Defective Production of Reactive Oxygen Intermediates by Tumor ...
Journal
Defective Production of Reactive Tumor-Associated Macrophages Pietro
Ghezzi,
Annalaura Istituto
Erroi,
Raffaefla
di Ricerche
Acero,
Farmacologiche
of Leukocyte
Oxygen Exposed Mario “Mario
Milan,
42:84-90
(1987)
Intermediates by to Phorbol Ester
Salmona, Negri,”
Biology
and Alberto
Mantovani
Italy
Macrophages were isolated from poorly immunogenic metastatic sarcomas (mFS6 and MN/MCA1) of C57BL16 origin. Tumor-associated macrophages (TAM) showed little release of superoxide when exposed to phorbol myristate acetate. When exposed to a phagocytic stimulus (zymosan), TAM released appreciable amounts of superoxide. TAM had a lower number of specific bInding sites for phorbol esters than resident or caseinateelicited peritoneal macrophages, but had normal NADPH-cytochrome C reductase. The tumor environment, possibly through previously demonstrated products of neoplastlc cells, may Influence the functional status of in situ macrophages and, thus, impair host anti-tumor and anti-microbial defense mechanisms. Key words;
tumor-associated
macrophages,
INTRODUCTION Reactive oxygen intermediates (ROI) play an important role in various functions of macrophages, such as microbicidal and tumoricidal activity [101. Conditioned medium of tumor cell lines and of certain nonmalignant cells has been shown to suppress the secretion of ROI by macrophages and their microbicidal activity [27,281. The in vivo relevance of these observations remains to be firmly established. Macrophages are a major component of the lymphoreticular infiltrate of neoplastic tissues [9,18]. The present study was designed to characterize the secretion of ROI by tumor-associated macrophages (TAM) in response to soluble (phorbol myristate acetate, PMA) or phagocytic stimuli (zymosan particles, Zy). We report that TAM from various transplanted malignant murine tumors are poor producers of ROI in response to PMA. Defective production of ROl was associated with reduced number of high affinity phorbol ester binding sites and with normal NADPH-cytochrome C reductase. The tumor environment, possibly through previously demonstrated in vitro products of neoplastic cells [27,281, might orient the functional status of in situ macrophages in a sense less favorable to the host.
MATERIALS Mice
AND METHODS
and Tumors
Male C57BL/6 (8 to 10 weeks old) mice were obtained from Charles River Breeding Laboratories, Calco, Italy. The chemically induced mFS6 and MN/MCA 1 sarcomas of C57BL/6 origin, free of contaminating lactic dehydrogenase virus, have been described in detail [16,17]. Both © 1987
Alan
R. Liss,
Inc.
phorbol
esters,
oxidative
metabolism
tumors are poorly immunogenic, macrophage content of 15-25% Peritoneal
metastatic, [16,17].
and have
a
Macrophages
Peritoneal exudate cells were obtained from unstimulated mice or mice injected 7 days previously with 1 ml of 6% caseinate (Merck, Darmstadt, Federal Rep. of Germany). The peritoneal cavity was washed with 4 ml of Eagle’s basal medium (BME, Eurobio, Paris, France). Lavage fluids from 4-8 mice were pooled, centrifuged at 400 x g for 5 mm, and resuspended in RPMI 1640 medium (GIBCO-Biocult, Glasgow, Scotland) with 50 g/ml gentamycin. Differential cell counts were made on cytocentrifuge smears, and 7.5 x i05 macrophages were seeded in I ml of medium in 16-mm wells (Costar, Cambridge, MA, or Nunc, Roskilde, Denmark). After incubation for 2 h, nonadherent cells were thoroughly washed off with jets of medium, and monolayers were more than 95% macrophages, as assessed by morphologic and functional criteria [16,17]. Tumor-Associated
Macrophages
TAM were isolated as previously described [7,8,16, 17]. Unless otherwise specified, tumors were obtained 15-20 days after transplantation. Tumors were minced with scissors and disaggregated by exposure for 45 mm at 37#{176}C to 0.3% collagenase (Sigma Chemicals, St. Louis, MO) in BME containing 10 g/ml DNase. Disaggregated Received Reprint ogiche
September
17, 1986; accepted
December
1, 1986.
requests: Alberto Mantovani, Istituto di Richerche “Mario Negri,” via Eritrea 62, 20157 Milan, Italy.
