Hydrogen peroxide release by rat peritoneal macrophages in the presence and absence of tumor cells

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 215, No. 2, May, pp. 355-366, 1982

Hydrogen

Peroxide Release by Rat Peritoneal Macrophages Presence and Absence of Tumor Cells’ EDWIN

L. THOMAS2

Departments of Biochemistry

MARVIN

AND

in the

FISHMAN

and Immundogy, St. Jude Children’s Research Hospital, Memphis, Tennessee 38101 Received

December

4, 1981

The ability of mineral oil-elicited rat peritoneal macrophages to release hydrogen peroxide (H202) to the extracellular medium was measured in the presence and absence of rat lymphoma cells grown in tissue culture, and in the presence of phorbol myristate acetate (PMA). Horseradish peroxidase (HRP)-catalyzed oxidation of scopoletin or phenol red was used to measure H202 release during incubation of cells in monolayer culture for periods up to 24 h. Macrophages appeared to release H202 with or without PMA, although PMA greatly increased the amount of H202 released in short (1 to 4 h) incubations. Tumor cells did not replace PMA as a triggering agent for H202 release. Instead, tumor cells inhibited H202 release. The probable basis for inhibition was competition between macrophages and tumor cells for the supply of oxygen (02). Tumor cells did not inhibit H202 release when the O2 concentration was held constant. The rates at which macrophages took up O2 and released H202 were proportional to the O2 concentration, as measured with the O2 electrode. Rates of H202 release could be calculated from the difference in the rate constants for O2 uptake measured in the presence of two different extracellular H20z-consuming systems (HRP-scopoletin vs catalase). PMA-stimulated uptake of O2 and release of H202 were highest in a small subpopulation of macrophages, obtained at the lowest-density position on gradients of bovine serum albumin. These cells also released H202 in the absence of PMA. Tumor cells had no effect on the rate constants for O2 uptake and H202 release by the unfractionated macrophages or the macrophage subpopulations.

Cells of the monocyte-macrophage lineage are thought to be important in the control of neoplastic proliferation (1). Many studies have shown that such cells have antitumor activity in vitro. Macrophage cytotoxicity appears to be selective against tumor cells, but such activity has been observed against normal cells, to a lesser degree. One possible mechanism of macrophage cytotoxicity is the release of toxic oxygen (0,) metabolites, including

superoxide anion (0;) and hydrogen peroxide (H202) (2). Macrophages can release 0; and H202 to the extracellular medium upon addition of a “triggering” agent such as phorbol myristate acetate (PMA)3 (2, 3). In one series of studies (4, 5), PMA increased the tumoricidal activity of mouse peritoneal macrophages. Tumor cells were reported to interfere with measurements a Abbreviations used: BSA, bovine serum albumin, EC:TC, effector cell-to-target cell ratio; FCS, fetal calf serum; HRP, horseradish peroxidase; LPS, bacterial lipopolysaccharide; PE, peritoneal exudate; PMA, phorbol myristate acetate; WGL, wheat germ lectin.

’ Supported by Grants DE 04235, AI 16795, CA 18672, CA 08480, and CA 21765 from the U. S. Public Health Service and by ALSAC. *Author to whom correspondence should be addressed.

355

0003-9861/82/060355-12$02.00/O Copyright All rights

0 1982 by Academic Press. Inc. of reproduction in any form reserved.

356

THOMAS

AND

of Hz02, but PMA did stimulate H202 release in the absence of tumor cells. It was assumed that tumor cells would not alter the rate of PMA-stimulated H202 release. With this assumption, the amount of H202 released and the toxicity of HzOz to the tumor cells were sufficient to account for tumoricidal activity. High effector:target (EC:TC) ratios were required, so that the amount of HzOz per target cell would be high. Other O2 metabolites were not required, and peroxidase enzymes were not required to amplify the toxicity of H202. In our studies on rat peritoneal macrophages elicited with mineral oil, antitumor activity was observed against syngeneic rat lymphoma cells ((6), and M. Fishman, unpublished results). Macrophages and tumor cells were incubated together in a complete medium with fetal calf serum (FCS) to support the growth of tumor cells. Within 24-48 h, tumoristatic action was observed at EC:TC ratios as low as l:l, and tumoricidal action at EC:TC ratios of 5:l and higher. PMA was not required for antitumor action. To study the possible role of H202 in this system, methods were developed to measure H202 release in the presence and absence of tumor cells. The aims were to determine whether H202 release accompanied antitumor action, and whether the presence of tumor cells was sufficient to trigger HzOz release. Tumor cells did not trigger HzOz release, although a slow rate of H202 release was obtained even in the absence of a triggering agent. This HzOz-releasing activity was highest in a small fraction of the macrophage population. Also, the results indicated that O2 concentration is an important variable in the expression of the ability to release HzOz. At high cell densities, competition between macrophages and tumor cells for the O2 supply may suppress Hz02 release. MATERIALS

Animals.

