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Reactive oxygen species are required for the phagocytosis of myelin by macrophages
Journal of Neuroimmunology 92 Ž1998. 67–75
Reactive oxygen species are required for the phagocytosis of myelin by macrophages Annette van der Goes a,) , Jantien Brouwer a , Karin Hoekstra a , Dirk Roos b, Timo K. van den Berg a , Christine D. Dijkstra a a
Department of Cell Biology and Immunology, Faculty of Medicine, Vrije UniÕersiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands b Central Laboratory of the Netherlands Red Cross Blood Transfusion SerÕice and Laboratory of Experimental and Clinical Immunology, Academic Medical Center, UniÕersity of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands Received 12 May 1998; revised 7 July 1998; accepted 7 July 1998
Abstract Reactive oxygen species ŽROS. are thought to be involved in the pathogenesis of multiple sclerosis ŽMS. and experimental allergic encephalomyelitis ŽEAE.. In this study we showed that the phagocytosis of myelin by macrophages triggers the production of ROS. We also demonstrated that ROS play a crucial role in the myelin phagocytosis. Blocking the ROS production with NADPH oxidase inhibitors Ž100 mM DPI or 10 mM Apocynin. essentially prevented the phagocytosis of myelin. Furthermore, scavenging of ROS with catalase . ŽH 2 O 2 . or mannitol ŽOHy. decreased the phagocytosis of myelin by macrophages, whereas superoxide dismutase ŽOy 2 did not show this effect. In addition, Lipoic acid ŽLA., a non-specific scavenger of ROS, also decreased the phagocytosis of myelin by macrophages. In our results, we demonstrate for the first time that ROS appear to play a regulatory role in the phagocytosis of myelin. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Macrophages; Phagocytosis; Reactive oxygen species; Myelin; Demyelination
1. Introduction Multiple sclerosis ŽMS. is characterized by the occurrence of inflammatory demyelinating lesions in the central nervous system ŽCNS.. In experimental allergic encephalomyelitis ŽEAE., an animal model for multiple sclerosis ŽMS., it has been shown that blood-derived macrophages play a crucial role in the development of clinical symptoms ŽHuitinga et al., 1990.. Macrophages contribute to demyelination by their ability to phagocytose and degrade myelin. Macrophages present in the lesions are filled with myelin, according to histological staining of lesion material with antibodies against myelin proteins ŽBruck ¨ et al., 1995.. The exact mechanism of the phagocytosis of myelin by macrophages is not yet fully understood ŽSmith, 1993; Mosley and Cuzner, 1996; Van der Laan et al., 1996.. ROS are produced both in MS and in EAE ŽGlabinsky et al., 1993; Guy et al., 1993; Lin et al., 1993.. Further) Corresponding author. Tel.: q31 20 4448079; fax: q31 20 4448081; e-mail: [email protected]
more, scavengers of ROS have been shown to decrease the severity of clinical symptoms in EAE ŽHartung et al., 1988; Guy et al., 1989; Ruuls et al., 1995.. The cells responsible for production of these ROS are thought to be the macrophages or activated microglia cells present in the lesions ŽGriot et al., 1989; Mayer et al., 1991; Okuda et al., 1995; Ruuls et al., 1995.. Apart from the direct measurement of ROS in MS and EAE, also their consequences, i.e., the damage of ROS on lipid membranes by lipid peroxidation have been demonstrated in MS and EAE ŽToshniwal and Zarling, 1992; Brett and Rumsby, 1993; Glabinsky et al., 1993.. It has been shown that ROS can damage both the myelin sheath ŽChia et al., 1983; Arduini et al., 1985; Konat and Wiggins, 1985. and the blood-brain barrier ŽRubanyi, 1988.. Here, we describe the role of ROS in the phagocytosis of myelin by macrophages. We used an in vitro myelin phagocytosis assay described earlier ŽVan der Laan et al., 1996.. Fluorescentlabeled myelin was fed to macrophages in the presence or absence of various ROS scavengers. One of the scavengers used in this study is Lipoic acid ŽLA.. LA is a known scavenger of various ROS, such as hydroxyl radicals ŽOH . .
0165-5728r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 1 7 5 - 1
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and hypochlorous acid ŽHOCl. ŽSuzuki et al., 1991; Yan et al., 1996.. LA is used as a therapeutic agent in the treatment of polyneuropathy of diabetes ŽNickander et al., 1996.. Furthermore, ROS production by macrophages after myelin phagocytosis was determined with either a nitrobluetetrazolium ŽNBT. assay or a dihydrorhodamine ŽDHR. assay. From our results we hypothesize that myelin can trigger the NADPH oxidase complex of macrophages, which results in the production of ROS. These ROS can not only damage the myelin sheath, but can also activate the macrophage to phagocytose the myelin. Scavengers of ROS can prevent the phagocytosis of myelin, which might lead to a decrease of macrophage activation and damage to myelin.
BSA standard curve. The standard was prepared by diluting a stock of 2 mgrml BSA. A total of 50 ml of each standard and sample was added to 1 ml Bicinchoninic acid ŽBCA; Pierce, IL, USA. reagent and incubated for 30 min at 378C. Absorbency was measured at 562 nm. Myelin was labeled with the lipophilic fluorescent dye 1.1X-dioctadecyl3,3,3X ,3X-tetramethylindocarbocyanine perchlorate ŽDiI; Molecular Probes, Eugene, USA. by incubation at a final concentration of 12.5 mgrml DiI on 1 mgrml myelin for 30 min at 378C. Excess DiI was removed by washing with sterile PBS Ž15 min at 15,000 rpm.. Labeled myelin was stored in aliquots in the dark at y208C ŽVan der Laan et al., 1996..
2. Materials and methods
Peritoneal macrophages were washed and resuspended in culture medium ŽRPMI 1640 containing 10% FCS, penicillin Ž100 IUrml., streptomycin Ž50 mgrml. and 1 mM glutamine. at a concentration of 1 = 10 6 cellsrml. An total of 0.5 ml cell suspension was added to a well of a 24-well plate containing a round coverglass ŽB s 10 mm. and was incubated for 90 min at 378Cr5% CO 2 . Subsequently, non-adherent cells were removed and the macrophages were incubated for another 90 min in 0.5 ml RPMI 1640 medium containing 2% NRS and myelin Ž15 mgrwell. or PMA,a potent protein kinase activator Ž1 mgrml.. Non-ingested or non-bound myelin was removed by washing the wells twice with RPMI 1640 medium. The cells were incubated in 0.5 ml RPMI 1640 medium containing 2% NRS and 0.5 mgrml Nitrobluetetrazolium ŽNBT; Sigma, St. Louis, USA.. NBT acts as a non-specific electron acceptor in biochemical pathways activated during the oxidative burst, producing a blue formazan precipitate within each reactive cell. After an incubation period of 60 min the cells were washed 3 = with cold PBS and were fixed in 0.5 ml methanol Žy208C. for 10 min at RT.