Farmacol-
Superoxide cells were centrifuged at 400 x g and resuspended in RPM! 1640 medium. The percentage of TAM in disaggregated cell suspensions, assessed as cells rosetting with antibody-coated erythrocytes, was 16-23% for the mFS6 sarcoma, 14-17% for the MN/MCA1 sarcoma; and 45% for the MS2 sarcoma. 7.5 X i05 TAM in 1 ml of RPM! 1644) mediwn were seeded in 16-mm wells and incubated for 60 mm at 37#{176}C,with gentle resuspension every 10-15 mm. Nonadherent cells were vigorously washed with jets of medium, and adherent cells were more than 95% macrophages, as assessed by morphology, uptake of neutral red, binding, and phagocytosis of antibody-coated erythrocytes [17]. Contaminating cells were (in order of frequency) tumor cells, unidentified small mononuclear cells, and polymorphs. In a number of experiments, to obtain purer TAM, adherent cells were cultured for 24 or 48 h in serum-free 199 medium, before further washing [8,17]. Under these conditions, more than 99% of the adherent cells were macrophages, using the above-mentioned criteria.
Production
by Tumor-Associated
Macrophages
PDBU was then added at concentrations to 200 nM in the presence and absence
85
ranging from 10 of a large excess
(30 tiM) of unlabelled PDBU (Sigma), to calculate the specific binding. After 30 mm, cells were washed three times with cold PBS containing 0.1 % BSA and then solubiized overnight in 0.8% Triton X-lOO. Radioactivity of the cell lysate was determined by liquid scintillation. Specific 3H-PDBU binding is expressed as pmol/ 106 cells and pmol/mg protein. Stability of 3H-PDBU was checked as follows: 3H-PDBU (100 nM) was incubated with or without macrophages, as described above. After 30 mm, supernatants were harvested, lyophilized, and dissolved in 0.2 ml of ethyl acetate. Ten d were applied to thin-layer chromatography (TLC) precoated silica gel plates (20 x 20 cm, Merck). TLC was developed with ethyl acetate, the plates were cut in 0.5-cm pieces, and radioactivity of each piece was determined. NADPH-Cytochrome
C Reductase
Cells prepared as described above were rinsed with PBS, disrupted by two cycles of freezing and thawing, Release of Superoxide Anion and finally resuspended in 1 ml of 0.05 M Na-phosphate O was measured by the superoxide dismutase-inhibbuffer, pH 7.4, containing 80 fIM cytochrome c. Absorbitable reduction of ferricytochrome c [12]. Briefly, macance was monitored at 550 nm in a recording spectrophorophage monolayers were rinsed and incubated at 37#{176}C tometer using a cuvette without macrophages as a with 1 ml of Hank’s balanced salt solution (HBSS) withreference. When a stable baseline was obtained, NADPH was added at a final concentration of 1mM. Data are out phenol red, containing 80 1iM cytochrome c (Type expressed as nmol cytochrome c reduced/mm x mg of ifi, Sigma) in the presence and absence of opsonized Zymosan (5 mg/mi) or PMA (routinely 150 ng/ml, protein. Sigma). At various times after incubation (routinely 60 mm), supernatants were harvested, and absorbance was RESULTS measured at 550 nm. The amount of cytochrome c reduced was calculated using an extinction coefficient for It has been reported that proteolytic enzymes such as reduced cytochrome c of 2 x l0 M cm. Three trypsin enhance the production of ROI by macrophages replicates per experimental group were used throughout. in response to PMA [11]. It was, therefore, important to Macrophages from three wells were used to determine assess in a preliminary series of experiments whether the the protein content of the monolayers by the method of enzyme used to disaggregate tumor tissues affected the Lowry et al [15], with bovine serum albumin (BSA) as production of ROI. As shown in Table I, exposure of standard. Three wells for each macrophage group were resident or caseinate-elicited macrophages to collagenase fixed and stained with Duff Quick (Harleco, Gibbstown, (0.3%) used