METHODS

rats were purchased from ARS Madison, Wisconsin. Peritoneal exudate (PE) cekk. Rats were injected intraperitoneally with 10 ml Klearol mineral oil (Ruger Chemical Co., Irvington, N. J.). Exudates were

Sprague-Dawley,

W/Fu

AND

FISHMAN collected 4-5 days after injection, and the oil and aqueous phases were separated as described previously (7). The yield of PE cells per animal was about 1 X lOa, of which 60-70% were macrophages. Except for experiments with cell subpopulations separated by density gradient centrifugation, all experiments were performed with the adherent population of PE cells. To separate adherent and nonadherent populations, 5 x lo7 cells were incubated in a 15 X 2.5-cm plastic tissue culture plate for 30 min at 37°C in 15 ml RPM1 1640 medium supplemented with 10% FCS. Nonadherent cells were poured off, and adherent cells were resuspended by adding 15 ml of 0.2% (w/v) EDTA in phosphate-buffered saline, pH 7.2, and incubating 20 min at 37°C. Equilibrium density gradient centri$ugation. Subpopulations of PE cells were separated by centrifugation on discontinuous bovine serum albumin (BSA) gradients prepared as described previously (7). The distribution of cells at the interfaces was similar to that described for rabbit PE cells (8). At the lowerdensity interfaces, 95-98% of the cells were macrophages. At the 20/30% BSA interface, 60-70% of the cells were macrophages, and at the 30/35% interface, 70-80% were lymphocytes. Neutrophils, erythrocytes, and dead cells were concentrated in the pellet. Tumor cells. G-l lymphoma cells syngeneic in W/ Fu rats were obtained from Dr. G. Shellan. The cell line was maintained as suspension cultures in RPM1 1640 medium with 10% FCS at 37°C in an atmosphere of 10% COs in air. Media and reagents. The RPM1 1640 medium with 10% FCS was used throughout. Solutions of horseradish peroxidase (HRP; 40 units * rng-‘; Sigma Chemical Co., St. Louis, MO.), wheat germ lectin (WGL; Calbiochem-Behring, San Diego, Calif.), lipopolysaccharide (LPS; Difco, Detroit, Mich.) and tuftsin diacetate (Sigma) were prepared in the medium. Catalase crystals (65,000 units * mg-‘; Boehringer-Mannheim, Indianapolis, Ind.) were washed by centrifugation in water, then dissolved at 15 age ml-’ in the medium. A 10 mM solution of scopoletin (Sigma) was prepared in 50 mM NasCOs, and immediately diluted to 0.4 mM in the medium. A 2 pM solution of PMA (Consolidated Midland Corp., Brewster, N. Y.) was prepared in dimethyl sulfoxide and stored at -20°C. H,O, release in monolayer culture. Cells were incubated in the medium containing HRP and the fluorescent compound scopoletin. HRP catalyzes the oxidation of scopoletin by H202 to nonfluorescent products, and H202 release is calculated from the decrease in fluorescence (9). A high scopoletin concentration (0.1 mM) was used, so that scopoletin was in excess of the amount of HsOa to be detected, and to overcome the interference by serum components (10) and the competition by other peroxidase substrates in the medium (11). Under these conditions,

HYDROGEN

PEROXIDE

oxidation of scopoletin was proportional to HzOz, but a lo-fold dilution of the incubation medium was required to obtain fluorescence proportional to scopoletin concentration. Assay mixtures contained cells, medium, 10 pg. ml-’ HRP and 0.1 mM scopoletin in l-ml total volume in wells of 2-cm* area on multiwell plates. When added, PMA was 2 X lo-* M and catalase was 1.5 pg. ml-‘. Plates were incubated at 37°C under 90% air10% COz in a humidified incubator. The fluid was removed from the wells and centrifuged to remove cells. Duplicate portions (0.3 ml) of the supernatant were diluted lo-fold with isotonic phosphate buffer, pH 7.4, and fluorescence measured with excitation at 365 nm and emission at 460 nm. Fluorescence was measured relative to medium with HRP-scopoletin incubated under the same conditions without cells. Release of Hz02 was calculated from standard curves prepared by adding known amounts of HzOz. A standard curve was prepared after each period of incubation using the medium with HRP-scopoletin that had been incubated without cells. The efficiency of HzOz-trapping by the HRP-scopoletin system was about 50% (mol scopoletin oxidized/mol HzOz = 0.5). Unreacted HzOz did not accumulate, indicating that the remaining HsOz was consumed in oxidation of other peroxidase substrates. At the cell densities used in these studies, macrophages or tumor cells or both did not alter the efficiency. Alternatively, 0.3 mM phenol red (phenolsulfonphthalein) was substituted for scopoletin (12). After cells were removed by centrifugation, supernatants were adjusted to pH 12.5 and absorbance measured at 610 nm relative to medium with HRP-phenol red that had been incubated without cells. Standard curves were prepared as described above. 0, uptake and HpOo release in stirred suspensions. Assays were performed at 37°C in 3 ml total volume in stirred, thermostatted chambers (2-cm diameter) with a Clarke-type 02 electrode. Assay mixtures contained cells, medium, and either 10 rg/ml HRP and 0.1 mM scopoletin or 1.5 pg. ml-’ catalase. The mixtures were rapidly warmed to 3’7°C with stirring while 95% air-5% COs was bubbled through the mixture, the electrode was inserted displacing the air space above the mixture, and Oz concentration was recorded continuously for 20 to 30 min. When PMA (2 x lo-’ M) was added, the addition was made immediately before inserting the electrode. In some experiments, after a period of incubation, the electrode was withdrawn, portions were removed, centrifuged, and diluted, and fluorescence was measured as described above. After each incubation, catalase activity was removed from the chamber and electrode surface by stirring 5 min with 0.1 M HCl. In some experiments, 0.3 mM phenol red was substituted for scopoletin. Incubations with constant Oz concentration were

