Negative feedback regulation of activated macrophages via Fas-mediated apoptosis

Tadaaki Niinobu, Keisuke Fukuo, Osamu Yasuda, Maki Tsubakimoto, Masaki Mogi, Hiroyuki Nishimaki, Shigeto Morimoto, and Toshio Ogihara

Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apoptosis is a critical event for eliminating activated macrophages. Here we show that Fas-mediated apoptosis may participate in the mechanism of negative feedback regulation of activated macrophages. Cytokine-activated macrophages released high levels of nitric oxide (NO) that induced apoptosis in macrophages themselves. This NO-induced macrophage apoptosis was inhibited by a Fas-Fc chimeric molecule that binds to Fas ligand (FasL) and prevents its interaction with endogenous cell surface Fas. High levels of NO stimulated the release of the soluble form of FasL that was inhibited by a matrix metalloproteinase inhibitor KB-8301. High levels of NO also upregulated the expression of Fas mRNA in macrophages. In addition, macrophages isolated from Fas-lacking mice were resistant to NO-induced apoptosis. Finally, inhibition of apoptosis by a caspase inhibitor augmented peroxide production from activated macrophages. These findings suggest that high levels of NO released from activated macrophages may promote the Fas-mediated macrophage apoptosis that may be a negative feedback mechanism for elimination and the downregulation of activated macrophages in the vessel wall.

inducible nitric oxide synthase; Fas ligand; metalloproteinase; lpr


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INFILTRATION OF MONOCYTES/MACROPHAGES into the vessel wall is critical for the progression of atherosclerosis (22). Recent pathological studies of human postmortem specimens have demonstrated that ruptured plaques have a larger extracellular lipid-rich core with an increased macrophage density and a reduced collagen and smooth muscle cell content in their fibrous cap compared with intact plaques (5, 7, 26). Human monocyte-derived macrophages were shown to induce collagen breakdown in fibrous caps of human atherosclerotic plaques (24), and tissue factor content is increased in unstable angina and correlates with areas of macrophages (16). In addition, the chemoattractant proteins (chemokines) monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 are found in human atheroma, and mice lacking receptors for these chemokines (3) or mice lacking MCP-1 itself (8) are less susceptible to atherosclerosis and have fewer monocytes in vascular lesions. These findings suggest that blockade of monocyte/macrophage recruitment is useful for the inhibition of atherogenesity.

Apoptosis is a physiological cell death pathway that removes damaged or unwanted cells. Recent pathological studies have shown that apoptosis was common in the atherosclerotic lesion, especially in vascular smooth muscle cells and inflammatory cells such as macrophages and T cells (2). Fas ligand (FasL; also called CD95 ligand or APO-1 ligand) is a cytokine that mediates apoptosis by binding to its receptor, Fas (also called CD95 or APO-1), through activating caspases (18). The Fas/FasL system is implicated in the elimination of activated T cells after they have responded to foreign antigens (12). Apoptosis regulates inflammatory cell survival, and its reduction contributes to the chronicity of an inflammatory process (27). On the basis of the expression of both Fas and FasL on macrophages (10), we hypothesized that Fas-mediated apoptosis may represent the mechanism for eliminating activated macrophages. High levels of nitric oxide (NO) released from activated macrophages have been postulated to play important roles in host defense mechanisms against tumor cells and organisms.

However, these high levels of NO also induce apoptosis in macrophages themselves (1, 23). In this study, we examined, therefore, whether NO-induced macrophage apoptosis is mediated through the Fas/FasL system. We also examined whether inhibition of apoptosis can modulate the production of reactive oxygen from macrophages. We found that high levels of NO stimulated the release of the soluble form of FasL (sFasL) and sensitized macrophages to Fas-mediated apoptosis and that inhibition of apoptosis augmented peroxide production from macrophages.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Interleukin-1beta (IL-1beta ), tumor necrosis factor-alpha (TNF-alpha ), interferon-gamma (INF-gamma ), and NG-monomethyl-L-arginine (L-NMMA) were purchased from Sigma Chemical (St. Louis, MO). Fas-Fc was generously provided by Dr. Shigekazu Nagata (Osaka Bioscience Institute, Osaka, Japan). KB-8301, a matrix metalloproteinase inhibitor, was donated by Kanebo (Osaka, Japan). N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) were purchased from Dojin Laboratory (Kumamoto, Japan). Hoechst 33258 was purchased from Flow Laboratories (McLean, VA). 2',7'-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR). DEVD-fmk (Z-Asp-Glu-Val-Asp-FMK), a caspase family inhibitor, was purchased from Medical and Biological Laboratories (Nagoya, Japan).

