Address reprint requests to Jim L. Kelley, PhD, Univer- sity ofTexas Health ..... 1981, 256:7105-7108. 8. Chait A, Iverius P-H, Brunzell JD: Lipoprotein lipase.
American Journal of Pathology, Vol. 131, No. 3, June 1988 Copyright C) American Association of Pathologists
Influence ofHypercholesterolemia and Cholesterol Accumulation on Rabbit Carrageenan Granuloma Macrophage Activation JIM L. KELLEY, PhD, C. ALAN SUENRAM, PhD,
From the Department ofPathology, University of Texas Health Science Center, San Antonio, Texas
M. MARIUS ROZEK, MD, and COLINJ. SCHWARTZ, MD
These experiments were designed to determine whether hypercholesterolemia and the accumulation of cholesterol or cholesteryl esters in rabbit carrageenan granuloma macrophages might influence selected markers of macrophage activation. Granulomas induced by subcutaneous injection of carrageenan into rabbits were harvested after 4, 14, and 28 days. Macrophages were isolated from granuloma tissues by collagenase digestion and cultured overnight. Secretion of lysosomal 68-glucuronidase, membrane 5'-nucleotidase, cellular plasminogen activator, and superoxide anion generation were measured as markers of activation. #l-Glucuronidase activity secreted into the media by granuloma macrophages from normocholesterolemic (NC) and hypercholesterolemic (HC) rabbits showed a trend toward an increase with time between 4 and 14 days in both groups. This was confirmed in a separate experiment with a significant increase by 14 days, together with a significantly greater secretion by NC macrophages and a significantly elevated level of cellular ,6-glucuronidase activity in NC relative to HC macrophages. Activity of the membrane ectoenzyme 5'-nucleotidase was minimal in lysates of NC or HC macrophages, in contrast to freshly isolated human
monocytes, indicating that both NC and HC granuloma macrophages were highly activated. Cellular plasminogen activator activity was significantly increased between 4 and 14 days, and was significantly greater in HC than in NC macrophages at 14 days. Stimulation of macrophages with phorbol myristate acetate increased superoxide anion generation by both NC and HC macrophages; however, no difference in superoxide anion generation was observed between macrophages from NC and HC rabbits. On the basis of the 5'-nucleotidase findings, it is concluded that both the NC and HC granuloma macrophages are highly activated, and further that hypercholesterolemia does not enhance macrophage generation ofsuperoxide anion, either spontaneously or as the result of phorbol myristate acetate stimulation. Although hypercholesterolemia results in macrophage activation in terms of an increased cellular plasminogen activator activity, the secretion of the lysosomal enzyme ,B-glucuronidase is diminished. Thus, hypercholesterolemia associated with macrophage cholesterol and cholesteryl ester accumulation has no consistent overall influence on activation, a finding of potential importance in the context of atherogenesis. (AmJ Pathol 1988, 131:539-546)
THE SUBCUTANEOUS carrageenan granuloma serves as an extravascular model of foam cell development in the rabbit. Granulomas are characterized by the accumulation of blood mononuclear phagocytes, which in cholesterol-fed (HC) rabbits develop the morphologic and biochemical characteristics of atheromatous intimal foam cells, with a striking accumulation of cholesteryl esters, primarily cholesterol oleate." 2 Tissue macrophages, largely derived from blood mononuclear phagocytes, are precursors of many of the foam cells in atherosclerotic plaques. These macrophages have many potential roles in atherogenesis,
especially in the context of an inflammatory process.
539
They serve as scavenger cells by virtue of their phagocytic capacity and play a key role in immunologic responses as processors of antigen and in lymphocyte activation.3'4 Activated macrophages synthesize and secrete many neutral and acidic hydrolases, which may modify components of the arterial wall or particiSupported by Grant Nos. HL-26890 and HL-07446. Accepted for publication January 29, 1988. Address reprint requests to Jim L. Kelley, PhD, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284.