357

RELEASE

performed in the same apparatus, but the electrode was not inserted, the chambers were closed at the top with stoppers, and the mixtures were stirred in contact with 15 ml of 95% air-5% COO.At intervals, portions were removed for fluorescence measurements as described above. RESULTS

Scopoletin Oxidation Cultures

in Monolayer

Figure 1 shows the time course of HzOz release during incubation of PE cells in monolayer culture, as calculated from loss of scopoletin fluorescence. Release of H202 was stimulated by PMA, and this stimulation was most apparent in incubations of 1 to 4 h. With PMA, the rate of H,Oz release decreased after 4 h. Without PMA, H202 release continued during incubations of up to 24 h, and at 24 h was usually 50 to 80% of the value obtained with PMA. These results suggested that PE cells did not require PMA for Hz02 release, but that PMA caused the cells to express their full HzOz-releasing capacity over a shorter time period. Figure 2 (top) shows that PMA-stimulated H202 release was roughly proportional to the number of PE cells, although Hz02 release reached a plateau at 2 to 4 x lo6 cells. ml-l in other experiments. Omitting HRP or adding catalase in place of HRP resulted in decreased ability to detect HzOz release. Figure 2 (bottom) shows that the apparent Hz02 release in the absence of PMA was also proportional to the number of PE cells. However, in

Time Ch)

FIG. 1. Time course of HzOz release. PE cells (2 X lo6 *ml-‘) were incubated with PMA (0) or without PMA (m) for the indicated periods of time.

THOMAS

358

AND FISHMAN

‘“Or 80

t

A

- 6Ot

/

PE Cells

(ml-‘X

i

IO?

FIG. 2. Hz02 release in the presence and absence of PMA. PE cells at the indicated cell density were incubated 24 h with PMA (top) or without PMA (bottom) in media containing HRP-scopoletin (O), scopoletin (m), or catalase and scopoletin (A).

contrast to results obtained with PMA, omitting HRP or adding catalase did not eliminate this apparent release of HzOz. These results suggested that the decrease in scopoletin fluorescence in the absence of PMA might be due to phenomena other than HzOz release. Also, part of the PMA-stimulated loss of fluorescence might not be due to HzOz. The loss of fluorescence from the medium was not due to accumulation of scopoletin within the cells, in that extraction of the cells to release any intracellular scopoletin did not alter the results. Scopoletin might be oxidized by O2 metabolites other than H202, such as other peroxides, the hydroxyl radical (HO * ) or singlet oxygen (0,‘). Adding superoxide dismutase had no effect, suggesting that scopoletin was not oxidized by 02. Scopoletin might also be chemically modified in reactions that do not involve H202 or other O2 metabolites. Under the conditions of these experiments, there was no significant loss of fluorescence when the medium with HRP and scopoletin was incubated 24 h without cells. Loss of fluorescence in the absence of cells or added Hz02 was observed only during incubations at pH 9-10. This effect probably did not contribute to loss of fluorescence in these experiments in that the pH tended to fall rather than rise when the cell den-

sity was high, and the results were not altered by adding additional buffering capacity to the medium. Also, the results were not due to production of substances that quenched fluorescence. The same levels of fluorescence were obtained when scopoletin was added to fresh medium, or to the medium in which cells had grown. Also, in experiments that resulted in a large loss of fluorescence (as in Fig. 1 at 24 h), it was possible to see the insoluble products of scopoletin oxidation in the wells. As described below, all or part of the decrease in fluorescence obtained without PMA had many of the same characteristics as the PMA-stimulated decrease, so that H202 was calculated from the total decrease in fluorescence, as in Figs. 1 and 2. The inability of catalase to completely eliminate the decrease does not prove that H202 was not involved, because catalase is a poor scavenger for HzOz at low H202 concentrations, and because catalase and other hemoproteins have weak peroxidase activity that might become significant during a 24-h incubation. The HRP-independent decrease in fluorescence, which could not be inhibited with catalase, was also obtained with tumor cells. However, the amount of this apparent H202 release was less than that obtained with PE cells, and PMA had little or no effect with tumor cells. Table I shows results of incubating PE and tumor cells separately and together. Tumor cells inhibited H202 release from PE cells, in the presence or absence of PMA. Because both PE and tumor cells appeared to release Hz02, it was not possible to determine whether the PE cells were completely inhibited and the tumor cells unaffected, or whether both cell populations were partially inhibited. Maximum inhibition was obtained at about 2:l initial ratio of tumor cells to PE cells. Inhibition by tumor cells was not due to interference with the assay for Hz02. Tumor cells had no effect on detection of exogenous HzOz, added before or after a 24-h incubation. Inhibition was not overcome by adding more HRP, scopoletin, or both. Also, inhibition of PMA-stimulated

HYDROGEN

TABLE INHIBITION

PEROXIDE

I

OF PE-CELL HzOz BY TUiOR CELLS

RELEASE

HsOs release (nmol * ml-‘) Cells

PMA

4.5 h

PE Tumor PE + tumor PE Tumor PE + tumor

-

12 7 15 40 13 14

+ + +

20-24

h

35 + 13 17+1 20+4 61 + 21 20+7 24f3

12 8 2 3 5 3 3

Note. PE cells and/or tumor cells at 2 X 106. ml-’ were incubated for 4.5 h (one experiment) or for periods of 20-24 h (n experiments), and HsOs release calculated from the decrease in scopoletin fluorescence.

