Proc. Nati. Acad. Sci. USA Vol. 77, No. pp. 414-417, January 1980 Cell Biology Induction of plasminogen activator secretion in macrophages by electrochemical stimulation of the hexose monophosphate shunt with methylene blue (nitro blue tetrazolium/methyl viologen/phagocytosis) JORG SCHNYDER AND MARCO BAGGIOLINI Wander Research Institute, Wander Ltd., P.O. Box 2747, CH-3001 Berne, Switzerland Communicated by Zanvil A. Cohn, October 5, 1979

ABSTRACT Resident peritoneal macrophages were ob- centrifugation, resuspended in Pi/NaCl at a concentration of tained from untreated mice and were cultured in medium 199 50 mg/ml, and autoclaved at 120°C (5). Latex beads, 1.01 ,um with or without 5% acid-treated fetal bovine serum. Three hours after harvesting, redox compounds-i.e., methylene blue, methyl (Serva, Heidelberg, W. Germany), were suspended in Pi/NaCl viologen, or nitro blue tetrazolium-were added to the cultures containing 0.01% Tween 80, washed three times by centrifu- of adherent cells. After 1 hr, the cells were washed and culturing gation, resuspended in Pi/NaCI/Tween 80, and sterilized by was continued in the absence of redox compounds. The effects UV irradiation (6). Sheep erythrocytes were washed five times of the redox compounds were tested by assaying for hexose in Pi/NaCl by centrifugation, resuspended in Pi/NaCl con- monophosphate (HMP) shunt activity and for plasminogen ac- taining 3% (wt/vol) formaldehyde, kept for 2 days at 4°C in tivator secretion, and the results were compared with the effects induced by phagocytic stimuli. Methylene blue caused a con- this medium, and then washed five more times in Pi/NaCJ (7). centration-dependent stimulation of the HMP shunt, whereas Phagocytosis experiments were performed exactly as described methyl viologen and nitro blue tetrazolium were ineffective. (1). Briefly, particles were suspended in a small volume of Shunt stimulation by methylene blue was followed, after a lag medium and were added to freshly established cultures of ad- of 2-4 days, by plasminogen activator secretion. The rate of herent cells. Phagocytosis was stopped after 1 hr by aspirating secretion was dependent on the methylene blue concentration the medium and washing three times with Culturing used. Methyl viologen and nitro blue tetrazolium were again Pi/NaCI. ineffective, whereas phagocytosis of zymosan or sheep eryth- was then continued in fresh medium for up to 10 days. rocytes, which stimulates the HMP shunt, induced plasminogen Pyridine Nucleotide Oxidation. The oxidation of NADH activator secretion at rates similar to those induced by meth- and NADPH was measured spectrophotometrically at 37°C. ylene blue. These results add further evidence to our hypothesis The reaction mixture consisted of 500 Al of 0.1 M sodium that the HMP shunt-dependent metabolic burst is involved in phosphate/HCI buffer at pH 7.0, 200 Ml of an aqueous solution macrophage activation. Because methylene blue mimics the of the redox 100 Ml of a 0.8 mM solution of NADH action of zymosan it appears that shunt stimulation by itself dye, and or initiates the activation process independently of phagocy- NADPH. Decrease in absorbance at 334 nm was recorded with tosis. an Eppendorf photometer (Eppendorf, Hamburg, W. Ger- many) equipped with a W+W linear-logarithmic recorder We have recently reported that mouse peritoneal macrophages (W+W Electronic, Munchenstein, Switzerland). become activated in culture after phagocytosis of zymosan, as HMP Shunt Assay. HMP shunt activity was always mea- indicated by the induction of plasminogen activator secretion. sured at the onset of culture by determining the amount of By testing the effects of various types of phagocytosable par- 14CO2 produced from [1-14C]glucose during 1 hr in the pres- ticles, we obtained evidence suggesting that the stimulus for ence of the agent to be tested as a possible stimulus (1). In some macrophage activation is related to the burst of hexose mono- experiments, the same technique was used on a reduced scale: phosphate (HMP) shunt activity rather than to the phagocytic 2.5 X 106 cells were plated on 33-mm dishes (Falcon Plastics, uptake per se or the