Cells. Male MRL/MPJ lpr/lpr mice and MRL/MPJ +/+ mice were purchased from breeding colonies at Japan SLC (Shizuoka, Japan). Resident peritoneal macrophages were collected from peritoneal cavities of each mouse and were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (75 U/ml), and streptomycin (0.05 µg/ml). The cells were then incubated for about 120 min at 37°C in a CO2 incubator to allow them to adhere to the plates. The nonadherent cells were then removed by three vigorous washings with prewarmed PBS solution. More than 95% of the adherent cells were judged to be macrophages by both Giemsa staining and carbon-particle uptake. These macrophages were cultured in RPMI 1640 medium and then used in the experiments.

Nitrite assay. The nitrite level in the medium, which is used as a reflection of NO production, was determined using Greiss reagent consisting of 1% sulfanilamide, 0.1% naphthylene-diamine-dihydrochloride, and 2% H3PO4. Absorbance at 560 nm was measured.

Apoptosis analysis. Apoptosis was monitored by measuring the population distribution of DNA content. The treated cells were harvested, suspended in 100 µl of PBS, fixed in 900 µl of ice-cold 100% ethanol, and then resuspended in staining buffer (1 mg/ml RNase, 20 mg/ml propidium iodide, and 0.01% Nonidet P-40). After staining, flow cytometric analysis was performed immediately in a Becton Dickinson FACScan. To analyze fragmented apoptotic nuclei, macrophages were fixed with methanol-acetic acid, 3:1 (vol/vol), and stained with fluorescent dye (Hoechst 33258, 10 µmol/l). After incubation for 30 min in the dark at 37°C, photographs were obtained with a Nikon EFD2 fluorescence microscope (×400).

Analysis of Fas mRNA. Total RNA from macrophages was extracted by a guanidine isothiocyanate/acid phenol method. RT-PCR was performed using a One Step RNA PCR kit from Takara Biomedicals (Tokyo, Japan). The primers used for amplification of the Fas mRNA were described below, respectively: Fas, upper primer sequence 5'-CATGTCTTCAGCAATTCTCGG-3', lower primer sequence 5'-GGCTGTGAACACTGTGTTCG-3'. The efficiency of the RT and the amount of RNA used in the RT-PCR were verified by detection of the mouse beta -actin mRNA with RT-PCR. One microgram of total RNA was reverse transcribed to cDNA by avian myeloblastosis virus RT (RTase) for 30 min at 50°C, and RTase was inactivated for 2 min at 94°C. The cDNA were then amplified with 5 units of Ampli Taq DNA polymerase and 0.4 mmol/l of both upstream and downstream primers in a volume of 50 µl. The 35 PCR cycles consisted of a denaturation step (94°C, 30 s), an annealing step (56°C, 30 s), and an elongation step (72°C, 2 min). The PCR products were analyzed on a 1.3% 40 mmol/l Trizma-base, 20 mmol/l sodium acetate 3H2O, 1 mmol/l EDTA Na2 2H2O agarose gel.

Immunoblot analysis of FasL expression. After incubation for 24 h with NOC-12 in the presence or absence of KB-8301, each supernatant was collected and cells were then treated with 500 µl of lysis buffer [50 mmol/l Tris·HCl (pH 8.0), 20 mmol/l EDTA, 1% SDS, and 100 mmol/l NaCl]. The supernatants (4 ml) from cells were concentrated to 100 µl with an Ultrafree-15 centrifugal filter device (Millipore, Bedford, MA). The cell lysates or concentrated supernatants were then melted by sample buffer (without 2-mercaptoethanol) and then analyzed by SDS-PAGE using a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore). After blocking, the membrane was incubated with an anti-mouse FasL goat polyclonal antibody (1:100; Santa Cruz Biotechnology). Membranes were then washed and incubated with a horseradish peroxidase-conjugated goat antibody. After washing, FasL expression was detected using enhanced chemiluminescence (Amersham International, Buckinghamshire, England).

Immunohistochemical studies. At 20 wk of age, mice were anesthetized with ether and killed by exsanguination. Specimens of kidneys were embedded in optimum cutting temperature compound and frozen on dry ice. Serial 10-µm-thick cryostat sections were collected on poly-D-lysine-coated slides and fixed in acetone. To examine cell surface markers of macrophages, we used the avidin-biotin-peroxidase complex technique. In brief, 10-µm-thick cryostat sections were incubated with monoclonal rat anti-mouse Mac-1 antigen (Immunotech) and then incubated with biotinylated rabbit anti-rat IgG (Dako). After they were combined with streptavidin peroxidase, sections were visualized with DAB substrates. In some experiments, sections were stained with hematoxylin and eosin.