Hz02 release was not due to competition for binding of PMA. Maximum HzOz release was obtained at 2 X lo-’ M PMA, and increasing the PMA concentration did not overcome the inhibition. Table II shows that qualitatively similar results were obtained in an experiment in which phenol red was substituted for scopoletin. PMA-stimulated oxidation of phenol red was obtained with PE cells, and this oxidation was complete within 4 h. Slow oxidation of phenol red was obtained with both PE and tumor cells without PMA, oxidation was not completely blocked by catalase, and tumor cells inhibited the oxidation obtained with or without PMA. However, values for HzOz release calculated from oxidation of phenol red were lower than those calculated from loss of scopoletin fluorescence (Table I). In other experiments, normal lymphnode cells were substituted for the tumor (lymphoma) cells. The lymph-node cells did not grow during 24 h in culture, did not release H202, and did not inhibit PEcell HzOz release. Similarly, when the tumor cells were preincubated with mitomycin to block their growth, these cells released less H202 and were less inhibitory. Therefore, HzOz release by target cells and inhibition of PE-cell H202 release

359

RELEASE

appeared to be associated with metabolism and growth. As described above, it is possible that part of the apparent H202 release detected in these experiments was due to other reactions. Nevertheless, inhibition of PEcell H202 release by tumor cells was observed regardless of the method used to calculate HzOz release (for example, from the total decrease in scopoletin fluorescence or increase in phenol red absorbance, from the difference in results obtained with or without PMA, or from the difference in results obtained with HRP or catalase). Therefore, experiments were undertaken to determine the basis for inhibition, using measures of O2 uptake to evaluate the effect of tumor cells on the O2 metabolism of PE cells. 0, Uptake and H202 Release in Stirred Suspensions Figure 3 (top) shows plots of O2 concentration vs time for PE cells with and without PMA, in media containing HRP-scopoletin or catalase. Without PMA, O2 uptake was slow, and was not altered by HRP-scopoletin or catalase. With PMA, the rate of O2 uptake was increased. Also, there was a difference in rates obtained TABLE INHIBITION

II

OF PE-CELL Hz02 BY TUMOR CELLS HsOs release HRPphenol red

Cells PE Tumor PE + tumor PE Tumor PE + tumor

PMA + + +

4h 4 2 1 39 4 21

24h 8 3 6 36 7 19

RELEASE

(nmol

. ml-‘)

Catalasephenol red 4h

24h

-

9 6 9

6 3 4

Note. PE cells and/or tumor cells at 2 X 106. ml-’ were incubated with HRP-phenol red or catalasephenol red for 4 or 24 h, and Ha02 release calculated from oxidation of phenol red.

360

THOMAS

AND FISHMAN

Time (mini FIG. 3. Effect of PMA on O2 uptake. PE cells at 2 X 106*ml-’ were incubated with PMA (closed symbols) or without PMA (open symbols) in media containing HRP-scopoletin (0, 0) or catalase (m, 0). The choice of zero time is arbitrary; usually about 2-3 min was allowed for the electrode response to stabilize and for the effect of PMA to be complete.

in media containing HRP-scopoletin or catalase. This difference is due to the differing stoichiometry of Hz02 consumption by the two HeOz-consuming systems. HRP

HzOz + XHB -

2 Hz0 + X,

catalase

2 Hz02 -

2 Hz0 + Oz.

Any H202 that is released to the medium containing HRP is reduced to water, with the oxidation of XH2 to X, where XHB represents scopoletin or other peroxidase substrates that compete with scopoletin for oxidation. In contrast, HzOz released to the medium containing catalase dismutates to yield 0.5 mol of O2 per mole of HzOz. Assuming that all the O2 taken up by the cells was converted to HzOz and released to the medium, the observed rate of O2 uptake would be half as fast in the presence of catalase as in the presence of HRP-sco-

poletin. In Fig. 3, the rate was about 37.5% lower with catalase, as calculated by the method below, so that about ‘75% of the Oe taken up was released as HaOa. The difference in rates demonstrates that PMA stimulated O2 uptake and Hz02 release. Also, the specificity of catalase confirms that the difference in rates was due to release of Hz02 rather than any other peroxide. However, the results do not rule out release of other O2 metabolites in addition to HzOz. Figure 3 (top) also shows that the rate of O2 uptake decreased with time. This effect was due to the decreasing O2 concentration, rather than to the declining ability of the cells to take up Oz. If the suspension was reaerated within the first 10 min of incubation, O2 uptake resumed at the original rate. Also, if the O2 concentration was lowered by bubbling Nz through the suspension, the rate of O2 uptake was the same as that observed when O2 uptake by the cells had lowered the O2 concentration to that level. Although the ability of cells to take up O2 did not decrease during the lo- to 20min period required to measure O2 uptake, the ability of cells to respond to PMA with increased O2 uptake did decrease during prolonged incubations. About 1 to 4 h of incubation was required to deplete cells of the ability to increase O2 uptake in response to PMA. This loss of response took place in the presence or absence of PMA, and was faster when the O2 concentration was high. When the cells were not incubated, they retained at least 60% of their ability to take up O2 at the PMA-stimulated rate after 24 h at 5°C. In the absence of added HRP-scopoletin or catalase, H202 did not accumulate to detectable levels in the medium. That is, adding catalase did not result in a detectable burst of O2 evolution, and adding HRP-scopoletin after cells were removed did not result in loss of fluorescence. The limits of detection were estimated as 5-10 ~.LMH202. Therefore, the Hz02 that was released was rapidly eliminated. Without added HRP-scopoletin or catalase, the rate of O2 uptake was intermediate between the rates observed with the