2.1. Animals Adult male WagRij rats ŽDutch Cancer Institute, Amsterdam, Netherlands. were kept under conventional circumstances. Rats were sacrificed and the resting peritoneal macrophages were isolated by rinsing the peritoneal cavity 3 = with 10 ml RPMI 1640 ŽGibco.. The yield was approximately 12.5 = 10 6 cells per rat. Fresh normal rat serum ŽNRS., a source of complement, was prepared by centrifugation of clotted peripheral blood of the rats. 2.2. Preparation and labelling of myelin Myelin was prepared from brain tissue of adult WagRij according to the method of Norton and Poduslo Ž1973., by means of sucrose density–gradient centrifugation. The purified fractions were stored in small portions Ž100 ml. at y708C. To determine the amount of proteins in the isolated myelin, a sample of the myelin was compared to a
2.3. NBT assay
Fig. 1. Microphotographs of an NBT assay on peritoneal macrophages, either unstimulated ŽA. or stimulated with myelin ŽB.. A formazan precipitate is seen in the cells, that have produced ROS.
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Finally, the cells were counter-stained for 10 min with nuclear fast red and embedded in aquamount. 2.4. DHR assay Peritoneal macrophages were isolated and resuspended as described above. The cells were incubated in 24-well plates for 90 min at 378Cr5% CO 2 Ž5 = 10 5 cellsrwell.. Non-adherent cells were removed and the macrophages were pre-incubated in 0.5 ml RPMI 1640 medium containing 2% NRS with or without diphenyleneiodonium ŽDPI; Sigma. or apocynin ŽSigma. at 378Cr5% CO 2 . After 25 min 0.5 mM 1,2,3-DHR ŽMolecular Probes. was added and the cells were pre-incubated for another 10 min. DHR acts
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as a non-specific electron donor, producing the fluorescent rhodamine. Myelin Ž15 mgrwell., serum-treated zymosan Ž25 mgrml. or latex beads ŽB s 0.5 mm. were added and the cells were incubated for another 90 min at 378Cr5% CO 2 . Non-ingested or non-bound particles were removed by washing of the wells twice with RPMI and once with PBS. The macrophages were incubated for 30 min on ice with PBSr5 mM EDTA and detached by vigorous pipetting. The cells were washed and resuspended in PBSr0.05% BSA. Fluorescence intensity ŽFL1. was determined in a FACScan flowcytometer ŽBecton Dickinson. equipped with an Argon laser with excitation 488 nm and calibrated with Calibrite beads ŽBeckton Dickinson.. The percentage of positive cells was used as a measure for the
Fig. 2. ŽA. ROS production by peritoneal macrophages measured with a DHR assay. Phagocytosis of myelin and serum-treated zymosan results in significant activation of the oxidative burst Ž w p F 0.001., whereas the activation of peritoneal macrophages by latex beads is much less. Shown are the percentage of positive cells " S.E.M. Ž n s 4–6.. ŽB. ROS production by peritoneal macrophages measured with a DHR assay. Peritoneal macrophages were pre-incubated with DPI Ž100 mM., Apocynin Ž10 mM. or Allopurinol Ž100 mM. and were stimulated with myelin in the presence of 2% NRS. The figure shows the percentage of positive cells " S.E.M. DPI Ž n s 5. and apocynin Ž n s 3. both significantly blocked the oxidative burst Ž) p F 0.001., whereas allopurinol Ž n s 2. showed no effect on ROS production.
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Fig. 3. Peritoneal macrophages are pre-incubated with DPI Ž1–10–100 mM. or apocynin Ž1–5–10 mM. and stimulated with myelin, serum-treated zymosan or latex beads in the presence of 2% NRS. Note that DPI and apocynin significantly decreased the phagocytosis of myelin in a dose-dependent manner. DPI and apocynin affected phagocytosis of zymosan and latex beads to a lesser extent. Data are presented as the mean percentages of binding and uptake of the fluorescent particles" S.D. Ž n s 2, which preclude statistical analysis..
activity of the NADPH oxidase complex of the macrophages. Statistical analysis of the results was performed with the Students t-test. 2.5. Phagocytosis experiment Peritoneal macrophages were isolated and resuspended as described above. The cells were incubated in 24-well plates for 90 min at 378Cr5% CO 2 Ž5 = 10 5 cellsrwell.. Non-adherent cells were removed and the macrophages were incubated for another 90 min in 0.5 ml of RPMIr2% NRS containing indicated concentrations of the ROS scavengers catalase ŽSigma., superoxide dismutase ŽSigma., mannitol or Lipoic acid ŽLA. ŽASTA Medica, Frankfurt, Germany. as well as myelin-DiI Ž15 mgrwell.; FITClabeled serum-treated zymosan Ž25 mgrml. or FITClabeled latex beads Ž2 mlrwell.. For experiments with DPI and apocynin Žsee Section 2.4., the cells were pre-treated with the indicated concentrations of DPI and apocynin for
30 min at 378Cr5% CO 2 in the presence of 2% NRS. Non-ingested or non-bound particles were removed by washing of the wells twice with RPMI 1640 medium and once with cold PBS. The macrophages were detached as described before. Fluorescence intensity was determined in a FACScan flowcytometer. The mean fluorescence was used as a measure for binding and uptake of labeled particles and was expressed as a percentage of phagocytosis. Statistical analysis of the results was performed with the Students t-test. 3. Results 3.1. ReactiÕe oxygen species (ROS) production in macrophages is induced by myelin phagocytosis 3.1.1. Nitrobluetetrazolium (NBT) assay In order to measure the effect of myelin on ROS production by macrophages, the NBT assay was per-
y. Fig. 4. The role of the different reactive oxygen species ŽH 2 O 2 , Oy produced during the oxidative burst in the phagocytosis of myelin. Catalase, 2 , OH superoxide dismutase ŽSOD. and mannitol were used in given concentrations in the presence of 2% NRS. Data are presented as the mean percentages of binding and uptake of the fluorescent myelin" S.E.M. of four experiments. w p F 0.005.
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formed. In absence of myelin only an occasional peritoneal macrophage was weakly positive for the blue formazan NBT reduction product ŽNBT-positive. ŽFig. 1A.. Following incubation of peritoneal macrophages with myelin in the presence of 2% NRS, the cells Ž70–80%. became NBT-positive ŽFig. 1B.. Peritoneal macrophages incubated with PMA all were NBT-positive Ždata not shown.. In case of myelin Žbut not in case of PMA. the blue precipitate was usually concentrated on one side of the cell, probably corresponding to the localization of phagocytosed particles.
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3.1.2. Dihydrorhodamine (DHR) assay To confirm and extend the effect of myelin on ROS production by macrophages, a quantitative measurement of the intracellular oxidative burst activity the dihydrorhodamine ŽDHR. assay was performed. As was seen with the NBT assay, the DHR assay also clearly showed a myelininduced oxidative burst in peritoneal macrophages ŽFig. 2A.. Apart from myelin, serum-treated zymosan was also able to trigger the oxidative burst, while latex beads were not able to get the peritoneal macrophages to produce ROS ŽFig. 2A.