to disaggregate tumors did not appreciably NJ), and the number of macrophages actually adhering modify O release in response to PMA or Zy. Higher was determined by counting 20 microscopic fields. Reconcentrations of collagenase (1.5 and 3%) reduced the sults are expressed as nmoles of O released per mg oxidative burst of macrophages subsequently exposed to protein or per 106 macrophages in a 60-mm incubation, PMA (data not shown). Table 2 and Figure 1 summarize unless otherwise specified. the results of a series of experiments in which we measured O production by TAM exposed to PMA (150 ng/ Binding Studies ml) or Zy (5 mg/mI). As previously described [21,28] resident peritoneal Binding of 3H-phorbol-12, 13-dibutyrate (PDBU, specific activity 10.5 Ci/mmol, Amersham) was measured macrophages responded poorly to PMA with 55.1 ± 3.3 in adherent macrophages in 16-mm wells, as previously (five experiments) nmol/mg protein. Inoculation of casdescribed [4]. Briefly, after removal of nonadherent cells, einate 7 days before the assay resulted in peritoneal adherent cells with a vigorous response to PMA (361.9 macrophages were incubated at 37#{176}Cfor 30 mm in phosphate-buffered saline (PBS) containing 0.1 % BSA ± 61.3 nmol/mg protein, eight experiments performed), (fraction V, Sigma) in a final volume of 0.2 ml. 3Hin agreement with previous data [21,28]. The Oj produc-
86
Ghezzi
et at
TABLE 1. Effect Macrophaqesa
of Enzymes
to Disaggregate
Tumors on Production
Resident Macrophages cxposed to
Stimulus
aResults
are mean
Caseinate-elicited
nmol/mg
protein
120.4 120.0
± 18.0 ± 35.6
8.9 ± 1.6 9.8 ± 2.0
± 15.! ± 61.8
8.7 7.9
None
Collagenase
macrophages nmol/ 106 cells
None PMA Zy
Medium
-
PMA Zy
115.1 104.4
± S.E.
of three experiments
of 02 by Normal
nmol/mg protein
351.4 147.5
-
performed.
macrophages nmol/ 106 cells
± 114.0 ± 53.9
30.6 ± 0.8 12.7 ± 0.9
± 86.3 ± 7.2
31.2 ± 2.8 11.4 ± 0.6
-
± 1.2 ± 4.7
Macrophage
445.8 162.0 monolayers
-
were exposed
to collagenase
(0.3%) for 45 mm and washed. bNot detectable.
TABLE 2. 0
Release
by Peritoneal
Macrophages
From mFS6 Sarcoma-Bearing O
Mice
Macrophages
Normal
aMice
were
examined
15 days
Caseinate-elicited
61.3 210.2
Resident Caseinate-elicited
193.6
after
tumor
65.5 inoculation.
proteinl
60 mm)
PMA
Resident
Tumorbearinga
(nmol/mg
Mice
Data
are mean
Zy 98.6 103.3 40.5 67.4
± 5.5 ± 4.1 ± 34.3 ± 38.5 ± S.E.
of experiments
± ± ± ±
performed
13 3.9 21.8 0 in
triplicate.
tion in response to Zy was not substantially altered by caseinate. When we studied mice 15 days after transplantation of poorly immunogenic metastatic tumors, the O production by resident or caseinate-elicited peritoneal macrophages in response to PMA or Zy was similar to that of normal control mice (Table 2). TAM isolated from the two tumors considered in this study had poor responsiveness to PMA, with values of 28.6 ± 13.7 and 38.2 ± 16.4 nmol/mg protein (five experiments) for the mFS6 and MN/MCA 1 sarcoma, respectively (p < 0.01 versus caseinate-elicited macrophages by Student’s t-test, Fig. 1A. The same was true when data were expressed per 106 cells). Zy elicited an appreciable O response in TAM with 90.7 ± 22.6 and 182.0 ± 41.9 nmol/mg protein for the mFS6 and MN/MCA 1 sarcoma, respectively. The results summarized in Figure 1 were obtained with PMA at a concentration of 150 ng/ml and an assay of 60 mm. The poor response of TAM to PMA was evident when different concentrations of PMA or different incubation times were used (Figs. 2, 3). In these experiments, O generation by TAM was always significantly lower (p < 0.01 by Student’s t-test) when compared with caseinate-elicited macrophages, regardless of PMA concentration (Fig. 2) or incubation time (Fig. 3). TAM were assayed routinely 2 weeks after tumor inoculation, but the poor responsiveness to PMA was also found when macrophages were examined 9 to 25 days after transplantation.