HYDROGEN

PEROXIDE

two HzOz-consuming systems. If all the HzOz was eliminated by cellular catalase, then the observed rate of Oz uptake would be the same in the presence or absence of added catalase. If all the HzOz was eliminated in peroxidase-mediated reactions, or if HzOz did accumulate in the medium, then the observed rate would be the same with or without HRP-scopoletin. The intermediate rate indicates participation of both catalase- and peroxidase-mediated reactions in the elimination of HzOz. Mammalian cells contain both catalase and glutathione peroxidase, which prevent accumulation of HzOz to levels higher than the micromolar range (13). Figure 3 (bottom) shows the data replotted as In Oz vs time. Linear plots were obtained, indicating that O2 uptake could be described by the equations O2 = uepkt, or In Oz = In a - kt, where a is the Oz concentration at the start of the incubation (0.2 mM), k is the rate constant, and t is time. This observation indicates that the rate of Oz uptake was proportional to the Oz concentration. Therefore, the enzymatic mechanism of Oz uptake was not saturated with respect to Oz in this range of Oz concentration, i.e., the Km for Oz was far in excess of 0.2 mM. Results obtained by measuring Oz concentration vs time were replotted as In Oz vs time, and linear regression analysis used to fit the best straight line to the data and to calculate the slope or rate constant, k. The coefficient of determination was always greater than 0.98, provided that the data were limited to the first 30 min of incubation and to Oz concentrations in the 0.2-0.1 mM range. At lower Oz concentrations (not shown), plots deviated from linearity indicating a faster rate than predicted from the equations above. This O2 uptake appeared to be due to an enzymatic mechanism with a low maximum rate and high affinity for Oz, which was saturated at low O2 concentrations. This high-affinity O2 uptake might be due to a differing mechanism of

RELEASE

361

O2 reduction by the same enzyme(s) involved in the low-affinity Oz uptake. Such behavior has been observed in Oz reduction by flavoproteins such as xanthine oxidase (14, 15). The yield of H202 per mole of Oz taken up was about the same at high or low O2 concentrations. Also, cyanide did not inhibit the PMA-stimulated rate of O2 uptake, measured without HRP-scopoletin or catalase. Therefore, mitochondrial reduction of Oz to water did not make a large contribution to Oz uptake. The presence of PMA, HRP-scopoletin, or catalase had no detectable effect on Oz uptake by the tumor cells. With tumor cells, or PE cells without PMA, the Oz concentration did not change greatly, so that the rate of Oz uptake could be equally well described as proportional to Oz, or constant with time. For convenience, all results were converted to logarithmic form. Table III summarizes results obtained in a number of experiments of this kind. To facilitate comparisons, results are presented as rates as well as rate constants. As described above, the rate of O2 uptake at an Oz concentration greater than 0.1 mM can be calculated as k. Oz, with the assumption that the saturable portion of O2 uptake makes a negligible contribution to O2 uptake at high Oz concentrations. Rates of O2 uptake are given as kl (a), where kl is the rate constant obtained with HRPscopoletin. This value is the calculated rate of Oz uptake in an air-saturated suspension (0.2 mM at 37”C), in the presence of a peroxidase-mediated HzOz-consuming system. (The same rate would be obtained in a system in which the released Hz02 is not consumed and accumulates in the medium.) Similarly, the rate of HzOz release at a particular Oz concentration can be calculated as 2(kl - k.J * (O,), where k2 is the rate constant obtained with catalase. Rates of HzOz release are given as the calculated rate in an air-saturated suspension, or 2(kl - b). (a). Rates calculated in this way were proportional to the amount of cells. Table III shows that PMA stimulated O2 uptake by PE cells but had little or no effect on tumor cells. The PMA-stimulated rate of HzOz release was about 70% of 02

362

THOMAS

AND TABLE

O2 UPTAKE Rate

Cells

PMA

h

PE Tumor PE + tumor PE Tumor PE + tumor

+ + +

0.0078 0.0082 0.0137 0.0375 0.0087 0.0453

FISHMAN III

AND HzOz

RELEASE

constants (min-‘)

nmol k-2

0.0086 0.0140 0.0244 0.0303

O2 uptake 1.6 1.6 2.7 7.5 1.7 9.1

f + * f f +

0.5 0.2 1.0 1.2 0.5 1.3

+ ml-’

* min-’ HzOz

release

n

-0.3

f 0.8

3 3 3 6 3 5

-0.1 + 0.4 5.2 rfI 1.6 6.0 f 1.6

Note. Rate constants and calculated rates for O2 uptake and HzOz release were obtained the text, with PE and/or tumor cells at 2 X 106*ml-‘, where n is the number of experiments preparations of cells used to calculate the mean values and standard deviation.