Fig. 5. ŽA. The ROS scavenger LA shows a dose-dependent effect on the binding and uptake of fluorescent myelin in the presence of 2% NRS. Results are shown from one representative experiment Ž n s 4.. Data are presented as the mean percentages of binding and uptake of the fluorescent myelin. ŽB. The effect of LA on the phagocytosis of various particles. Peritoneal macrophages were activated with myelin, serum-treated zymosan or latex beads in the presence of LA Ž1 mgrml. and 2% NRS. Although LA significantly decreased the phagocytosis of all particles Ž w p F 0.05., the effect of LA on the binding and uptake of myelin is more significant Ž p - 0.0001. than on the binding and uptake of zymosan Ž p s 0.003. or latex beads Ž p s 0.05..
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Previous studies have shown that the NADPH oxidase complex plays a major role in ROS production in macrophages. To determine whether this enzyme is involved in the myelin induced ROS production the NADPH oxidase complex was blocked with DPI or apocynin. Both are specific inhibitors of the NADPH oxidase complex. DPI blocks a flavocytochrome ŽTakao et al., 1996., whereas apocynin blocks the assembly of the NADPH oxidase complex ŽStolk et al., 1994.. Furthermore Allopurinol, a specific inhibitor of xanthine oxidase, was used to determine whether xanthine oxidase is involved in the myelin induced ROS production. Both inhibitors of the NADPH oxidase complex decreased the oxidative burst activity to background levels ŽFig. 2B.. Allopurinol did not show this decrease of the oxidative burst activity ŽFig. 2B.. 3.2. The role of ROS on phagocytosis of myelin by macrophages 3.2.1. NADPH oxidase complex inhibitors The above show that the oxidative burst is activated by binding andror phagocytosis of myelin by macrophages. The induction of an oxidative burst in macrophages after phagocytosis is a well-described phenomenon ŽJohnston and Kitagawa, 1985; Winrow et al., 1993.. However, nothing is known about the requirement of ROS for the phagocytosis of myelin by macrophages. In the above experiments we showed that the oxidative burst in rat macrophages could be blocked by the NADPH oxidase inhibitors DPI Ž100 mM. and apocynin Ž10 mM.. In order to investigate the role of NADPH oxidase complex inhibitors in myelin phagocytosis the bindingrinternalization of DiI-labeled myelin was investigated in the presence of these inhibitors. Pre-treatment of peritoneal macrophages with DPI or apocynin in indicated concentrations resulted in a concen-
tration dependent inhibition of myelin phagocytosis ŽFig. 3.. Similar results, although perhaps somewhat less profound, were obtained when the NADPH oxidase complex inhibitors were tested on serum-treated zymosan and latex beads phagocytosis. Allopurinol did not block the phagocytosis of myelin, serum-treated zymosan or latex beads Ždata not shown.. 3.2.2. ReactiÕe oxygen species (ROS) scaÕengers Inhibiting the NADPH oxidase complex can block the phagocytosis of myelin, as shown in Fig. 3. The next step was to determine which product of the NADPH oxidase complex is involved in the phagocytosis of myelin. The products of the NADPH oxidase complex can be scavenged with superoxide dismutase ŽSOD., which converts . Ž . superoxide ŽOy 2 into hydrogen peroxide H 2 O 2 , catalase, a scavenger of hydrogen peroxide ŽH 2 O 2 . or mannitol, a scavenger of hydroxyl radicals ŽOHy. . The scavengers were added to the peritoneal macrophages. SOD did not decrease the phagocytosis of myelin by macrophages, whereas catalase and mannitol in a dose-dependent manner decreased the phagocytosis to background levels ŽFig. 4.. 3.3. The effect of Lipoic acid on the phagocytosis of myelin Lipoic acid ŽLA. is a non-specific scavenger of ROS. It is able to scavenge not only OHy and HOCl, but also other ROS. The reduced form of LA, dihydrolipoic acid ŽDHLA., is also involved in the vitamin E cycle and has been described as an inhibitor of NFk B. LA decreased the phagocytosis of myelin in a dose dependent manner ŽFig. 5A.. To compare the effect of LA on the phagocytosis of myelin with that of other particles, macrophages were
Fig. 6. LA shows a time-dependent effect on the binding and uptake of fluorescent myelin. Myelin was incubated with peritoneal macrophages in the presence of 2% NRS. Right from the beginning of the experiment LA Ž1 mgrml. was added to a well every 15 min. The longer LA had been present in the media, the larger the inhibition of binding and uptake of fluorescent myelin Ž n s 3.. w p F 0.001.
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incubated with myelin-DiI, FITC-labeled serum-treated zymosan or FITC-labeled latex beads in the presence of LA Ž1 mgrml. ŽFig. 5B.. Although LA decreased the phagocytosis of both serum-treated zymosan and latex beads, the inhibition of the phagocytosis of myelin by LA was more pronounced. We also investigated the time-dependency of this process. Peritoneal macrophages were incubated with myelin and after different intervals LA Ž1 mgrml. was added to the wells. Fig. 6 shows the time dependent effect of LA on the phagocytosis of myelin. The phagocytosis of myelin by peritoneal macrophages decreases with the time that LA is present in the media. This shows that a complete inhibition requires the continuous presence of LA, which is compatible with a possible role as a scavenger of ROS.