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KELLEY ET AL
pate in arterial remodeling. They also secrete a mitogen, macrophage derived growth factor (MDGF),5 which may regulate smooth muscle and endothelial cell proliferation.6 Macrophages undoubtedly play a significant role in lipoprotein metabolism, with secretion of lipoprotein lipase,7'8 the substrate of which is primarily triglyceride-rich lipoproteins and apolipoprotein E-containing lipoproteins.9 They are also capable of endocytosis and catabolism of lipoproteins leading to massive cholesteryl ester accumulation." 2 Activated macrophages generate reactive oxygen intermediates as part of a bactericidal or tumoricidal function.'" In atherosclerosis, reactive oxygen intermediates such as superoxide anion or hydrogen peroxide may lead to lipid peroxidation. Peroxidation of cell membrane lipids may alter membrane function or induce injury, whereas oxidative modification of lipoproteins in the interstitium leads to their uptake by the scavenger receptor pathway. In addition, lipid peroxides in atheromatous plaques may predispose to thrombus formation by inhibiting prostacyclin for-
mation."' In response to a variety of stimuli, including chronic inflammatory agents such as carrageenan, macrophages exhibit changes in a large number of biochemical parameters such as the synthesis and secretion of enzymes and the expression of cellular receptors. However, the influence of hypercholesterolemia on macrophage activation is unknown. Changes in the levels of serum and granuloma tissue lipids during the evolution of the carrageenan granuloma in normocholesterolemic (NC) and HC rabbits used in this experiment have been reported separately.2 Briefly, serum cholesterol in HC rabbits increased from day 4 to day 14 and remained elevated at 28 days, while that in NC rabbits remained low throughout. In both granuloma tissue and isolated macrophages from HC rabbits, total, free, and esterified cholesterol increased from day 4 to day 14, while from day 14 to 28, free cholesterol remained constant with the increase in total cholesterol largely accounted for by an increase in esterified cholesterol.2 This experiment was designed to explore the influence of hypercholesterolemia and cholesterol (free and esterified) accumulation on several markers that have been useful indicators of macrophage activation-differentiation. At 14 days, NC macrophages secreted significantly more 3-glucuronidase into the medium compared to HC macrophages and, in addition, intracellular ,B-glucuronidase activity was significantly greater in NC macrophages consistent with an increased secretion. 5'-Nucleotidase activity was similar in NC and HC macrophages, an observation that indicates that both groups are highly activated. Cellular
AJP * June 1988
plasminogen activator activity was significantly elevated between 4 and 14 days in both groups, and was significantly greater in HC compared to NC macrophages at 14 days. There was no impairment in the ability ofeither group to generate superoxide anion in response to phorbol myristate acetate (PMA).
Materials and Methods Animals Male New Zealand white rabbits from 2.0 to 4.0 kg in weight were used.
Granuloma Induction Carrageenan (Gelcarin HMR, FMC Corporation, Marine Colloids Division, Rockland, Me) was prepared as a 1% (wt/vol) solution in physiologic saline (0.85% NaCl, wt/vol) and sterilized in an autoclave at 250 C for 30 minutes. Rabbits were anesthetized and injected with carrageenan as described previously." 2 Fifteen milliliters of the warmed (37 C), sterile carrageenan solution was injected subcutaneously at two sites near the midline of the abdominal wall. Experimental Design The design of the study was the same as that reported previously. 2 NC rabbits were fed ad libitum with rabbit pellets (Wayne Feed Division, Continental Grain Company, Chicago, Ill), and HC rabbits were fed ad libitum a rabbit pellet diet supplemented with 0.3% cholesterol and 6% peanut oil. The supplemented diet was prepared by dissolving the cholesterol (0.25 g, USP grade) in 6.0 ml warm peanut oil and mixing the warm cholesterol-peanut oil solution evenly into 75 g of rabbit pellets. The diets were prepared weekly and stored at 4 C. The cholesterol diet was started 4-6 weeks prior to carrageenan injection and continued until death. At 4, 14, and 28 days after carrageenan injection, rabbits were anesthetized and the granulomas were excised. Small portions (0.1-0.5 g) of the total granuloma tissue (10-15 g) were set aside for morphologic studies and lipid analysis" 2; the remainder was used for macrophage isolation and culture.