uptake. In another experiment, phenol red was substituted for scopoletin. With PMA and 2 X lo6 PE cells-ml-‘, the observed rates of O2 uptake were 5.5 with HRPphenol red, 5.2 with phenol red, and 3.4 with catalase or catalase-phenol red. The calculated rate of HzOz release was 4.2 nmol - min-’ *ml-’ or 76% of O2 uptake. Without PMA, rates of O2 uptake were 1.0 with HRP-phenol red, and 0.9 with phenol red, catalase, or catalase-phenol red. The calculated rate of H202 release was 0.2 nmol * min-’ -ml-’ or 20% of O2 uptake. Table III also shows that the calculated rates of O2 uptake by PE and tumor cells together were about equal to the sum of the rates obtained with PE and tumor cells incubated separately. Therefore, tumor cells did not stimulate or inhibit 0s uptake by PE cells. Without PMA, no HzOz release from PE cells was detected during the loto 20-min period required to measure O2 uptake. Also, no HzOz was detected when PE and tumor cells were incubated together without PMA. Therefore, tumor cells did not replace PMA as a triggering agent for H202 release. In the presence of PMA, the rate of Hz02 release was about the same with PE cells alone, or with PE and tumor cells together. Therefore, tumor cells did not stimulate or inhibit the PMA-stimulated Hz02 release. Although tumor cells did not inhibit PEcell H202 release when measured as a func-

as described in with separate

tion of O2 concentration, tumor cells did inhibit when H202 release was measured as a function of time. Because the tumor cells consumed Oa, the PE cells were exposed to a lower average O2 concentration when tumor cells were present. After measuring O2 uptake for 30 min, the electrode was withdrawn and portions of the medium diluted for measurements of H202 release from the scopoletin fluorescence. With tumor and PE cells both at 2 X 106s ml-‘, the amount of H202 release in 30 min was inhibited by about 20%. If competition for 0s accounts for inhibition of HzOz release, then there should be no inhibition when the O2 concentration is held constant. Results in Fig. 4 show that no inhibition was observed when PE and tumor cells were incubated together under continuous aeration to maintain the 0s concentration at 0.2 mM. Figure 4 also shows that the total amount of H202 released was much less than would be calculated from the rates given in Table III. For example if the rate of HzOz release was 5 nmol - ml-’ - min-‘, then total HzOz release in 1 h at constant 0.2 mM O2 should be 300 nmol- ml-l, or about six- to sevenfold greater than observed. These results show that the ability of PMA-stimulated cells to release Hz02 (or the ability to respond to PMA) was exhausted when the O2 concentration was high. Therefore, the calculated rates of O2

HYDROGEN

PEROXIDE

FIG. 4. HZ02 release wtih constant 02. PE cells at 2 x 106. ml-’ were incubated alone (0) or with 2 x 106. ml-’ tumor cells (0) with HRP-scopoletin and PMA, in stirred suspensions in contact with 95% air5% COa. At the indicated times, portions were removed and centrifuged, and relative fluorescence of the diluted supernatants was measured.

uptake and Hz02 release are maximum possible rates, rather than rates that can be sustained for long periods. The amount of HzOz released during 1 h incubation with continuous aeration may provide an estimate of the maximum possible amount of H202 that can be released. The short incubation periods used in O2 uptake studies might not be sufficient to demonstrate an effect of tumor cells on the O2 metabolism of PE cells. Also, stirring would disrupt cell-cell contacts that might be required for such an effect. Therefore, experiments were carried out in which PE cells were first placed in monolayer culture either alone or with an equal number of tumor cells. After 3 h, the cells were resuspended, and O2 uptake was measured in stirred suspensions. After 3 h in culture, the ability of the PE cells to respond to PMA with increased O2 uptake and H202 release had declined, but those cells which were in contact with tumor cells were not inhibited relative to the control PE cells. For PE and tumor cells together, the calculated rates of PMA-stimulated Oa uptake and Hz02 release were 4.0 and 1.8 nmol * ml-‘. min-I, respectively. Macrophage

Populations

The calculated rates of O2 uptake and Hz02 release provided a method to com-

363

RELEASE

pare the activity of different cell populations. Table IV compares PE cells from animals injected with mineral oil, or with Coqnebacterium parvum, or both. The cells elicited with mineral oil were the most responsive to PMA, and PMA was the only substance observed to stimulate O2 uptake by these cells. Other agents (including LPS, WGL, or tuftsin) that may stimulate HzOz release from PE cells elicited with other agents or in other animals (16, 17) did not stimulate the mineral oilelicited rat PE cells. Similar results were obtained in experiments performed in monolayer culture. Table V shows rates obtained with PEcell subpopulations after fractionation on a gradient of BSA. The PE cells obtained at the lower-density positions (Bands A, B, C) had higher rates of O2 uptake and H202 release, and were more responsive to PMA. The A, B, and C band cells together represent only about 25% of the macroTABLE EFFECT

IV

OF ELICITING AGENT ON O2 UPTAKE AND HzOz RELEASE nmol

Eliciting

agent

C. parwm C. parvum + mineral Mineral

oil

Triggering agent PMA -

oil

PMA PMA WGL LPS Tuftsin

* ml-‘.

min-’

O2 uptake

I-Mb release

5.4 0.9 3.2

1.2 0.6 1.7

1.6 7.8 1.3 0.9 0.7

0.2 5.6 -

Note. C. powum-elicited exudates were obtained 7 days after injection of C. portrum (Burroughs-Wellcome, Research Triangle Park, N. C.) at 7 mg * kg-’ body wt. Alternatively, C. parvum and mineral oil were injected 7 days and 4-5 days, respectively, prior to collection of the exudate. Mineral oil-elicited exudates were obtained as under Materials and Methods. Calculated rates of Oa uptake and Ha02 release were obtained as described in the text, with PE cells at 2 x 10’. ml-‘, and no triggering agent, or with 2 x 10-s M PMA, 10 pg. ml-’ WGL, 0.3 mg * ml-’ LPS, or 10 pg. ml-’ tuftsin.