4. Discussion ROS are thought to be involved in the pathogenesis of EAE and MS ŽHartung et al., 1988; Guy et al., 1989; Glabinsky et al., 1993; Hansen et al., 1995; Malfroy et al., 1997.. ROS are not only able to damage CNS tissue, such as endothelial cells of the blood-brain barrier ŽRubanyi, 1988., but they can also damage the myelin sheath ŽChia et al., 1983; Arduini et al., 1985; Konat and Wiggins, 1985.. In this study we show that ROS are involved in the phagocytosis of myelin by macrophages. Several enzymes are capable of producing ROS, such as xanthine oxidase and NADPH oxidase. NADPH oxidase is a membrane-bound multi-component electron transfer chain involving a flavocytochrome. The switch for activation of the NADPH oxidase complex is induced by a conformational change in the flavocytochrome. Diphenyleneiodonium ŽDPI. inhibits the NADPH oxidase complex at the level of the flavocytochrome ŽTakao et al., 1996., whereas apocynin inhibits the assembly of the different components of the NADPH oxidase complex ŽStolk et al., 1994.. Because both NADPH oxidase inhibitors DPI and apocynin decreased the amount of ROS produced by the macrophages and the xanthine oxidase inhibitor allopurinol did not inhibit the production of ROS ŽFig. 2B., it is likely that only the NADPH oxidase complex is the main if not the only source of ROS in our cells. This process is called the respiratory or oxidative burst ŽBabior, 1984.. Although we did not measure ROS production in macrophages stimulated with latex beads, DPI and apocynin inhibited the phagocytosis of latex beads. It is probable that even small extracellularly non-detectable amounts of ROS are able to trigger signal transduction. Scavenging of the ROS produced by the NADPH oxidase complex with catalase or mannitol decreased the phagocytosis of myelin by macrophages, whereas superoxide dismutase ŽSOD. did not show this effect ŽFig. 4A.. Therefore, we conclude that hydrogen peroxide and hy-
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droxyl radicals are the ROS involved in the phagocytosis of myelin. The question whether hypochlorous acid is involved in the phagocytosis of myelin remains to be resolved. In several in vivo studies it has been shown that catalase can decrease the severity of the clinical signs in EAE of Lewis rats ŽRuuls et al., 1995. and that it can decrease the demyelination in EAN in guinea-pigs ŽGuy et al., 1989.. In both studies, SOD had little effect, although Hartung et al. Ž1988. showed an effect of both catalase and SOD in EAN in Lewis rats. Guy et al. Ž1989. have previously proposed that this effect of SOD could actually be due to preservatives, present in the SOD, which may function as antioxidants. The role of catalase and SOD on the phagocytosis of bacteria has been examined by Takao et al. Ž1996.. These investigators showed that the murine peritoneal macrophage cell line IC-21 was able to phagocytose bacteria in the presence of catalase Ž100–500 Urml. or SOD Ž100–500 Urml., while the killing of the bacteria was inhibited by the addition of SOD and catalase. We observed no effect of catalase in these concentrations, but instead needed concentrations of 5000 Urml to inhibit the phagocytosis of myelin in our system. This is probably due to the fact that ROS are produced continuously during the full 90 min of the experiment. Our observation that a complete blocking of phagocytosis required scavengers to be present throughout the experiment is in line with this ŽFig. 6.. To be able to scavenge the ROS produced over the whole experiment high concentrations of scavengers may be necessary. We also used Lipoic acid ŽLA. to scavenge ROS produced during the phagocytosis of myelin ŽFig. 4B.. The exact mechanism by which LA influence myelin phagocytosis is not extremely clear at the moment. LA can be reduced to dihydrolipoic acid ŽDHLA., both LA and DHLA are antioxidants Žfor a review, see Packer et al., 1995.. LA scavenges hydroxyl radicals, hypochlorous acid, nitric oxide hydrogen peroxide and singlet oxygen. It also chelates transition metals. DHLA is an even more potent scavenger. DHLA is able to protect membranes to lipid peroxidation by regenerating vitamin E. Furthermore, LA and DHLA have an effect on signal transduction by inhibiting NFk B activation. In a cultured human T lymphocyte Jurkat cell line LA administration resulted in intracellular DHLA formation ŽHandelman et al., 1994.. Thus, also in our system, LA and DHLA may be present both intracellularly and extracellularly. How are ROS involved in the phagocytosis? Macrophages as well as microglia produce ROS during phagocytosis ŽWilliams et al., 1994; Mosley and Cuzner, 1996.. Phagocytosis does not need to be completed, because contact between the phagocyte and the stimulating particle is enough to stimulate the oxidative burst ŽGoldstein et al., 1975.. Furthermore, the literature to date has shown that ROS can play an essential role in signal transduction Žfor a review, see Lander, 1997.. From our
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results it appears that the products of the oxidative burst can not only damage the myelin sheath, but can also activate the macrophage to phagocytose the myelin. Gamaley et al. Ž1994. showed that mouse peritoneal macrophages activated with 10 mM H 2 O 2 phagocytosed twice as much as without pre-incubation with H 2 O 2 . We have shown in this study that LA was relatively potent in the inhibition of myelin phagocytosis as compared to serum-treated zymosan or latex beads phagocytosis ŽFig. 5B.. This may be due to the fact that ROS can modify both macrophages and myelin, and both can be prevented by addition of LA. In the future, it will be interesting to test the effect of LA on demyelination in vivo.
Acknowledgements The authors wish to thank Christa Homburg and Petra Hilarius for their help with the NBT assay, and Prof. A. Bast for valuable discussion concerning LA. This work was supported financially by the Dutch foundation ‘Vrienden MS Research’, project MS 95-202.
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Hansen, L.A., Willenborg, D.O., Cowden, W.B., 1995. Suppression of hyperacute and passively transferred experimental autoimmune encephalomyelitis by the anti-oxidant, butylated hydroxyanisole. J. Neuroimmunol. 62, 69–77. Hartung, H.P., Schafer, B., Heininger, K., Toyka, K.V., 1988. Suppres¨ sion of experimental autoimmune neuritis by the radical scavengers superoxide dismutase and catalase. Ann. Neurol. 23, 453–460. Huitinga, I., Rooijen, N.v., de Groot, C.J.A., Uitdehaag, B.M.J., Dijkstra, C.D., 1990. Suppression of experimental allergic encephalomyelitis after elimination of macrophages. J. Exp. Med. 172, 1025–1033. Johnston, R.B., Kitagawa, S., 1985. Molecular basis for the enhanced respiratory burst of activated macrophages. Fed. Proc. 44. Konat, G.W., Wiggins, R.C., 1985. Effect of reactive oxygen species on myelin membrane proteins. J. Neurochem. 45, 1113–1118. Lander, H.M., 1997. An essential role for free radicals and derived species in signal transduction. FASEB J. 11, 118–124. Lin, R.F., Lin, T.S., Tilton, R.G., Cross, A.H., 1993. Nitric oxide localized to spinal cords of mice with experimental allergic encephalomyelitis: an electron paramagnetic resonance study. J. Exp. Med. 178, 643–648. Malfroy, B., Doctrow, S.R., Orr, P.L., Tocco, G., Fedoseyeva, E.V., Benichou, G., 1997. Prevention and suppression of autoimmune encephalomyelitis by EUK-8, a synthetic catalytic scavenger of oxygen-reactive metabolites. Cell. Immunol. 