Granuloma Macrophage Isolation and Culture The excised granuloma tissue was crudely minced in a small volume of warm collagenase solution (Type I, Cooper Biomedical, Inc., 494,000 units/liter) prepared in DMEM at pH 7.4. Sufficient collagenase so-
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lution was added, to make a final volume of 25 ml for each 5 g tissue, in a large plastic petri dish and incubated, with rocking, in a humidified 37 C incubator with 5% Co2. After 1 hour, the incubated tissue mince was filtered sequentially through a coarse and medium mesh filter (Nitex Screening Fabric, HD3-500 and HC3-183, respectively, Tetko, Inc., Elmsford, NY), collecting the filtrate in 50 ml polypropylene centrifuge tubes (Corning Glass Works, Corning, NY). The undigested material was transferred from the coarse filter to a large petri dish, and 25 ml fresh collagenase solution was added for a second incubation at 37 C. After the second incubation, the incubated tissue mince was again filtered. The cell-rich filtrates were centrifuged for 15 minutes at 2500 rpm in a Beckman TJ-6 centrifuge (Beckman Instruments, Palo Alto, Calif.). The supernatants were aspirated and the pellets resuspended in 50 ml GBSS and recentrifuged at 2500 rpm for 15 minutes. The pellets were resuspended in 20-40 ml warm GBSS and filtered through a fine mesh (HD3-85, Tetko) to remove fibrous material. The volume was adjusted to 50 ml and the macrophages repelleted. Cell pellets were resuspended in Dulbecco's Modified Eagle Medium (DMEM) + 10% fetal calf serum (FCS), plated in 35 mm or 60 mm dishes (Primaria, Becton-Dickenson & Co., Oxnard, CA) at 3-4 X 106 cells per ml, then incubated at 37 C in a humidified 95% air: 5% CO2 atmosphere. After 2 hours, nonattached cells were removed and the dishes were rinsed twice with GBSS and the macrophages cultured overnight in DMEM + 10% acid-treated FCS. Conditioned medium was harvested and macrophages were rinsed twice with PBS. A volume of 1 ml 0. 1% Triton X- 100 was added to each plate to lyse the cells at room temperature for 30 minutes. The plates were then scraped with a rubber policeman and rinsed with another 1.0 ml Triton X- 100, which was combined with the lysates. Lysates were either assayed immediately or frozen at -20 C. Analyses
Plasminogen Activator Plasminogen activator(s) were measured in conditioned media and cell lysates by the '251-fibrin plate assay described by Gordon." Fibrinogen was freed of contaminating plasminogen by passage over a lysinesepharose column according to the procedure of Deutsch and Mertz'2 and was labeled with 1251 by the ICI method of McFarlane'3 as modified by Bilheimer et al.'4 Unlabeled fibrinogen was mixed with radiolabeled 1251I-fibrinogen in distilled water to yield 0.5-1.0 X 105 precipitable dpm and 20 jig fibrinogen per 0.3
GRANULOMA MACROPHAGE ACTIVATION
541
ml. The fibrinogen solution was added to 24 well cluster plates at 0.3 ml per well and dried for 48 hours. The plates were activated by incubation for 2 hours at 37 C with 1.0 ml DMEM + 10% FCS per well, followed by rinsing each well twice with PBS or HBSS. Plasminogen activator activity was measured by adding 100 1l of sample to a 300 ,u reaction mixture containing 6.25 ,tg plasminogen and 125 jig bovine serum albumin (BSA) in 0.1 M Tris-HCl, pH 8.0. The assay included controls without plasminogen and without a putative activator. The plates were incubated at 37 C, and aliquots of 100 ,1. were removed from each well at 4 hours. Plots of radioactivity released versus time were superimposable on similar curves for urokinase. Further, the amounts of radiolabel released over time by the macrophage lysates were highly correlated with amounts released by urokinase standards (r = 0.999). Therefore, the results are reported as urokinase units.
