysaccharide(Re mutant)
(b)
Receptor-independent Phorbol myristateacetate(PMA) Fluoride (+A13+) Arachidonate Ca2+ ionophores(ionomycinor A23187)
Hancock, Hendersonand Jones, unpublished. Ineffectiveas activators were: FMLP, TNI],IL-lct, INFct,INFI3and opsonizedzymosan. The maximumrate of O~ production,given by PMA, was about 10 nmol/min/mgprotein. *Lipopolysaccharid¢may not require a receptor. We have found that arachidonic acid is a good activator of the EBV-B lymphocyte oxidase (Table 1) just as it is for neutrophils (Badwey and Karnovsky, 1986; Henderson et al., 1989). It is possible that arachidonic acid has an effect which is quite specific, acting as a second messenger within the membrane. Some support for this view is given by experiments with a variety of inhibitors of phospholipase A2, such as RO 4639, each of which inhibits O1 production by EBV B lymphocytes; the inhibition is overcome by the addition of arachidonate to the assay suspension. An involvement of a G protein in activation is suggested by the stimulatory activity of F-, together with A13+.
90
O.T.G. Jones et al.
Time Course of 0~, Generation EBV lymphocytes treated with a variety of stimuli release O~ over periods of 40-50 min. The response to PMA is shown in Fig. 1. The rate of O~ secretion continues to increase for about 30 rain. and then slowly declines over the next 30 mins. This contrasts with the response of neutrophils which reaches a maximum at about 7 min. and then declines sharply. This suggests that the system of down regulation in EBV B lymphocytes is quite different. The integrated yield of O½ from activated EBV B lymphocytes is very considerable: it is similar to that from activated macrophages and could have significant biological effects.
Inhibitors of O~ Generation by EBV B Lymphocytes The effects of some potential inhibitors on O~ generation are summarised in Table 2. In addition to those compounds already mentioned, it is noteworthy that KCN at lmM was without effect in inhibiting the PMA induced respiratory burst although it was a potent inhibitor of lymphocyte respiration. This shows that the lymphocyte O1 generating oxidase is quite distinct from the mitochondrial electron transport system. Staurosporine, an inhibitor of protein kinase C (Tamaoki et al., 1986) inhibited the oxidase after activation by PMA, IL-1B, HAGG or by LPS. Forskolin, which increases intracellular cAMP did not activate the lymphocyte oxidase, indeed it appeared to diminish the effectiveness of PMA; such down regulation by cAMP has been found for the neutrophil respiratory burst (Nakagawara and Minikami, 1975). Diphenylene iodonium is (Cross and Jones, 1986) a good inhibitor of phagocyte NADPH oxidase in vivo and in vitro. Table 2. Effects of potential inhibitors on O~ generation by EBV-B lymphocytes Compound tested
Stimulus
Effect
Staurosporine
PMA LPS IL- 11] HAGG
Inhibition Inhibition Inhibition Inhibition
DPI CN" RO 4639 Forskolin PCMB
All stimuli PMA PMA PMA PMA
Inhibition No inhibition Inhibition Inhibition No inhibition
Hancock, Henderson and Jones, unpublished
It was equally as effective against the EBV B lymphocyte oxidase when added to cells activated by all the stimuli listed in Table 1. It did not inhibit the respiratory activity of the lymphocytes.
The Possible Functions of O~ Secreted by B kymphocytes The biological role of the O~ released by B lymphocytes is uncertain. There are reports that oxygen free radicals stimulate fibroblast proliferation (Murrell etal., 1989a) and antioxidants
Oxygen Radical Production
91
inhibit proliferation of T lymphocytes (Chaudri et al., 1986; Chaudri et al., 1988). Further, superoxide is produced by many cell types, including endothelial cells (Rosen and Freeman 1984; Gorog et al., 1988), fibroblasts (Meier et al., 1989; Murrell et al., 1989b), and platelets (Marcus, 1979). It is possible that superoxide radicals released by cell plasma membranes act as secondary messengers, affecting adjacent cells and perhaps initiating proliferation or transformation (see a review by Saran and Bors, 1989). They have a lifetime sufficient to act over short distances such as 55-3000 nm. In the case of B lymphocytes it is possible that radicals could also contribute to antigen processing, by oxidising susceptible amino acid residues, as well as to antigen-induced lymphocyte proliferation.
References Andersson, J. and A. Coutinho, W. Lernhardt, F. Melchers, (1977). Clonal growth and maturation to immunoglobulin section in vitro of every growth-inducible B-lymphocyte. Cell 10, 27-34. Badwey, J.A. and M.L. Kamovsky, (1986). Production of superoxide by phagocytic leukocytes: paradigm for stimulus response phenomena. Current Tooics in Cellular Regulation 28, 183-208.
A
Badwey, J.A. and M.L. Kamovsky, (1980). Active oxygen species and the function of phagocytic leukocytes. Arm, Rev. Bi0¢hem. 49, 695-726. Chaudri, G. and N.H. Hunt, I.A. Clark, R. Ceredig, (1988). Antioxidants inhibit proliferation and cell surface expression of receptors for interleukin-2 and transferrin in T lymphocytes stimulated with phorbol myristate acetate and ionomycin. Cell Immunol. 115,204-213. Chaudri, G. and I.A. Clark, N.H. Hunt, W.BH. Cowden, R. Ceredig, (1986). Effects of antioxidant on primary alloantigen-induced T cell activation and proliferation. J. Immungl. 137, 2646-2652. Cross, A.R. (1987). The inhibitory effects of some iodinium compounds on the supemxide generating system of neutrophils and their failure to inhibit diaphorase activity. Bioqhem. Pharmaqol. 36,489-493. Cross, A.R. and O.T.G. Jones (1986). The effect of the inhibitor diphenylene iodonium on the superoxide generating system of neutrophils. Bi0fhem, L 237 111-116. Cross, A.R. and J.F. Parkinson, O.T.G. Jones, (1985). Mechanism of the superoxide producing oxidase of neutrophils; 02 is necessary for the fast reduction of cytochrome b-245 by NADPH. Biochem. J. 226, 881-884. Cross, A.R. and O.T.G. Jones, R. Garcia, A.W. Segal, (1982). The association of FAD with the cytochrome b of human neutrophils. Biochem. J. 208, 759-763 Cross, A.R. and O.T.G. Jones, A.M. Harper, Segal, A.W. (1981) Oxidation-reduction properties of the cytochrome b found in the plasma membrane fraction of human neutrophils. Bipeh¢m, J. 194,599-606. De Franco, A.L. (1987). Molecular aspects of B-lymphocyte activation. Ann. Rev. Cell Biol. 3, 143-178. Hancock, J.T. and F.E. Maly, O.T.G. Jones, (1989). Properties of the superoxide-generating oxidase of Blymphocyte cell lines. Determination of Michaelis parameters. Biochem J..2~i2, 373-375.
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O . T . G . Jones et al.
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