177, 62–68. Mayer, M., Urbanek, K., Hajduch, ´ ˆ M., 1991. Luminol-dependent chemiluminiscence of monocytes and granulocytes in multiple sclerosis: the effect of autologous thrombocytes. Folia Biol. 37, 213–223. Mosley, K., Cuzner, M.L., 1996. Receptor-mediated phagocytosis of myelin by macrophages and microglia: effect of opsonization and receptor blocking agents. Neurochem. Res. 21, 481–487. Nickander, K.K., McPhee, B.R., Low, P.A., Tritschler, H., 1996. Alphalipoic acid: antioxidant potency against lipid peroxidation of neural tissues in vitro and implication for diabetic neuropathy. Free Radic. Biol. Med. 21, 631–639. Norton, W.T., Poduslo, S.E., 1973. Myelination in rat brain: method of myelin isolation. J. Neurochem. 21, 749–757. Okuda, Y., Nakatsuji, Y., Fyjimura, H., Esumi, H., Ogura, T., Yanagihara, T., Sakoda, S., 1995. Expression of the inducible isoform of nitric oxide synthase in the central nervous system of mice correlates with the severity of actively induced experimental allergic encephalomyelitis. J. Neuroimmunol. 62, 103–112. Packer, L., Witt, E.H., Tritschler, H.J., 1995. Alpha-lipoic acid as a biological antioxidant. Free Radic. Biol. Med. 19, 227–250. Rubanyi, G.M., 1988. Vascular effects of oxygen-derived free radical. Free Radic. Biol. Med. 4, 107–120. Ruuls, S.R., Bauer, J., Sontrop, K., Huitinga, I., ’t Hart, B.A., Dijkstra, C.D., 1995. Reactive oxygen species are involved in the pathogenesis of experimental allergic encephalomyelitis. J. Neuroimmunol. 56, 207–217. Smith, M.E., 1993. Phagocytosis of myelin by microglia in vitro. J. Neurosci. Res. 35, 480–487. Stolk, J., Hilterman, T.J.N., Dijkman, J.H., Verhoeven, A.J., 1994. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am. J. Respir. Cell Mol. Biol. 11, 95–102. Suzuki, Y.J., Tsuchiya, M., Packer, L., 1991. Thioctic acid and dihydrolipoic acid are novel antioxidants which interact with reactive oxygen species. Free Radic. Res. Comms. 15, 255–263. Takao, S., Smith, E.H., Wang, D., Chan, C.K., Bulkley, G.B., Klein, A.S., 1996. Role of reactive oxygen metabolites in murine peritoneal macrophage phagocytosis and phagocytic killing. Am. J. Physiol. 271, C1274–C1284. Toshniwal, P.K., Zarling, E.J., 1992. Evidence for increased lipid peroxidation in multiple sclerosis. Neurochem. Res. 17, 205–207. Van der Laan, L.J.W., Ruuls, S.R., Weber, K.S., Lodder, I.J., Dopp, ¨ E.A., Dijkstra, C.D., 1996. Macrophage phagocytosis of myelin in vitro determined by flow cytometry: phagocytosis is mediated by CR3
A. Õan der Goes et al.r Journal of Neuroimmunology 92 (1998) 67–75 and induces production of TNF-alpha and nitric oxide. J. Neuroimmunol. 70, 145–152. Williams, K., Ulvestad, E., Waage, A., Antel, J.P., McLaurin, J., 1994. Activation of adult human derived microglia by myelin phagocytosis in vitro. J. Neurosci. Res. 38, 433–443. Winrow, V.R., Winyard, P.G., Morris, C.J., Blake, D.R., 1993. Free
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radical in inflammation: second messengers and mediators of tissue destruction. Br. Med. Bull. 49, 506–522. Yan, L.J., Traber, M.G., Kobuchi, H., Matsugo, S., Tritschler, H.J., Packer, L., 1996. Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation. Arch. Biochem. Biophys. 327, 330–334.
Reactive oxygen species are required for the phagocytosis of myelin by macrophages Annette van der Goes a,) , Jantien Brouwer a , Karin Hoekstra a , Dirk Roos b, Timo K. van den Berg a , Christine D. Dijkstra a a
Department of Cell Biology and Immunology, Faculty of Medicine, Vrije UniÕersiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands b Central Laboratory of the Netherlands Red Cross Blood Transfusion SerÕice and Laboratory of Experimental and Clinical Immunology, Academic Medical Center, UniÕersity of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands Received 12 May 1998; revised 7 July 1998; accepted 7 July 1998
Abstract Reactive oxygen species ŽROS. are thought to be involved in the pathogenesis of multiple sclerosis ŽMS. and experimental allergic encephalomyelitis ŽEAE.. In this study we showed that the phagocytosis of myelin by macrophages triggers the production of ROS. We also demonstrated that ROS play a crucial role in the myelin phagocytosis. Blocking the ROS production with NADPH oxidase inhibitors Ž100 mM DPI or 10 mM Apocynin. essentially prevented the phagocytosis of myelin. Furthermore, scavenging of ROS with catalase . ŽH 2 O 2 . or mannitol ŽOHy. decreased the phagocytosis of myelin by macrophages, whereas superoxide dismutase ŽOy 2 did not show this effect. In addition, Lipoic acid ŽLA., a non-specific scavenger of ROS, also decreased the phagocytosis of myelin by macrophages. In our results, we demonstrate for the first time that ROS appear to play a regulatory role in the phagocytosis of myelin. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Macrophages; Phagocytosis; Reactive oxygen species; Myelin; Demyelination
1. Introduction Multiple sclerosis ŽMS. is characterized by the occurrence of inflammatory demyelinating lesions in the central nervous system ŽCNS.. In experimental allergic encephalomyelitis ŽEAE., an animal model for multiple sclerosis ŽMS., it has been shown that blood-derived macrophages play a crucial role in the development of clinical symptoms ŽHuitinga et al., 1990.. Macrophages contribute to demyelination by their ability to phagocytose and degrade myelin. Macrophages present in the lesions are filled with myelin, according to histological staining of lesion material with antibodies against myelin proteins ŽBruck ¨ et al., 1995.. The exact mechanism of the phagocytosis of myelin by macrophages is not yet fully understood ŽSmith, 1993; Mosley and Cuzner, 1996; Van der Laan et al., 1996.. ROS are produced both in MS and in EAE ŽGlabinsky et al., 1993; Guy et al., 1993; Lin et al., 1993.. Further) Corresponding author. Tel.: q31 20 4448079; fax: q31 20 4448081; e-mail: [email protected]
more, scavengers of ROS have been shown to decrease the severity of clinical symptoms in EAE ŽHartung et al., 1988; Guy et al., 1989; Ruuls et al., 1995.. The cells responsible for production of these ROS are thought to be the macrophages or activated microglia cells present in the lesions ŽGriot et al., 1989; Mayer et al., 1991; Okuda et al., 1995; Ruuls et al., 1995.. Apart from the direct measurement of ROS in MS and EAE, also their consequences, i.e., the damage of ROS on lipid membranes by lipid peroxidation have been demonstrated in MS and EAE ŽToshniwal and Zarling, 1992; Brett and Rumsby, 1993; Glabinsky et al., 1993.. It has been shown that ROS can damage both the myelin sheath ŽChia et al., 1983; Arduini et al., 1985; Konat and Wiggins, 1985. and the blood-brain barrier ŽRubanyi, 1988.. Here, we describe the role of ROS in the phagocytosis of myelin by macrophages. We used an in vitro myelin phagocytosis assay described earlier ŽVan der Laan et al., 1996.. Fluorescentlabeled myelin was fed to macrophages in the presence or absence of various ROS scavengers. One of the scavengers used in this study is Lipoic acid ŽLA.. LA is a known scavenger of various ROS, such as hydroxyl radicals ŽOH . .