5'-Nucleotidase The activity of 5'-nucleotidase was measured by the procedure of Avrum and Wallach"5 using the ammonium salt of 3H-adenosine monophosphate (AMP) as substrate. Macrophage lysates were incubated with the 3H-AMP for 60 minutes and the reaction was stopped by adding ZnSO4. Unhydrolyzed AMP and protein were precipitated with Ba(OH)2. Aliquots of the supernatant solution containing the hydrolyzed 3H-adenosine were counted in a scintillation spectrometer. Dilutions of human serum (Sigma) were used as standards and lysates of isolated human monocytes served as positive controls. Data were expressed as nmol AMP hydrolyzed/min/,tg DNA.
f3-Glucuronidase f3-Glucuronidase activity was assayed by the fluorimetric procedure of Woollen and Walker.'6 Enzyme activity was measured in conditioned media and in cell lysates of cultured granuloma macrophages. The reaction was terminated after 2 hours of incubation at 37 C by addition of glycine-NaCl-NaOH buffer, pH 13.0. The fluorescent product 4-methylumbelliferone was measured using a Perkin-Elmer LS-3 fluorescence spectrometer (excitation, 364 nm; emission, 448 nm).
Superoxide Anion Generation Superoxide anion generation was measured as the superoxide dismutase inhibitable chemical reduction of ferricytochrome c according to the method of Johnston et al.'7 After an overnight incubation, macrophages were washed twice with GBSS, the last wash being completely aspirated. Six milliliters of ferricytochrome c (1 mg/ml) in GBSS containing 0.1% dex-
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KELLEY ET AL
AJP * June 1988
Table 1 -f-Glucuronidase Activity in Conditioned Media From Cultured Granuloma Macrophages 4-day
14-day
28-day
216.73 ± 82.99 8 196.92 ± 35.89 8 0.8297
1064.25 ± 642.27
372.07 ± 228.09 6 186.99 ± 56.66 6 0.41 25
Animals NC n HC n P
7 435.78 ± 153.34 7
0.3295
buu
o
400-
XI
D NC D HC
Pz .046 T
U C
0) 0 ox
z 0 01
300 -
a
E .0
200 to _
NC: normocholesterolemic (chow-fed); HC: hypercholesterolemic (cholesterol-fed). Values are the mean ± SE and are expressed as pmol substrate hydrolyzed/hour/mg DNA.
ua
100-
C-L 0
I1 14
4
28
L
Days after Carrageenan Injection
trose, 4 mM NaHCO3, and 10 mM Hepes, pH 7.07.2, were added to each plate followed by the addition of 6 Al PMA (1 mg/ml in ethanol/dimethyl sulfoxide) and/or 180 ,ul superoxide dismutase or equivalent volumes of GBSS as required to obtain equal volumes of reaction mixture. The plates were then incubated at 37 C, and 750 ,AI aliquots were removed at appropriate intervals and their absorbance measured at 550 nm. Data were expressed an nmol cytochrome c reduced/ min/jig DNA. DNA DNA in cell lysates were measured by the fluorimetric procedure of Sorger and Germinario'8 using the fluorimetric reagent diamidinophenylindole.
Statistics Data were analyzed by analysis of variance (ANOVA) followed by Student's t test where appropriate. Serum cholesterol data were analyzed by ANOVA for a repeated design. Superoxide anion generation over time was examined by regression analysis. 2500
-
2000-
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p -.0176
a NC U HC
z
1500
-
1000
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Q
0
500
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I
I
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Secreted
Cellular
---
Figure 1-j3-Glucuronidase activity measured in conditioned media and cell lysates of cultured 1 4-day NC and HC granuloma macrophages. Statistical comparisons were made using the Student's t test. Results are the mean ±
SE.
Figure 2-Plasminogen activator activity measured in cell lysates of granuloma macrophages isolated from NC and HC rabbits at 4, 14, and 28 days after carrageenan injection. Results are the mean ± SE. N = 4 pairs at 4 days, n = 4 pairs at 14 days, and n = 6 pairs at 28 days. Data were analyzed by ANOVA and the paired Student's t test.