364

THOMAS TABLE

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V

OF PE-CELL POPULATION IN Oa UPTAKE AND HzOz RELEASE

HETEROGENEITY

nmol * ml-’ * min-’ Band A-B

Percentage of total cells” 3

C

11

D

53

Eb

33

PMA

02 uptake

KG release

+ + + +

6.6 14.6 4.3 8.0 1.5 3.8 0.6 1.9

1.2 10.9 0.6 2.9 0.3 0.9 0.3

Note. PE cells were separated into subpopulations A (50% BSA interface), B (Wll%), C (11/20%), D (20/30%), and E (30/35%). Calculated rates of Oa uptake and HaOa release were obtained as described in the text with 4 X lo5 cells * ml-’ (pooled A and B), 1 X lo6 cells. ml-’ (C), 4 X lo6 cells * ml-’ (D), and 2 X 10 cells. ml-’ (E). Results are normalized to 2 X lo6 cells. ml-‘. a Percentage of total cells was calculated excluding those cells which were pelleted. *Results from a separate experiment.

phage population, but account for 48% of 0s uptake and 66% of HsOs release by the macrophages. Also, in three experiments on the lowest-density subpopulation, HsOs release was consistently detected in the absence of PMA. In other experiments, tumor cells at a 1:l ratio to the PE-cell subpopulations did not alter the rate constants for 0s uptake and Hz02 release by the PE cells. DISCUSSION

The release of toxic 0s metabolites may be one of the mechanisms of macrophage cytotoxicity, or may serve as an indicator that cytotoxic activities are being expressed. PMA might take the place of a tumor-specific signal, triggering the expression of cytotoxic activities in the absence of tumor cells. If macrophages can recognize tumor cells, and PMA takes the place of the recognition signal, then the presence of tumor cells should have the

FISHMAN

same triggering effect as PMA. In a recent review (2), unpublished results were cited to the effect that tumor cells stimulated 0, release from mouse peritoneal macrophages. In other studies (4, 5), PMA was required for HzOz-mediated antitumor activity, suggesting that tumor cells did not trigger H202 release. In results presented here, tumor cells did not replace PMA as a triggering agent for rat peritoneal macrophages. Therefore, the results suggest that contact with tumor cells would not be sufficient to cause high rates of HsOs release from these macrophages in vivo, and that some other signal or triggering agent (such as an antibody (18, 19)) would be required. On the other hand, it appeared that Hz02 was released at low rates in the absence of PMA or tumor cells. That is, there was a slow decrease in scopoletin fluorescence in the absence of PMA. Also, Hz02 release in the absence of PMA could be detected by the method of measuring 0s uptake rates, when the PE cells were fractionated and the most active cells were concentrated and examined separately. If these results indicate cytotoxic activity, then they suggest that cytotoxic activity is expressed continuously rather than only in the presence of tumor cells. Rather than stimulating H202 release, tumor cells inhibited Hz02 release under the conditions used to measure antitumor activity. However, no evidence was obtained for a direct effect of tumor cells on the 0s metabolism of the macrophages. Instead, the probable basis for inhibition was the competition between the macrophages and proliferating tumor cells for the supply of Oz. The macrophage Hz02releasing mechanism was not saturated with respect to O2 even in air-saturated suspensions, so that changes in 0s concentration resulted in large changes in the rate of H202 release. This low-affinity 0s uptake appeared different from the highaffinity process reported for 0; release from human polymorphonuclear leukocytes (20). Another report indicated that macrophage 0; release was not proportional to cell density in assays performed in monolayer culture (21), which may in-

HYDROGEN

PEROXIDE

dicate a decreasing O2 supply with increasing cell density. Competition for Oz may in part account for variability in results reported for cytotoxicity assays. The Oz concentration at the unstirred surface of a layer of metabolizing cells may be very low, resulting in suppression of Oz-dependent cytotoxicity. Competition for Oz would be minimized at low cell densities, high EC:TC ratios, and short incubations. Also, H20z-releasing activity was highest in a subpopulation of the macrophages, although all the PE-cell subpopulations took up Oz. Removing the less active cells might favor 02-dependent cytotoxicity. Differences in cytotoxic activity of activated, elicited, and resident macrophage populations (22-24) might be due to differing ratios of the macrophage subpopulations. Macrophage populations are heterogeneous with regard to surface receptors, enzyme content, and phagocytic or cytotoxic activities (25, 26). Previous studies showed that PE cells could be fractionated on discontinuous BSA gradients to yield macrophage subpopulations differing in their state of activation or maturation (68). The macrophages obtained at the lowdensity position are larger, less phagocytic, contain less peroxidase and more hydrolase activity, and are more active in antitumor assays ((6~8), and M. Fishman, unpublished results). In this report, O2 uptake, HzOz release, and responsiveness to PMA were highest in this small subpopulation. To our knowledge, this is the first report that macrophage populations are heterogeneous with regard to their Oz metabolism, although unpublished results were cited to the effect that such populations contain cells that differ in the intracellular location of 0; generation (2). It should be noted that the assay for HzOz release based on scopoletin fluorescence gave ambiguous results, particularly during prolonged incubations (4-24 h). That is, a portion of the decrease in fluorescence was not blocked by catalase. Similar results were observed in another study (lo), and the phenomenon was described as “disappearance of reduced scopoletin without oxidation.” In that study,