intracellular fate of the particles (1). Cockeysville, MD) and nonadherent cells were washed off 3 We have now studied the effects of HMP shunt stimulation hr later. The cultures were then supplied with 1.5 ml of fresh induced by methylene blue instead of phagocytosis and have medium containing labeled glucose (1 uCi per dish; 1 Ci = 3.7 found that it equally leads to macrophage activation. As in our X 1010 becquerels) and the redox compound or other agents to earlier studies (1, 2), macrophage activation was assessed by be tested. After 1 hr, the 14CO2 released was determined (1). testing for plasminogen activator secretion (3, 4). In contrast to our former reports, results are expressed as the total amounts of CO2 formed. For this purpose, 14CO2 values MATERIALS AND METHODS were corrected for the amount escaping during incubation in Macrophage Cultures. Macrophages obtained from un- the culture dishes, which was determined to be 55.8 b 1.5 treated male OF, mice (Sandoz, Basel, Switzerland) weighing percent (mean d SEM) of the total 14CO2 that is recovered 20-24 g were cultured in medium 199 with or without 5% when the same reaction mixture is tested in gas-tight ves- acid-treated fetal bovine serum according to established tech- sels-i.e., Warburg-type flasks. The medium containing the niques (2). redox compounds was filtered (Millipore. 0.22 um pore size) Phagocytosis. Zymosan (Koch-Light Laboratories, Coln- before use. All these experiments were performed in the dark brook, Buckinghamshire, England) was boiled in phosphate- (Forma CO2 incubator, model 3156, Forma Scientific, Marietta, buffered saline (Pi/NaCI) for 1 hr, washed three times by OH). Chemicals. The following reagents were used: f3-NADPH The publication costs of this article were defrayed in part by page tetrasodium salt and f3-NADH disodium salt (Boehringer charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: HMP, hexose monophosphate; Pi/NaCl, phosphate- this fact. buffered saline. 414 Downloaded by guest on September 26, 2021 Cell Biology: Schnyder and Baggiolini Proc. Natl. Acad. Sci. USA 77 (1980) 415 Table 1. Oxidation of pyridine nucleotides by redox compounds Table 3. HMP shunt stimulation in nonelicited mouse peritoneal Concentration (MM) macrophages by methylene blue and by phagocytosis at which pyridine CO2 liberation from glucoset nucleotides are oxidized at Stimulus* [1-14C]- [6-14C]- E0* 1 Mmol/min at pH 7 Compound V NADH NADPH None 1.30 + 0.10 (23) 0.47 ± 0.02 (9) Methylene blue (0.1 mM) 12.75 + 0.51 (11) 1.64 + 0.19 (9) Methyl viologen -0.45 >250 >250 Zymosan 17.99 i 1.23 (12) 0.94 ± 0.01 (6) NADPH -0.32 Sheep erythrocytes 12.82 ± 1.48 (6) 0.77 + 0.02 (6) >25 Nitro blue tetrazolium 0 >25 * Methylene blue or particles were added to the cultures for 1 hr and Methylene blue 0.01 6.3 7.0 the 14CO2 formed during this time from labeled glucose was deter- * E' is the standard redox potential at pH 7. mined. Particle-to-cell ratios were 8:1 for zymosan and 5:1 for sheep erythrocytes. t Nanomoles of CO2 per 106 cells; mean values + SEM; in parentheses, number of assays. Mannheim); methyl viologen, pure analytical reagent and nitro blue tetrazolium chloride, pure analytical reagent (Koch-Light after phagocytosis of particles that stimulate the HMP shunt Laboratories); methylene blue B and H202 (E. Merck, Darm- (1). We therefore tested the effect of methylene blue under stadt, W. Germany); D-[1-14C]glucose (New England Nuclear); similar experimental conditions. Nonelicited mouse peritoneal D-[6-14C]glucose (The Radiochemical Centre, Amersham, macrophages were put in culture and the adherent cells were England); catalase from bovine liver, type V glucose oxidase challenged 3 hr later with either zymosan or 10MM methylene from Aspergillus niger, and type I superoxide dismutase from blue for 1 hr. Both treatments resulted in a marked increase in bovine blood (Sigma). Reagents used for culturing macrophages plasminogen activator secretion as compared to the control (Fig. were described in a previous paper (2). 