Results Secretion of f3-glucuronidase activity by cultured granuloma macrophages isolated from NC and HC rabbits at either, 4, 14, or 28 days after carrageenan injection (Table 1) was measured. A large mean increase was noted in secreted f3-glucuronidase activity between 4 and 14 days and a subsequent decrease between 14 and 28 days; the activity secreted by NC macrophages was about twice that of HC macrophages. However, ANOVA by diet groups and time failed to show statistical significance. Therefore, we performed a separate experiment using 14-day granuloma macrophages from NC and HC rabbits and observed a significantly greater activity secreted by NC than by HC macrophages (Figure 1, P = 0.0176). Additionally, intracellular ,B-glucuronidase activity was significantly greater in NC macrophages (P = 0.01 12) than in HC macrophages. Activity of 5'-nucleotidase measured in cell lysates was at the lower limits of sensitivity of the assay in both NC and HC macrophages compared to that measured in isolated human monocytes at 2 hours (0.42 ± 0.09 nmol/min/,ug DNA) and 24 hours (7.44 ± 3.72 nmol/min/,ug DNA) of culture. The DNA content of the cultured macrophages indicated that sufficient numbers of cells were present to measure activity. The results show that little 5'-nucleotidase activity was present in granuloma macrophages from either NC or HC rabbits, consistent with a similar and near-maximal level of macrophage activation in both groups. Cellular plasminogen activator activity in both NC and HC macrophages was significantly elevated at 14 days compared to 4-day granuloma macrophages (Figure 2, P = 0.0030). HC macrophages at 14 days showed significantly greater plasminogen activator
GRANULOMA MACROPHAGE ACTIVATION
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activity than NC macrophages (P = 0.046). There was no significant difference in activity between 14 and 28 days. Additionally, we measured the level of PA activity secreted by 14-day NC and HC macrophages (n = 4 pairs) in a separate experiment and found no significant difference. Regression analysis of superoxide anion generation over time by diet and stimulation with PMA showed significant effects due to PMA stimulation but not to diet. Stimulation of 14-day granuloma macrophages with PMA increased the amount of superoxide anion generated over time compared to nonstimulated macrophages (Figure 3) in both diet groups. However, there was no difference in superoxide anion released by either NC or HC macrophages at 14 days irrespective of PMA stimulation. Superoxide anion generation by 28-day granuloma macrophages was similar to that of 14-day macrophages (data not shown). Regression analyses were undertaken to examine possible relationships between several parameters of activation and granuloma tissue lipid content and distribution. The lipid compositional data2 are summarized in Table 2. These data include only those animals for which activation parameters were measured and are averaged over time. In granulomas from HC animals there was a positive correlation between granuloma-free cholesterol and secreted f-glucuronidase activity (P = 0.0422, r = 0.470). There was no significant relationship among the enzymes measured and HC granuloma esterified cholesterol or phospholipid contents. In granulomas from NC animals, cellular plasminogen activator activity was significantly correlated with tissue phospholipid content (P = 0.0434, r 0.546). Discussion
Peripheral blood monocytes are recruited to the arterial intima during atherogenesis and in experimental hyperlipidemia, and are now recognized as progenitors of many of the foam cells in the atheromatous plaque. Intimal monocyte-derived macrophages have a number of potential roles in atherogenesis, particularly in the context of atherosclerosis as an inflammatory process.'9 In addition to their phagocytic potential and role in immunologic processes,3'4 intimal macrophages are clearly involved in the catabolism of lipoproteins,2,7,8 a function inherent in the evolution of cholesteryl ester-rich foam cells. Under the influence of dietary hypercholesterolemia, subcutaneous granuloma macrophages develop the morphologic appearance of plaque foam cells' while accumulating considerable amounts of cholesteryl esters in which oleic acid is the predominant fatty acid.2 Activated
c ._
543
4 z 0
0 -
CL)
c 6._
E
0
x
0
Cl)
0
X~~~
0
NC
E c
0
10
20
30
40
50
60
70
80
90
Time (minutes) Figure 3-Superoxide anion generation by 14-day granuloma macrophages. Superoxide anion generation by NC and HC macrophages was measured over a period of 60 minutes, with and without PMA stimulation. Statistical comparisons were made by regression analysis over time. Values are the mean and the error bars represent the SE; n = 10 for NC macrophages and n = 9 for HC macrophages.