Assay for peroxide production. Production of peroxide by the cells was quantified by flow cytometry using the peroxide-sensitive dye DCFH-DA (9). Cells treated with cytokines in the presence or absence of a caspase inhibitor were incubated with DCFH-DA (5 µmol/l). After 30 min, fluorescence intensity, which is directly proportional to peroxide content, was then analyzed by flow cytometry on the FL1 channel.

Statistical analysis. The differences between means were evaluated using Student's t-test. Significant levels were established when P values were <0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Large amounts of NO-induced macrophage apoptosis is partially mediated through activation of the Fas/FasL system. Incubation of macrophages with IFN-gamma (400 U/ml), TNF-alpha (40 ng/ml), and IL-1beta (10 ng/ml) induced high levels of NO release in a time-dependent manner (Fig. 1A). Incubation for 72 h with these cytokines induced apoptosis in macrophages. However, L-NMMA, an inhibitor of NO synthase, partially suppressed the cytokine-induced apoptosis (Fig. 1B). NOC-12, a NO donor, at a high concentration of 0.5 mg/ml, also induced macrophage apoptosis (Fig. 1C). Nuclear staining with Hoechst 33258 showed condensed and fragmented apoptotic nuclei in NOC-12-treated cells (Fig. 2), suggesting that large amounts of NO released from macrophages induce apoptosis in macrophage themselves. To address the mechanism of NO-induced macrophage apoptosis, we next examined the effect of a Fas-Fc chimeric molecule that binds to FasL and prevents its interaction with endogenous cell surface Fas (19) on macrophage apoptosis.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Cytokine-activated macrophages release high levels of nitric oxide (NO) that induce Fas-Fc-inhibitable macrophage apoptosis. A: kinetic changes of NO production from macrophages after stimulation with cytokines [400 U/ml interferon-gamma (IFN-gamma ) + 40 ng/ml tumor necrosis factor-alpha (TNF-alpha ) + 10 ng/ml interleukin-1beta (IL-1beta )] in the presence or absence of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 3 mmol/l) were examined by measuring nitrite concentrations in the supernatant as described in the text. B: cells were incubated for 72 h with these cytokines in the presence or absence of L-NMMA or a Fas-Fc molecule (10 µg/ml). A quantitative measurement of apoptosis was then performed by flow cytometric analysis as described in the text. C: cells were incubated for 48 h with N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) with or without a Fas-Fc molecule (10 µg/ml). Values are means ± SD of 4 individual experiments, each containing 2 replicates. * P < 0.05, significantly different from control; *** P < 0.05, significantly different from cells treated with NOC-12 alone.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2.   Nuclear morphology of NO-treated macrophages. Macrophages were incubated for 48 h with vehicle (A) or NOC-12 (0.5 mg/ml) (B). Cells were then stained with Hoechst 33258 (10 µmol/l) as described in the text (×400).

Incubation with a Fas-Fc chimeric molecule partially inhibited macrophage apoptosis induced by cytokines and NOC-12 (Fig. 1, B and C), indicating that NO-induced macrophage apoptosis may be partially mediated through the Fas-mediated pathway.

High levels of NO upregulates the Fas/FasL expression in macrophages. We next examined whether NO modulates the Fas/FasL expression in macrophages. RT-PCR analysis showed that incubation for 2 and 4 h with 0.5 mg/ml NOC-12 induced an increase in the levels of Fas mRNA in macrophages (Fig. 3A). In addition, immunoblot analysis with an anti-FasL antibody showed that NOC-12 stimulated the release of sFasL from macrophages into the supernatant (Fig. 3B). KB-8301, a metalloproteinase inhibitor, inhibited this sFasL release. Although NOC-12 alone did not affect the levels of the cellular FasL, NOC-12-induced upregulation of the cellular FasL was unmasked in the presence of KB-8301 (Fig. 3C).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3.   High levels of NO upregulate the Fas/Fas ligand (FasL) system. A: Fas mRNA expression in macrophages treated with NOC-12 (0.5 mg/ml) was examined by RT-PCR method as described in the text. The amount of the soluble form of FasL released into the supernatant (B) or the cellular FasL expression in macrophages (C) was determined after incubation for 24 h with NOC-12 (0.5 mg/ml), KB-8301 (10 µmol/l), a metalloproteinase inhibitor, or vehicle by immunoblot analysis as described in the text.