1). As in earlier experiments, secretion occurred after a lag period (which in this case was somewhat shorter than usual). RESULTS Fig. 2 shows that the rate of plasminogen activator secretion Pyridine nucleotide oxidation and stimulation of the depended on the methylene blue concentration to which the HMP shunt by methylene blue cells were exposed. At the two higher concentrations (100 and Pyridine nucleotides were readily oxidized by methylene blue, 300 ,uM), the methylene blue pulse induced levels of plasmin- but not by two other redox compounds that we have studied, ogen activator secretion similar to those for zymosan phago- methyl viologen and nitro blue tetrazolium (Table 1). In ac- cytosis (see Fig. 1 and Table 5). The experiments presented in cordance with these results, methylene blue also stimulated the Fig. 1 were performed in the absence and those presented in HMP shunt of macrophages in culture in a concentration- Fig. 2 in the presence of serum. Methylene blue was ictive dependent manner, presumably by reoxidizing the NADPH under both conditions. When serum was present, however, formed, whereas both methyl viologen and nitro blue tetrazo- higher concentrations were needed, presumably because the lium were ineffective (Table 2). As shown in Table 3, the dye is partially trapped by binding to serum proteins. Plas- stimulatory effect of methylene blue compared well with that minogen activator secretion was also induced when methylene of phagocytosis. An approximately 10-fold enhancement of blue was supplied to the media during the entire culture period 4CO2 formation from 1-14C-labeled glucose was obtained with (Fig. 3). Under these conditions, secretory responses similar to 0.1 mM methylene blue and during phagocytosis of zymosan those in the earlier experiments were obtained with consider- or sheep erythrocytes. All three treatments also enhanced 14CO2 ably lower concentrations of the dye (0.3-1.5 MuM). In Fig. 3, formation from 6-'4C-labeled glucose, but to a considerably smaller extent. Induction of plasminogen activator secretion by methylene blue In previous experiments we had shown that the production and secretion of plasminogen activator was induced in macrophages

Table 2. HMP shunt stimulation in nonelicited mouse peritoneal macrophages by redox compounds Redox HMP shunt activityt compound,* Methylene Nitro blue Methyl ,uM blue tetrazolium viologen 0 1.50 + 0.13 (32) 1 1.27 + 0.02 (3) 3 1.68 0.05 (3) -10 4.98 i 0.15 (8) 1.08 + 0.02 (3) 1.17 ± 0.01 (3) 30 8.60 ± 0.45 (6) 4 6 Time in 100 11.46 ± 0.54 (26) 1.19 + 0.03 (9) 0.95 ± 0.05 (6) culture, days 300 12.01 ± 0.85 (6) FIG. 1. Induction of plasminogen activator secretion in noneli- cited macrophages by zymosan phagocytosis and by methylene blue. * The redox compounds were added to the cultures for 1 hr and the Units are defined in ref. 2. Zymosan, eight particles per cell (u- *), 14CO2 formed during this time from 1-14C-labeled glucose was de- or methylene blue, 10 AM (U----), was added at the beginning of termined. culturing and eliminated 1 hr later by washing the cell monolayers. t Nanomojes ofCO2 per 106 cells; mean values I SEM; in parentheses, Serum-free media were used throughout. o, Control values. Each number of assays. value is the average from triplicate cultures. Downloaded by guest on September 26, 2021 416 Cell Biology: Schnyder and Baggiolini Proc. Natl. Acad. Sci. USA 77 (1980)

Time in u I culture, days 0 Fl(. 2. of Induction plasminogen activator secretion in noneli- - 25- Methyl viologen cited macrophages by various concentrations of methylene blue. At 4) the beginning of the experiments, the cells were exposed to the indi- cated concentrations of the redox compound for 1 hr. The cells were then washed and cultured in a medium containing 5% acid-treated .-V0 fetal bovine serum. 0, Control values in the absence of methylene blue. Each value is the average from triplicate cultures. the effects of methylene blue are compared with those of