0165-5728r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 1 7 5 - 1
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and hypochlorous acid ŽHOCl. ŽSuzuki et al., 1991; Yan et al., 1996.. LA is used as a therapeutic agent in the treatment of polyneuropathy of diabetes ŽNickander et al., 1996.. Furthermore, ROS production by macrophages after myelin phagocytosis was determined with either a nitrobluetetrazolium ŽNBT. assay or a dihydrorhodamine ŽDHR. assay. From our results we hypothesize that myelin can trigger the NADPH oxidase complex of macrophages, which results in the production of ROS. These ROS can not only damage the myelin sheath, but can also activate the macrophage to phagocytose the myelin. Scavengers of ROS can prevent the phagocytosis of myelin, which might lead to a decrease of macrophage activation and damage to myelin.
BSA standard curve. The standard was prepared by diluting a stock of 2 mgrml BSA. A total of 50 ml of each standard and sample was added to 1 ml Bicinchoninic acid ŽBCA; Pierce, IL, USA. reagent and incubated for 30 min at 378C. Absorbency was measured at 562 nm. Myelin was labeled with the lipophilic fluorescent dye 1.1X-dioctadecyl3,3,3X ,3X-tetramethylindocarbocyanine perchlorate ŽDiI; Molecular Probes, Eugene, USA. by incubation at a final concentration of 12.5 mgrml DiI on 1 mgrml myelin for 30 min at 378C. Excess DiI was removed by washing with sterile PBS Ž15 min at 15,000 rpm.. Labeled myelin was stored in aliquots in the dark at y208C ŽVan der Laan et al., 1996..
2. Materials and methods
Peritoneal macrophages were washed and resuspended in culture medium ŽRPMI 1640 containing 10% FCS, penicillin Ž100 IUrml., streptomycin Ž50 mgrml. and 1 mM glutamine. at a concentration of 1 = 10 6 cellsrml. An total of 0.5 ml cell suspension was added to a well of a 24-well plate containing a round coverglass ŽB s 10 mm. and was incubated for 90 min at 378Cr5% CO 2 . Subsequently, non-adherent cells were removed and the macrophages were incubated for another 90 min in 0.5 ml RPMI 1640 medium containing 2% NRS and myelin Ž15 mgrwell. or PMA,a potent protein kinase activator Ž1 mgrml.. Non-ingested or non-bound myelin was removed by washing the wells twice with RPMI 1640 medium. The cells were incubated in 0.5 ml RPMI 1640 medium containing 2% NRS and 0.5 mgrml Nitrobluetetrazolium ŽNBT; Sigma, St. Louis, USA.. NBT acts as a non-specific electron acceptor in biochemical pathways activated during the oxidative burst, producing a blue formazan precipitate within each reactive cell. After an incubation period of 60 min the cells were washed 3 = with cold PBS and were fixed in 0.5 ml methanol Žy208C. for 10 min at RT.
2.1. Animals Adult male WagRij rats ŽDutch Cancer Institute, Amsterdam, Netherlands. were kept under conventional circumstances. Rats were sacrificed and the resting peritoneal macrophages were isolated by rinsing the peritoneal cavity 3 = with 10 ml RPMI 1640 ŽGibco.. The yield was approximately 12.5 = 10 6 cells per rat. Fresh normal rat serum ŽNRS., a source of complement, was prepared by centrifugation of clotted peripheral blood of the rats. 2.2. Preparation and labelling of myelin Myelin was prepared from brain tissue of adult WagRij according to the method of Norton and Poduslo Ž1973., by means of sucrose density–gradient centrifugation. The purified fractions were stored in small portions Ž100 ml. at y708C. To determine the amount of proteins in the isolated myelin, a sample of the myelin was compared to a
2.3. NBT assay
Fig. 1. Microphotographs of an NBT assay on peritoneal macrophages, either unstimulated ŽA. or stimulated with myelin ŽB.. A formazan precipitate is seen in the cells, that have produced ROS.
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Finally, the cells were counter-stained for 10 min with nuclear fast red and embedded in aquamount. 2.4. DHR assay Peritoneal macrophages were isolated and resuspended as described above. The cells were incubated in 24-well plates for 90 min at 378Cr5% CO 2 Ž5 = 10 5 cellsrwell.. Non-adherent cells were removed and the macrophages were pre-incubated in 0.5 ml RPMI 1640 medium containing 2% NRS with or without diphenyleneiodonium ŽDPI; Sigma. or apocynin ŽSigma. at 378Cr5% CO 2 . After 25 min 0.5 mM 1,2,3-DHR ŽMolecular Probes. was added and the cells were pre-incubated for another 10 min. DHR acts
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as a non-specific electron donor, producing the fluorescent rhodamine. Myelin Ž15 mgrwell., serum-treated zymosan Ž25 mgrml. or latex beads ŽB s 0.5 mm. were added and the cells were incubated for another 90 min at 378Cr5% CO 2 . Non-ingested or non-bound particles were removed by washing of the wells twice with RPMI and once with PBS. The macrophages were incubated for 30 min on ice with PBSr5 mM EDTA and detached by vigorous pipetting. The cells were washed and resuspended in PBSr0.05% BSA. Fluorescence intensity ŽFL1. was determined in a FACScan flowcytometer ŽBecton Dickinson. equipped with an Argon laser with excitation 488 nm and calibrated with Calibrite beads ŽBeckton Dickinson.. The percentage of positive cells was used as a measure for the
Fig. 2. ŽA. ROS production by peritoneal macrophages measured with a DHR assay. Phagocytosis of myelin and serum-treated zymosan results in significant activation of the oxidative burst Ž w p F 0.001., whereas the activation of peritoneal macrophages by latex beads is much less. Shown are the percentage of positive cells " S.E.M. Ž n s 4–6.. ŽB. ROS production by peritoneal macrophages measured with a DHR assay. Peritoneal macrophages were pre-incubated with DPI Ž100 mM., Apocynin Ž10 mM. or Allopurinol Ž100 mM. and were stimulated with myelin in the presence of 2% NRS. The figure shows the percentage of positive cells " S.E.M. DPI Ž n s 5. and apocynin Ž n s 3. both significantly blocked the oxidative burst Ž) p F 0.001., whereas allopurinol Ž n s 2. showed no effect on ROS production.