Superoxide was the ROl measured routinely, but the poor responsiveness to PMA was also evident when H2O2 production was assessed by the horseradish peroxidase/ scopoletin fluorometric method [3] (data not shown). In the experiments described so far, macrophages were evaluated immediately upon isolation from mice. TAM contained a small but appreciable (
Defective Production of Reactive Tumor-Associated Macrophages Pietro
Ghezzi,
Annalaura Istituto
Erroi,
Raffaefla
di Ricerche
Acero,
Farmacologiche
of Leukocyte
Oxygen Exposed Mario “Mario
Milan,
42:84-90
(1987)
Intermediates by to Phorbol Ester
Salmona, Negri,”
Biology
and Alberto
Mantovani
Italy
Macrophages were isolated from poorly immunogenic metastatic sarcomas (mFS6 and MN/MCA1) of C57BL16 origin. Tumor-associated macrophages (TAM) showed little release of superoxide when exposed to phorbol myristate acetate. When exposed to a phagocytic stimulus (zymosan), TAM released appreciable amounts of superoxide. TAM had a lower number of specific bInding sites for phorbol esters than resident or caseinateelicited peritoneal macrophages, but had normal NADPH-cytochrome C reductase. The tumor environment, possibly through previously demonstrated products of neoplastlc cells, may Influence the functional status of in situ macrophages and, thus, impair host anti-tumor and anti-microbial defense mechanisms. Key words;
tumor-associated
macrophages,
INTRODUCTION Reactive oxygen intermediates (ROI) play an important role in various functions of macrophages, such as microbicidal and tumoricidal activity [101. Conditioned medium of tumor cell lines and of certain nonmalignant cells has been shown to suppress the secretion of ROI by macrophages and their microbicidal activity [27,281. The in vivo relevance of these observations remains to be firmly established. Macrophages are a major component of the lymphoreticular infiltrate of neoplastic tissues [9,18]. The present study was designed to characterize the secretion of ROI by tumor-associated macrophages (TAM) in response to soluble (phorbol myristate acetate, PMA) or phagocytic stimuli (zymosan particles, Zy). We report that TAM from various transplanted malignant murine tumors are poor producers of ROI in response to PMA. Defective production of ROl was associated with reduced number of high affinity phorbol ester binding sites and with normal NADPH-cytochrome C reductase. The tumor environment, possibly through previously demonstrated in vitro products of neoplastic cells [27,281, might orient the functional status of in situ macrophages in a sense less favorable to the host.
MATERIALS Mice
AND METHODS
and Tumors
Male C57BL/6 (8 to 10 weeks old) mice were obtained from Charles River Breeding Laboratories, Calco, Italy. The chemically induced mFS6 and MN/MCA 1 sarcomas of C57BL/6 origin, free of contaminating lactic dehydrogenase virus, have been described in detail [16,17]. Both © 1987
Alan
R. Liss,
Inc.
phorbol
esters,
oxidative
metabolism
tumors are poorly immunogenic, macrophage content of 15-25% Peritoneal
metastatic, [16,17].
and have
a
Macrophages
Peritoneal exudate cells were obtained from unstimulated mice or mice injected 7 days previously with 1 ml of 6% caseinate (Merck, Darmstadt, Federal Rep. of Germany). The peritoneal cavity was washed with 4 ml of Eagle’s basal medium (BME, Eurobio, Paris, France). Lavage fluids from 4-8 mice were pooled, centrifuged at 400 x g for 5 mm, and resuspended in RPMI 1640 medium (GIBCO-Biocult, Glasgow, Scotland) with 50 g/ml gentamycin. Differential cell counts were made on cytocentrifuge smears, and 7.5 x i05 macrophages were seeded in I ml of medium in 16-mm wells (Costar, Cambridge, MA, or Nunc, Roskilde, Denmark). After incubation for 2 h, nonadherent cells were thoroughly washed off with jets of medium, and monolayers were more than 95% macrophages, as assessed by morphologic and functional criteria [16,17]. Tumor-Associated
Macrophages
TAM were isolated as previously described [7,8,16, 17]. Unless otherwise specified, tumors were obtained 15-20 days after transplantation. Tumors were minced with scissors and disaggregated by exposure for 45 mm at 37#{176}C to 0.3% collagenase (Sigma Chemicals, St. Louis, MO) in BME containing 10 g/ml DNase. Disaggregated Received Reprint ogiche
September
17, 1986; accepted
December
1, 1986.
requests: Alberto Mantovani, Istituto di Richerche “Mario Negri,” via Eritrea 62, 20157 Milan, Italy.