methyl viologen and nitro blue tetrazolium, two redox com- 25-- Nitro blue tetrazolium pounds that neither oxidize pyridine nucleotides (Table 1) nor stimulate the HMP shunt (Table 2). Both compounds, even at comparatively high concentrations, failed to induce plasmin- ogen activator secretion. At 0.1 mM methyl viologen was ap- parently cytotoxic, because large amounts of lactate dehydro- ___ genase and ,B-glucuronidase were released into the medium (not shown). .--. . . .~~~~~~~a I I In additional experiments, we obtained some information 0 2 4 6 8 10 on the possible mechanism of methylene blue action. As shown Time in culture, days FIl(. 3. Effects of methylene blue, methyl viologen, and nitro blue Table 4. Effects of enzymes that metabolize superoxide or tetrazolium on plasminogen activator secretion by nonelicited mac- hydrogen peroxide on macrophage activation by methylene blue rophages. Methylene blue, 1.5 yM (0-u) or 0.3 ,uM (- - -- *), and methyl viologen, 10 uM (----), were present in the respective media Plasminogen during the entire experiment. Nitro blue tetrazolium, 100 gM HMP shunt activator ( ----), was added to the cells at the onset of culture and eliminated Stimulus* Addition* activityt secretiont by washing after 1 hr. o, Control values. Each value is the average from triplicate cultures. None - 1.05 0.62 H202 in Table 4, catalase and, to a lesser extent, superoxide dismutase 0.01 mM - 1.47 0.13 attenuated the respiratory burst, but only catalase inhibited the 0.1 mM - 3.32 - effect of on activator secretion. 1.0 mM - 4.23 - methylene blue plasminogen None Catalase 0.84 0.50 Addition of H202 to the cultures induced an increase in HMP Methylene blue - 10.72 10.40 shunt activity in a concentration-dependent fashion. At the Methylene blue Catalase 4.90 4.81 lowest and barely effective concentration, H202 did not induce plasminogen activator secretion. At higher concentrations, this None - 1.35 0.72 could not be tested because H202 was cytotoxic. H202 gener- None SOD 0.87 0.96 ation by glucose oxidase and glucose (60-900 nmol/min) re- Methylene blue - 10.29 5.0 sulted in a stimulation of the HMP shunt, which was prevented Methvlene blue SOD 6.26 6.71 by catalase (data not in Table 4, because no corrections were made for consumption of labeled glucose by glucose oxidase). * The stimuli were added to the cultures for 1 hr and were eliminated by washing; culturing was then continued for up to 10 days. The Here too, no plasminogen activator secretion was induced at methylene blue concentration was 0.1 mM. Catalase (3 mg/ml) or low rates of H202 formation, whereas at higher rates cell via- superoxide dismutase (SOD, 400 jg/ml) was added to the cultures bility was impaired. 1 hr before methylene blue and was eliminated with the dye washing. Addition of methylene blue at the concentration used in this Results from two separate experiments. work had no apparent effect on cell viability. Lysozyme se- t Nanomoles of CO2 per 106 cells formed during 1 hr in the presence cretion and release of lactate dehydrogenase were similar to of the stimulus or in the respective controls; mean values from triplicate cultures. those in control cultures. Microscopic examination revealed Amount of plasminogen activator secreted between day 4 and day occasional blue dots in 10-15% of the cells after incubation with 7. Mean values (units per 10fi cells) from triplicate cultures. the dye and subsequent washing; the dots disappeared within § This stimulus was toxic to the cells, which did not survive culturing a day. This suggests that methylene blue may remain tempo- for several days. rarily adsorbed to a portion of the cells. Dead cells, on the other Downloaded by guest on September 26, 2021 Cell Biology: Schnyder and Baggiolini Proc. Natl. Acad. Sci. USA 77 (1980) 417 hand, could be readily recognized by their uniform light-blue actually interrelated, they show a striking apparent correlation staining. between the degree of shunt stimulation in response to different concentrations of methylene blue and the rate of plasminogen DISCUSSION activator secretion obtained, which suggests the existence of a We have shown that treatment of nonelicited macrophages with functional link between the two processes. methylene blue results in the stimulation of glucose oxidation The possible role of the metabolic burst as initiating event through the HMP shunt and in the induction of plasminogen in macrophage activation is supported by results obtained in activator