365

RELEASE

with 2 PM scopoletin and 2.5 X lo6 leukocytes * ml-‘, the minimum loss of fluorescence was about 10% in 30 min, and was about twice as high (18%) when the cells were ingesting particles. These results correspond to a loss of 0.2-0.4 PM scopoletin in 30 min. If this phenomenon could continue at the same rate for 24 h, the loss would be lo-20 PM. With comparable numbers of PE cells and 0.1 mM scopoletin, we observed a 20-40 PM loss in 24 h, and about a 20 PM loss with tumor cells. Therefore, these results are quantitatively comparable to the earlier study. However, it is not clear that all of this decrease in fluorescence occurred without oxidation, in that the decrease resembled the PMAstimulated, catalase-blocked decrease in most respects, except for the slower rate. The results do suggest that the fluorescent assay should not be used uncritically to measure H202 release, particularly in longer incubations. Scopoletin and phenol red were also used in the assay for H202 release which is based on differences in observed rates of O2 uptake. The use of scopoletin in this assay is fundamentally different from that in the fluorescent assay. That is, it was used as an electron donor to favor peroxidase-mediated reduction of HzOz to water. The requirements for such a donor are that it be nontoxic in both reduced and oxidized forms, and not reduced by 0;. If scopoletin is oxidized by agents other than Hz02, or if it is lost in nonoxidative reactions, then those reactions should not influence the difference in O2 uptake rates. Also, only lo-20 min was required to measure O2 uptake which should minimize any problems in the use of scopoletin (10). ACKNOWLEDGMENTS We thank M. Morrison and G. Schonbaum for helpful discussions, Kate P. Bates, M. Margaret Jefferson, and Gail Crawford for technical assistance and Pat Nicholas for manuscript preparation. REFERENCES 1. HIBBS, J. B., JR., CHAPMAN, H. A., AND WEINBERG, J. B. (1978) J. Reticuloendothel. Sot. 24, 549570.

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2. BADWAY, J. A., AND KARNOVSKY, M. L. (1980) Annu. Rev. B&hem 49,695-726. 3. NATHAN, C. F., AND ROOT, R. K. (1977) J. Eq. Med. 146,1648-1662. 4. NATHAN, C. F., BRUKNER, L. H., SILVERSTEIN, C. S., AND COHN, Z. A. (1979) J. Exp. Med 149, 84-99. 5. NATHAN, C. F., SILVERSTEIN, C. S., BRUKNER, L. H., AND COHN, Z. A. (1979) J. Exp. Med 149, 100-113. 6. WEINBERG, D. S., FISHMAN, M., AND VEIT, B. (1978) Cell. ImmunoL 38, 94-104. 7. RICE, S. G., AND FISHMAN, M. (1974) Cell. ImmunoL 11,130-145. 8. FISHMAN, M., AND WEINBERG, D. S. (1979) Cell. ImmunoL 45,437-445. 9. PERSCHKE, H., AND BRODA, E. (1961) Nature (lhrukm) 190, 257-258. 10. ROOT, R. K., METCALF, J., OSHINO, N., ANDCHANCE, B. (1975) J. Clin Invest. 55, 945-955. 11. BOVERIS, A., MARTINO, E., AND STOPPANI, A. 0. M. (1977) AnaL Biochem. 80,145-158. 12. PICK, E., AND KEISARI, Y. (1980) J. ZmmunoL Methods 38,161-170. 13. CHANCE, B., SIES, H., AND BOVERIS, A. (1979) PhysioL Rev. 59, 527-605. 14. FRIDOVICH, I. (1970) J. BioL Chem 245,4053-4057.

FISHMAN

15. SINGER, T. P., AND EDMONDSON, D. E. (1974) in Molecular Oxygen in Biology: Topics in Molecular Oxygen Research (Hayaishi, O., ed.), pp. 315-337, North-Holland, Amsterdam. 16. TOMIOKA, H., ANDSAITO, H. (1980) Ideck Immun. 28, 336-343. 17. PABST, M. J., AND JOHNSTON, R. B., JR. (1980) J. Exp. Med. 151,101-114. 18. NATHAN, C. F., BRLJKNER, L., KAPLAN, G., UNKELESS, J., AND COHN, Z. (1980) J. Exp. Med 152, 183-197. 19. NATHAN, C. F., AND COHN, Z. (1980) J. Exp. Med. 152,198-208. GABIG, T. G., BEARMAN, S. I., AND BABIOR, B. M. 20’ (1979) Blood 53,1133-1139. n. LI. JOHNSTON, R. B., JR. (1978) Fed Proc. 37, 27592764. 22. NORTH, R. J. (1978) J. ZmmunoL 121.806-809. 23. KARNOVSKY, M. L. (1978) J. ImmunoL 121, 809813. 24. COHN, Z. A. (1978) J. ImmunoL 121, 813-816. 25. FISHMAN, M., AND ADLER, F. L. (1970) in Mononuclear Phagocytes (van Furth, R., ed.), pp. 581-594, Blackwell, Oxford. 26. WALKER, W. S. (1976) J. ReticuloendotheL Sot 20, 57-65.