Farmacol-
Superoxide cells were centrifuged at 400 x g and resuspended in RPM! 1640 medium. The percentage of TAM in disaggregated cell suspensions, assessed as cells rosetting with antibody-coated erythrocytes, was 16-23% for the mFS6 sarcoma, 14-17% for the MN/MCA1 sarcoma; and 45% for the MS2 sarcoma. 7.5 X i05 TAM in 1 ml of RPM! 1644) mediwn were seeded in 16-mm wells and incubated for 60 mm at 37#{176}C,with gentle resuspension every 10-15 mm. Nonadherent cells were vigorously washed with jets of medium, and adherent cells were more than 95% macrophages, as assessed by morphology, uptake of neutral red, binding, and phagocytosis of antibody-coated erythrocytes [17]. Contaminating cells were (in order of frequency) tumor cells, unidentified small mononuclear cells, and polymorphs. In a number of experiments, to obtain purer TAM, adherent cells were cultured for 24 or 48 h in serum-free 199 medium, before further washing [8,17]. Under these conditions, more than 99% of the adherent cells were macrophages, using the above-mentioned criteria.
Production
by Tumor-Associated
Macrophages
PDBU was then added at concentrations to 200 nM in the presence and absence
85
ranging from 10 of a large excess
(30 tiM) of unlabelled PDBU (Sigma), to calculate the specific binding. After 30 mm, cells were washed three times with cold PBS containing 0.1 % BSA and then solubiized overnight in 0.8% Triton X-lOO. Radioactivity of the cell lysate was determined by liquid scintillation. Specific 3H-PDBU binding is expressed as pmol/ 106 cells and pmol/mg protein. Stability of 3H-PDBU was checked as follows: 3H-PDBU (100 nM) was incubated with or without macrophages, as described above. After 30 mm, supernatants were harvested, lyophilized, and dissolved in 0.2 ml of ethyl acetate. Ten d were applied to thin-layer chromatography (TLC) precoated silica gel plates (20 x 20 cm, Merck). TLC was developed with ethyl acetate, the plates were cut in 0.5-cm pieces, and radioactivity of each piece was determined. NADPH-Cytochrome
C Reductase
Cells prepared as described above were rinsed with PBS, disrupted by two cycles of freezing and thawing, Release of Superoxide Anion and finally resuspended in 1 ml of 0.05 M Na-phosphate O was measured by the superoxide dismutase-inhibbuffer, pH 7.4, containing 80 fIM cytochrome c. Absorbitable reduction of ferricytochrome c [12]. Briefly, macance was monitored at 550 nm in a recording spectrophorophage monolayers were rinsed and incubated at 37#{176}C tometer using a cuvette without macrophages as a with 1 ml of Hank’s balanced salt solution (HBSS) withreference. When a stable baseline was obtained, NADPH was added at a final concentration of 1mM. Data are out phenol red, containing 80 1iM cytochrome c (Type expressed as nmol cytochrome c reduced/mm x mg of ifi, Sigma) in the presence and absence of opsonized Zymosan (5 mg/mi) or PMA (routinely 150 ng/ml, protein. Sigma). At various times after incubation (routinely 60 mm), supernatants were harvested, and absorbance was RESULTS measured at 550 nm. The amount of cytochrome c reduced was calculated using an extinction coefficient for It has been reported that proteolytic enzymes such as reduced cytochrome c of 2 x l0 M cm. Three trypsin enhance the production of ROI by macrophages replicates per experimental group were used throughout. in response to PMA [11]. It was, therefore, important to Macrophages from three wells were used to determine assess in a preliminary series of experiments whether the the protein content of the monolayers by the method of enzyme used to disaggregate tumor tissues affected the Lowry et al [15], with bovine serum albumin (BSA) as production of ROI. As shown in Table I, exposure of standard. Three wells for each macrophage group were resident or caseinate-elicited macrophages to collagenase fixed and stained with Duff Quick (Harleco, Gibbstown, (0.3%) used to disaggregate tumors did not appreciably NJ), and the number of macrophages actually adhering modify O release in response to PMA or Zy. Higher was determined by counting 