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Fig. 3. Peritoneal macrophages are pre-incubated with DPI Ž1–10–100 mM. or apocynin Ž1–5–10 mM. and stimulated with myelin, serum-treated zymosan or latex beads in the presence of 2% NRS. Note that DPI and apocynin significantly decreased the phagocytosis of myelin in a dose-dependent manner. DPI and apocynin affected phagocytosis of zymosan and latex beads to a lesser extent. Data are presented as the mean percentages of binding and uptake of the fluorescent particles" S.D. Ž n s 2, which preclude statistical analysis..
activity of the NADPH oxidase complex of the macrophages. Statistical analysis of the results was performed with the Students t-test. 2.5. Phagocytosis experiment Peritoneal macrophages were isolated and resuspended as described above. The cells were incubated in 24-well plates for 90 min at 378Cr5% CO 2 Ž5 = 10 5 cellsrwell.. Non-adherent cells were removed and the macrophages were incubated for another 90 min in 0.5 ml of RPMIr2% NRS containing indicated concentrations of the ROS scavengers catalase ŽSigma., superoxide dismutase ŽSigma., mannitol or Lipoic acid ŽLA. ŽASTA Medica, Frankfurt, Germany. as well as myelin-DiI Ž15 mgrwell.; FITClabeled serum-treated zymosan Ž25 mgrml. or FITClabeled latex beads Ž2 mlrwell.. For experiments with DPI and apocynin Žsee Section 2.4., the cells were pre-treated with the indicated concentrations of DPI and apocynin for
30 min at 378Cr5% CO 2 in the presence of 2% NRS. Non-ingested or non-bound particles were removed by washing of the wells twice with RPMI 1640 medium and once with cold PBS. The macrophages were detached as described before. Fluorescence intensity was determined in a FACScan flowcytometer. The mean fluorescence was used as a measure for binding and uptake of labeled particles and was expressed as a percentage of phagocytosis. Statistical analysis of the results was performed with the Students t-test. 3. Results 3.1. ReactiÕe oxygen species (ROS) production in macrophages is induced by myelin phagocytosis 3.1.1. Nitrobluetetrazolium (NBT) assay In order to measure the effect of myelin on ROS production by macrophages, the NBT assay was per-
y. Fig. 4. The role of the different reactive oxygen species ŽH 2 O 2 , Oy produced during the oxidative burst in the phagocytosis of myelin. Catalase, 2 , OH superoxide dismutase ŽSOD. and mannitol were used in given concentrations in the presence of 2% NRS. Data are presented as the mean percentages of binding and uptake of the fluorescent myelin" S.E.M. of four experiments. w p F 0.005.
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formed. In absence of myelin only an occasional peritoneal macrophage was weakly positive for the blue formazan NBT reduction product ŽNBT-positive. ŽFig. 1A.. Following incubation of peritoneal macrophages with myelin in the presence of 2% NRS, the cells Ž70–80%. became NBT-positive ŽFig. 1B.. Peritoneal macrophages incubated with PMA all were NBT-positive Ždata not shown.. In case of myelin Žbut not in case of PMA. the blue precipitate was usually concentrated on one side of the cell, probably corresponding to the localization of phagocytosed particles.
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3.1.2. Dihydrorhodamine (DHR) assay To confirm and extend the effect of myelin on ROS production by macrophages, a quantitative measurement of the intracellular oxidative burst activity the dihydrorhodamine ŽDHR. assay was performed. As was seen with the NBT assay, the DHR assay also clearly showed a myelininduced oxidative burst in peritoneal macrophages ŽFig. 2A.. Apart from myelin, serum-treated zymosan was also able to trigger the oxidative burst, while latex beads were not able to get the peritoneal macrophages to produce ROS ŽFig. 2A.
Fig. 5. ŽA. The ROS scavenger LA shows a dose-dependent effect on the binding and uptake of fluorescent myelin in the presence of 2% NRS. Results are shown from one representative experiment Ž n s 4.. Data are presented as the mean percentages of binding and uptake of the fluorescent myelin. ŽB. The effect of LA on the phagocytosis of various particles. Peritoneal macrophages were activated with myelin, serum-treated zymosan or latex beads in the presence of LA Ž1 mgrml. and 2% NRS. Although LA significantly decreased the phagocytosis of all particles Ž w p F 0.05., the effect of LA on the binding and uptake of myelin is more significant Ž p - 0.0001. than on the binding and uptake of zymosan Ž p s 0.003. or latex beads Ž p s 0.05..
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Previous studies have shown that the NADPH oxidase complex plays a major role in ROS production in macrophages. To determine whether this enzyme is involved in the myelin induced ROS production the NADPH oxidase complex was blocked with DPI or apocynin. Both are specific inhibitors of the NADPH oxidase complex. DPI blocks a flavocytochrome ŽTakao et al., 1996., whereas apocynin blocks the assembly of the NADPH oxidase complex ŽStolk et al., 1994.. Furthermore Allopurinol, a specific inhibitor of xanthine oxidase, was used to determine whether xanthine oxidase is involved in the myelin induced ROS production. Both inhibitors of the NADPH oxidase complex decreased the oxidative burst activity to background levels ŽFig. 2B.. Allopurinol did not show this decrease of the oxidative burst activity ŽFig. 2B.. 3.2. The role of ROS on phagocytosis of myelin by macrophages 3.2.1. NADPH oxidase complex inhibitors The above show that the oxidative burst is activated by binding andror phagocytosis of myelin by macrophages. The induction of an oxidative burst in macrophages after phagocytosis is a well-described phenomenon ŽJohnston and Kitagawa, 1985; Winrow et al., 1993.. However, nothing is known about the requirement of ROS for the phagocytosis of myelin by macrophages. In the above experiments we showed that the oxidative burst in rat macrophages could be blocked by the NADPH oxidase inhibitors DPI Ž100 mM. and apocynin Ž10 mM.. In order to investigate the role of NADPH oxidase complex inhibitors in myelin phagocytosis the bindingrinternalization of DiI-labeled myelin was investigated in the presence of these inhibitors. Pre-treatment of peritoneal macrophages with DPI or apocynin in indicated concentrations resulted in a concen-
tration dependent inhibition of myelin phagocytosis ŽFig. 3.. Similar results, although perhaps somewhat less profound, were obtained when the NADPH oxidase complex inhibitors were tested on serum-treated zymosan and latex beads phagocytosis. Allopurinol did not block the phagocytosis of myelin, serum-treated zymosan or latex beads Ždata not shown.. 3.2.2. ReactiÕe oxygen species (ROS) scaÕengers Inhibiting the NADPH oxidase complex can block the phagocytosis of myelin, as shown in Fig. 3. The next step was to determine which product of the NADPH oxidase complex is involved in the phagocytosis of myelin. The products of the NADPH oxidase complex can be scavenged with superoxide dismutase ŽSOD., which converts . Ž . superoxide ŽOy 2 into hydrogen peroxide H 2 O 2 , catalase, a scavenger of hydrogen peroxide ŽH 2 O 2 . or mannitol, a scavenger of hydroxyl radicals ŽOHy. . The scavengers were added to the peritoneal macrophages. SOD did not decrease the phagocytosis of myelin by macrophages, whereas catalase and mannitol in a dose-dependent manner decreased the phagocytosis to background levels ŽFig. 4.. 3.3. The effect of Lipoic acid on the phagocytosis of myelin Lipoic acid ŽLA. is a non-specific scavenger of ROS. It is able to scavenge not only OHy and HOCl, but also other ROS. The reduced form of LA, dihydrolipoic acid ŽDHLA., is also involved in the vitamin E cycle and has been described as an inhibitor of NFk B. LA decreased the phagocytosis of myelin in a dose dependent manner ŽFig. 5A.. To compare the effect of LA on the phagocytosis of myelin with that of other particles, macrophages were
Fig. 6. LA shows a time-dependent effect on the binding and uptake of fluorescent myelin. Myelin was incubated with peritoneal macrophages in the presence of 2% NRS. Right from the beginning of the experiment LA Ž1 mgrml. was added to a well every 15 min. The longer LA had been present in the media, the larger the inhibition of binding and uptake of fluorescent myelin Ž n s 3.. w p F 0.001.