macrophages, and presumably those within the intima or plaque, secrete a spectrum of neutral and acid hydrolases and also generate reactive oxygen intermediates.20 Hydrolases secreted by activated macrophages have considerable pathogenic potential, both in terms of arterial injury and in reparative or remodeling roles. The present studies were undertaken on the premise that cholesterol and/or cholesteryl ester accumulation in macrophages and foam cells might modulate or induce activation, and thus enhance their secretory or other functions. In this report we have therefore examined the influence of dietary hypercholesterolemia and the resulting cholesterol (free and esterified) accumulation in granuloma macrophages on their production of ,B-glucuronidase, 5'-nucleotidase, plasminogen activator(s), and superoxide anion generation, each of which provides an index of macrophage activation-differentiation. Increased secretion of lysosomal fl-glucuronidase is a characteristic marker of macrophage activation.2' 22 We found no statistically significant difference in secreted fl-glucuronidase activity between NC and HC granuloma macrophages at 4, 14, and 28 days (Table Table 2-Total, Free, and Esterified Cholesterol and Phospholipid Contents of Granulomas From NC and HC Rabbits
Cholesterol Animals NC HC
Total
Free
Esterified
Phospholipid
846± 72 2679 ±737
815± 74 1645 ± 330
53± 15 1742 ± 718
3.9±0.4 4.4 ± 0.5
NC: normocholesterolemic (chow-fed); HC: hypercholesterolemic
(cholesterol-fed). Values are the mean ± SE and are expressed as 9g/g tissue wet weight with 12 animals in each group. These data2 are averaged over time.
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KELLEY ET AL
1), although there was a trend toward an increased ,Bglucuronidase secretion by 14-day NC macrophages. In a separate experiment we confirmed the observation that 14-day NC macrophages secreted greater fglucuronidase activity than HC macrophages, and we also observed a significantly greater intracellular ,Bglucuronidase activity in NC macrophages. These results are consistent with either an impairment by hypercholesterolemia of processes that result in secretion of lysosomal enzymes by activated macrophages and/or a direct influence on lysosomal f-glucuronidase activity. Whether other lysosomal enzymes are similarly affected by hypercholesterolemia has yet to be determined. Activation of macrophages is typically associated with a reduced activity of the membrane ectoenzyme 5'-nucleotidase.23 Granuloma macrophages isolated from both NC and HC rabbits exhibited similarly low or unmeasurable levels of 5'-nucleotidase activity, a finding that indicates a high level of activation in both dietary groups. Clearly, as the NC macrophages are fully activated in terms of their reduced 5'-nucleotidase activity, we cannot exclude the possibility that hypercholesterolemia and/or cholesterol or cholesteryl ester accumulation in a population of less activated cells might augment the level of activation. Hypercholesterolemia clearly did not inhibit activation as measured by 5'-nucleotidase activity. Although the biologic roles of plasminogen activator(s) are not fully understood, these neutral proteases are thought to be involved in a variety of proteolytic events associated with tissue degradation and remodeling24 including fibrinolysis and cellular migration. In the context of inflammation, plasminogen activators are enhanced by inflammatory agents25'26 and are suppressed by anti-inflammatory glucocorticoids.27 At least two types of plasminogen activators are known, namely, tissuetype and urokinasetype. The primary structures of these enzymes are known and appear to be the products of different genes.28130 Human monocyte-derived macrophages and monocytelike cells of the histiocytic lymphoma cell line U937 have been shown to secrete the urokinasetype plasminogen activator.3' For this reason granuloma macrophage plasminogen activator activity was compared to urokinase activity and is reported as urokinase units. Definitive identification of the type of plasminogen activator synthesized by rabbit carrageenan granuloma macrophages will ultimately depend upon its immunologic and molecular characterization. Fourteen days after granuloma induction the activity of plasminogen activator was significantly greater (P = 0.046) in macrophage cell lysates isolated from HC relative to those from NC rabbits, a difference that
AJP * June 1988