secretion. Other redox compounds such as methyl other laboratories. Various soluble agents such as concanavalin viologen and nitro blue tetrazolium that do not influence the A (9), phorbol myristate acetate (9), and products of mitogen- HMP shunt also fail to induce plasminogen activator secretion. treated lymphocytes (10, 11) have been reported to induce In a recent study on the biochemical effects of phagocytosis in plasminogen activator secretion in macrophages, and HMP cultured macrophages (1), we observed a similar parallelism shunt stimulation is a common early effect of all these agents between shunt stimulation and induction of plasminogen ac- (12, 13). In our own experiments on nonelicited mouse mac- tivator secretion. The effects of methylene blue and of different rophages, similar degrees of shunt stimulation were obtained phagocytic stimuli are summarized for comparison in Table with 10 nM phorbol myristate acetate and with 100 ,M 5. methylene blue. Production and secretion of plasminogen activator is a The mechanism by which methylene blue acts on the ma- characteristic property of activated macrophages (3, 4, 8). Our crophage is unknown. It probably reoxidizes the NADPH results suggest that-independently of its cause-a rise in HMP produced by the HMP shunt either directly by entering the cell shunt activity may be involved in initiating the activation or at the distance by coupling to electron-transferring inter- process. The triggering nature of the metabolic burst is indi- mediates in the cell membrane or through the extracellular cated by the fact that it is sufficient to expose the cells to a generation of H202. We can exclude that methylene blue or stimulus-e.g., zymosan (1), sheep erythrocytes (1) or meth- impurities present form precipitates that can be phagocytosed ylene blue-for the short period of 1 hr at the onset of culturing and stimulate the cells by this mechanism because the dye so- in order to obtain a secretory response that manifests itself 2-4 lutions and the media were sterilized by Millipore filtration. days later. Although our experiments do not prove that the metabolic burst and the secretion of plasminogen activator are We thank Ms. Agathe Wild and Mr. Eugen Jenni for their excellent technical assistance and Dr. Beatrice Dewald for critically reviewing the manuscript. Table 5. Stimulation of HMP shunt and induction of plasminogen activator secretion by methylene blue and 1. Schnyder, J. & Baggiolini, M. (1978) J. Exp. Med. 148, 1449- phagocytosable particles 1457. 2. Schnyder, J. & Baggiolini, M. (1978) J. Exp. Med. 148, 435- Plasminogen 450. HMP shunt activator 3. Unkeless, J. C., Gordon, S. & Reich, E. (1974) J. Exp. Med. 139, Stimulus* activityt secretion* 834-850. 4. Gordon, S., Unkeless, J. C. & Cohn, Z. A. (1974) J. Exp. Med. 140, None (control) 1.33 1.7 995-1010. Latex beads (40:1) 1.64 1.7 5. Weissmann, G., Dukor, P. & Zurier, R. B. (1971) Nature (Lon- Zymosan (8:1) 14.00 22.8 don) New Biol. 231, 131-135. Sheep erythrocytes (5:1) 13.99 56.2 6. Ulrich, F. & Zilversmit, D. B. (1970) Am. J. Physiol. 218, Methylene blue 1118-1127. 10 'M 3.44 2.8 7. Rabinovitch, M (1967) Proc. Soc. Exp. Biol. Med. 124, 396- 30,uM 6.16 7.3 399. 100 PM 9.86 17.4 8. Cohn, Z. A. (1978) J. Immunol. 121, 813-816. * The stimuli were present during 1 hr, after which time culturing was 9. Vassalli, J.-D., Hamilton, J. & Reich, E. (1977) Cell 11, 695- continued in the absence ofstimuli for up to 10 days. Particle-to-cell 705. ratios are given in parentheses. 10. Vassalli, J.-D. & Reich, E. (1977) J. Exp. Med. 145, 429-437. t Nanomoles ofCO2 per 106 cells formed during 1 hr in the presence 11. Klimetzek, V. & Sorg, C. (1977) Eur. J. Immunol. 7, 185-187. of the stimulus or in the controls; mean values from triplicate cul- 12. Romeo, D., Zabucchi, G. & Rossi, F. (1973) Nature (London) tures. See ref. 1 for details. New Biol. 243, 111-112. Amount of plasminogen activator secreted between day 4 and day 13. Lazdins, J. K., Kuhner, A. L., David, J. R. & Karnovsky, M. L. 7 (units per 106 cells). Mean values from triplicate cultures. (1978) J. Exp. Med. 148, 746-758. Downloaded by guest on September 26, 2021