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incubated with myelin-DiI, FITC-labeled serum-treated zymosan or FITC-labeled latex beads in the presence of LA Ž1 mgrml. ŽFig. 5B.. Although LA decreased the phagocytosis of both serum-treated zymosan and latex beads, the inhibition of the phagocytosis of myelin by LA was more pronounced. We also investigated the time-dependency of this process. Peritoneal macrophages were incubated with myelin and after different intervals LA Ž1 mgrml. was added to the wells. Fig. 6 shows the time dependent effect of LA on the phagocytosis of myelin. The phagocytosis of myelin by peritoneal macrophages decreases with the time that LA is present in the media. This shows that a complete inhibition requires the continuous presence of LA, which is compatible with a possible role as a scavenger of ROS.
4. Discussion ROS are thought to be involved in the pathogenesis of EAE and MS ŽHartung et al., 1988; Guy et al., 1989; Glabinsky et al., 1993; Hansen et al., 1995; Malfroy et al., 1997.. ROS are not only able to damage CNS tissue, such as endothelial cells of the blood-brain barrier ŽRubanyi, 1988., but they can also damage the myelin sheath ŽChia et al., 1983; Arduini et al., 1985; Konat and Wiggins, 1985.. In this study we show that ROS are involved in the phagocytosis of myelin by macrophages. Several enzymes are capable of producing ROS, such as xanthine oxidase and NADPH oxidase. NADPH oxidase is a membrane-bound multi-component electron transfer chain involving a flavocytochrome. The switch for activation of the NADPH oxidase complex is induced by a conformational change in the flavocytochrome. Diphenyleneiodonium ŽDPI. inhibits the NADPH oxidase complex at the level of the flavocytochrome ŽTakao et al., 1996., whereas apocynin inhibits the assembly of the different components of the NADPH oxidase complex ŽStolk et al., 1994.. Because both NADPH oxidase inhibitors DPI and apocynin decreased the amount of ROS produced by the macrophages and the xanthine oxidase inhibitor allopurinol did not inhibit the production of ROS ŽFig. 2B., it is likely that only the NADPH oxidase complex is the main if not the only source of ROS in our cells. This process is called the respiratory or oxidative burst ŽBabior, 1984.. Although we did not measure ROS production in macrophages stimulated with latex beads, DPI and apocynin inhibited the phagocytosis of latex beads. It is probable that even small extracellularly non-detectable amounts of ROS are able to trigger signal transduction. Scavenging of the ROS produced by the NADPH oxidase complex with catalase or mannitol decreased the phagocytosis of myelin by macrophages, whereas superoxide dismutase ŽSOD. did not show this effect ŽFig. 4A.. Therefore, we conclude that hydrogen peroxide and hy-
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droxyl radicals are the ROS involved in the phagocytosis of myelin. The question whether hypochlorous acid is involved in the phagocytosis of myelin remains to be resolved. In several in vivo studies it has been shown that catalase can decrease the severity of the clinical signs in EAE of Lewis rats ŽRuuls et al., 1995. and that it can decrease the demyelination in EAN in guinea-pigs ŽGuy et al., 1989.. In both studies, SOD had little effect, although Hartung et al. Ž1988. showed an effect of both catalase and SOD in EAN in Lewis rats. Guy et al. Ž1989. have previously proposed that this effect of SOD could actually be due to preservatives, present in the SOD, which may function as antioxidants. The role of catalase and SOD on the phagocytosis of bacteria has been examined by Takao et al. Ž1996.. These investigators showed that the murine peritoneal macrophage cell line IC-21 was able to phagocytose bacteria in the presence of catalase Ž100–500 Urml. or SOD Ž100–500 Urml., while the killing of the bacteria was inhibited by the addition of SOD and catalase. We observed no effect of catalase in these concentrations, but instead needed concentrations of 5000 Urml to inhibit the phagocytosis of myelin in our system. This is probably due to the fact that ROS are produced continuously during the full 90 min of the experiment. Our observation that a complete blocking of phagocytosis required scavengers to be present throughout the experiment is in line with this ŽFig. 6.. To be able to scavenge the ROS produced over the whole experiment high concentrations of scavengers may be necessary. We also used Lipoic acid ŽLA. to scavenge ROS produced during the phagocytosis of myelin ŽFig. 4B.. The exact mechanism by which LA influence myelin phagocytosis is not extremely clear at the moment. LA can be reduced to dihydrolipoic acid ŽDHLA., both LA and DHLA are antioxidants Žfor a review, see Packer et al., 1995.. LA scavenges hydroxyl radicals, hypochlorous acid, nitric oxide hydrogen peroxide and singlet oxygen. It also chelates transition metals. DHLA is an even more potent scavenger. DHLA is able to protect membranes to lipid peroxidation by regenerating vitamin E. Furthermore, LA and DHLA have an effect on signal transduction by inhibiting NFk B activation. In a cultured human T lymphocyte Jurkat cell line LA administration resulted in intracellular DHLA formation ŽHandelman et al., 1994.. Thus, also in our system, LA and DHLA may be present both intracellularly and extracellularly. How are ROS involved in the phagocytosis? Macrophages as well as microglia produce ROS during phagocytosis ŽWilliams et al., 1994; Mosley and Cuzner, 1996.. Phagocytosis does not need to be completed, because contact between the phagocyte and the stimulating particle is enough to stimulate the oxidative burst ŽGoldstein et al., 1975.. Furthermore, the literature to date has shown that ROS can play an essential role in signal transduction Žfor a review, see Lander, 1997.. From our
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results it appears that the products of the oxidative burst can not only damage the myelin sheath, but can also activate the macrophage to phagocytose the myelin. Gamaley et al. Ž1994. showed that mouse peritoneal macrophages activated with 10 mM H 2 O 2 phagocytosed twice as much as without pre-incubation with H 2 O 2 . We have shown in this study that LA was relatively potent in the inhibition of myelin phagocytosis as compared to serum-treated zymosan or latex beads phagocytosis ŽFig. 5B.. This may be due to the fact that ROS can modify both macrophages and myelin, and both can be prevented by addition of LA. In the future, it will be interesting to test the effect of LA on demyelination in vivo.
Acknowledgements The authors wish to thank Christa Homburg and Petra Hilarius for their help with the NBT assay, and Prof. A. Bast for valuable discussion concerning LA. This work was supported financially by the Dutch foundation ‘Vrienden MS Research’, project MS 95-202.
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