persisted at 28 days, although at this time it was no longer statistically significant. Recently, Saito et al132 reported that tissuetype rather than urokinasetype plasminogen activator was expressed in blood monocytes isolated from HC rabbits. This intriguing difference may indicate that monocytes and monocyte-derived macrophages may synthesize both types of plasminogen activator and that cholesterol feeding may influence the synthesis of the two types differently. Alternatively, the high level of activation of granuloma macrophages may have contributed to the different responses to cholesterol feeding. Further, because both plasminogen activator inhibitors, as well as plasminogen activator itself, are synthesized and secreted by macrophages,31'33-36 the level of plasminogen activator measured intracellularly may reflect increased synthesis of inhibitors in NC macrophages or less synthesis of inhibitors in HC macrophages. Notwithstanding these various possibilities, hypercholesterolemia is associated with an enhanced plasminogen activator activity in granuloma macrophages and this activity correlates positively with the granuloma macrophage content of free but not esterified cholesterol. Superoxide anion generation by macrophages could result in the oxidative modification of interstitial and membrane lipids, both possibly leading to altered membrane function or injury. Specifically, the oxidative modification of lipoproteins, including low density lipoproteins (LDLs), results in alterations such that they are taken up by macrophages via the scavenger receptor pathway.37'38 Further, oxidized LDL is cytotoxic to cells cultured in vitro.29'40 Additionally, it has been shown that lipid peroxides are potent inhibitors of prostacyclin (PGI2) generation by platelets, arterial wall microsomes, and fresh vascular tissue.4'42 Such an inhibition of PGI2 generation by lipid peroxides within atheromatous plaques could facilitate thrombogenesis.'° In this context it is of interest that PGI2 generation is decreased in rabbit,43 and human44 atherosclerotic arteries, and that cultured smooth muscle cells isolated from rabbit atherosclerotic plaques generate less PGI2 than control smooth muscle cells.45 PGI2 also increases the activity of acid and neutral cholesterol esterases,46,47 probably through the action of cyclic AMP,48 providing yet another pathway through which lipid peroxidation might indirectly influence the atherogenic sequence. In the current study superoxide anion generation by both NC and HC macrophages was found to be similar (Figure 3), a result consistent with the recent observations of Rogers et al49 employing rat peritoneal macrophages. Stimulation of NC and HC macrophages with PMA increased superoxide anion generation significantly, but equally, showed no influence of
GRANULOMA MACROPHAGE ACTIVATION
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hypercholesterolemia and cholesterol or cholesteryl ester accumulation on the generation of reactive oxygen species. The observation of a positive correlation between activation parameters and tissue-free cholesterol rather than cholesteryl ester is consistent with the overall observation that both NC and HC macrophages are highly activated and that these activation parameters are not consistently influenced by the cholesteryl ester accumulation. The positive correlation of tissue-free cholesterol and phospholipid contents likely reflects changes in membrane composition during granuloma differentiation. In this study we have examined the influence of hypercholesterolemia and cholesterol (free and esterified) accumulation on several aspects of granuloma macrophage function. Activity of the ectoenzyme 5'nucleotidase was not detected in the lysates of NC or HC macrophages, putative evidence that the carrageenan-containing granuloma macrophages are highly activated. The level of intracellular plasminogen activator activity was significantly greater in HC than in NC macrophages at 14 days. Secreted and intracellular f-glucuronidase activity was greater in NC than in HC macrophages at 14 days, suggesting that hypercholesterolemia and cholesterol (free and esterified) accumulation may depress lysosomal enzyme function. Hypercholesterolemia and its associated cholesterol accumulation failed to augment superoxide anion generation by HC macrophages, whereas stimulation with PMA increased the generation of superoxide anion equally in both NC and HC macrophages.
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120:391-401 3. Unanue ER: The macrophage as regulator of lymphocyte function. Hosp Prac [off] 1979, 14:61-64 4. Pierce CW: Macrophages: Modulators of immunity. Am J Pathol 1980, 98:10-28 5. Martin BM, Gimbrone MA, Unanue ER, Cotran RS: Stimulation of nonlymphoid mesenchymal cell proliferation by a macrophage derived growth factor. J Immunol 1981, 126:1510-1515 6. Thakral KK, Goodson WH, Hunt TK: Stimulation of wound blood vessel growth by wound macrophages. J Surg Res 1979, 26:430-436
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Acknowledgments The authors wish to thank Peter A. Binkley and Linda M. Kelly for their expert technical assistance.