20 microscopic fields. Reconcentrations of collagenase (1.5 and 3%) reduced the sults are expressed as nmoles of O released per mg oxidative burst of macrophages subsequently exposed to protein or per 106 macrophages in a 60-mm incubation, PMA (data not shown). Table 2 and Figure 1 summarize unless otherwise specified. the results of a series of experiments in which we measured O production by TAM exposed to PMA (150 ng/ Binding Studies ml) or Zy (5 mg/mI). As previously described [21,28] resident peritoneal Binding of 3H-phorbol-12, 13-dibutyrate (PDBU, specific activity 10.5 Ci/mmol, Amersham) was measured macrophages responded poorly to PMA with 55.1 ± 3.3 in adherent macrophages in 16-mm wells, as previously (five experiments) nmol/mg protein. Inoculation of casdescribed [4]. Briefly, after removal of nonadherent cells, einate 7 days before the assay resulted in peritoneal adherent cells with a vigorous response to PMA (361.9 macrophages were incubated at 37#{176}Cfor 30 mm in phosphate-buffered saline (PBS) containing 0.1 % BSA ± 61.3 nmol/mg protein, eight experiments performed), (fraction V, Sigma) in a final volume of 0.2 ml. 3Hin agreement with previous data [21,28]. The Oj produc-
86
Ghezzi
et at
TABLE 1. Effect Macrophaqesa
of Enzymes
to Disaggregate
Tumors on Production
Resident Macrophages cxposed to
Stimulus
aResults
are mean
Caseinate-elicited
nmol/mg
protein
120.4 120.0
± 18.0 ± 35.6
8.9 ± 1.6 9.8 ± 2.0
± 15.! ± 61.8
8.7 7.9
None
Collagenase
macrophages nmol/ 106 cells
None PMA Zy
Medium
-
PMA Zy
115.1 104.4
± S.E.
of three experiments
of 02 by Normal
nmol/mg protein
351.4 147.5
-
performed.
macrophages nmol/ 106 cells
± 114.0 ± 53.9
30.6 ± 0.8 12.7 ± 0.9
± 86.3 ± 7.2
31.2 ± 2.8 11.4 ± 0.6
-
± 1.2 ± 4.7
Macrophage
445.8 162.0 monolayers
-
were exposed
to collagenase
(0.3%) for 45 mm and washed. bNot detectable.
TABLE 2. 0
Release
by Peritoneal
Macrophages
From mFS6 Sarcoma-Bearing O
Mice
Macrophages
Normal
aMice
were
examined
15 days
Caseinate-elicited
61.3 210.2
Resident Caseinate-elicited
193.6
after
tumor
65.5 inoculation.
proteinl
60 mm)
PMA
Resident
Tumorbearinga
(nmol/mg
Mice
Data
are mean
Zy 98.6 103.3 40.5 67.4
± 5.5 ± 4.1 ± 34.3 ± 38.5 ± S.E.
of experiments
± ± ± ±
performed
13 3.9 21.8 0 in
triplicate.
tion in response to Zy was not substantially altered by caseinate. When we studied mice 15 days after transplantation of poorly immunogenic metastatic tumors, the O production by resident or caseinate-elicited peritoneal macrophages in response to PMA or Zy was similar to that of normal control mice (Table 2). TAM isolated from the two tumors considered in this study had poor responsiveness to PMA, with values of 28.6 ± 13.7 and 38.2 ± 16.4 nmol/mg protein (five experiments) for the mFS6 and MN/MCA 1 sarcoma, respectively (p < 0.01 versus caseinate-elicited macrophages by Student’s t-test, Fig. 1A. The same was true when data were expressed per 106 cells). Zy elicited an appreciable O response in TAM with 90.7 ± 22.6 and 182.0 ± 41.9 nmol/mg protein for the mFS6 and MN/MCA 1 sarcoma, respectively. The results summarized in Figure 1 were obtained with PMA at a concentration of 150 ng/ml and an assay of 60 mm. The poor response of TAM to PMA was evident when different concentrations of PMA or different incubation times were used (Figs. 2, 3). In these experiments, O generation by TAM was always significantly lower (p < 0.01 by Student’s t-test) when compared with caseinate-elicited macrophages, regardless of PMA concentration (Fig. 2) or incubation time (Fig. 3). TAM were assayed routinely 2 weeks after tumor inoculation, but the poor responsiveness to PMA was also found when macrophages were examined 9 to 25 days after transplantation.
Superoxide was the ROl measured routinely, but the poor responsiveness to PMA was also evident when H2O2 production was assessed by the horseradish peroxidase/ scopoletin fluorometric method [3] (data not shown). In the experiments described so far, macrophages were evaluated immediately upon isolation from mice. TAM contained a small but appreciable (