Macrophages isolated from lpr mice are resistant to NO-induced apoptosis. To further confirm the mechanistic link between NO-induced apoptosis and the Fas/FasL system, we next examined the levels of NO-induced apoptosis in macrophages isolated from lpr mice that lack functional Fas (18) compared with macrophages isolated from wild-type mice. Macrophages from lpr mice were resistant to NOC-12-induced apoptosis compared with those from wild-type mice (Fig. 4). In addition, immunohistochemical examination showed that macrophages were much more accumulated in a perivascular space of the renal artery in lpr mice compared with wild-type mice (Fig. 5). These findings indicate that Fas-mediated apoptosis may be important in the regulation of macrophage elimination in the vessel wall.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Macrophages isolated from Fas-lacking mice are resistant to NO-induced apoptosis. Macrophages isolated from wild-type mice or Fas-lacking lpr mice were treated for 48 h with NOC-12 at the indicated concentrations. Apoptotic cells were then quantitated as described in the text. Values are means ± SD of 4 individual experiments, each containing 2 replicates. * P < 0.05, significantly different from control; ** P < 0.05, significantly different from wild-type mice.



View larger version (84K):
[in this window]
[in a new window]
 
Fig. 5.   Macrophages are much more accumulated in a perivascular space of the renal artery in lpr mice compared with wild-type mice. Immunostaining with anti-macrophage antibody Mac-1 (1:200) was examined in a 20-wk-old male wild-type mouse (A) or a Fas-lacking lpr mouse (B) as described in the text (×80).

Caspase inhibitor augments peroxide production from activated macrophages. To address whether apoptosis can modulate the levels of reactive oxygen production from macrophages, we next measured the levels of peroxide production from cytokine-activated macrophages in the presence or absence of an inhibitor of the caspase cascade that is a downstream apoptotic signal for not only the Fas/FasL system but also other apoptotic inducers (18). Consistent with previous reports, incubation for 24 h with IL-1beta , TNF-alpha , and IFN-gamma induced an increase in the levels of peroxide production from macrophages (Fig. 6A). However, the progression of cytokine-induced apoptosis at 48 h was associated with a decrease in the levels of peroxide production (Fig. 6B). Interestingly, in the presence of 20 µmol/l DEVD-fmk, a caspase family inhibitor, cytokine-induced peroxide production was augmented and restored (Fig. 6, A and B).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6.   Caspase inhibitor augments peroxide production from activated macrophages. Relative peroxide concentrations in the cells were quantified by flow cytometry using the peroxide-sensitive dye 2',7'-dichlorofluorescin diacetate (DCFH-DA). Cells were cultured for 24 h (A) and 48 h (B) with cytokines (400 U/ml IFN-gamma  + 40 ng/ml TNF-alpha  + 10 ng/ml IL-1beta ) in the presence or absence of a caspase family inhibitor DEVD-fmk (20 µmol/l) and then incubated with DCFH-DA (5 µmol/l) for a further 30 min of each treatment. Intracellular peroxide concentrations were then quantitated as described in the text.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent histological studies have demonstrated that the vulnerable plaque from patients with acute coronary syndromes contains many more monocytes/ macrophages compared with the stable plaque (17). Because monocytes/macrophages release many inflammatory mediators including cytokines, proteinases, and free radicals such as NO and superoxide, suppression of monocyte/macrophage accumulation may be a useful therapeutic strategy for the prevention of acute coronary syndromes. Although pathological accumulation of monocytes/macrophages has been attributed to the migration and proliferation of these cells, inhibition of apoptosis may also participate in the mechanism of monocyte/macrophage accumulation. It has been shown that monocyte/macrophage apoptosis is controlled by a variety of inflammatory cytokines and bacterial products (13, 21). However, the precise mechanism of macrophage apoptosis is not well known. Large amounts of NO released from cytokine-activated macrophages exert two separate effects on macrophages, stimulating effector functions while simultaneously inducing apoptosis (1, 23). In addition to well-established proapoptotic effects of NO, low levels of NO function as an important inhibitor of apoptosis by interference with signal transduction pathways that control apoptotic cell death (14, 15). The present study demonstrated that NO-induced macrophage apoptosis is partially mediated through activation of the Fas/FasL system. Kiener et al. (11) reported that monocyte-derived macrophages that express both Fas and FasL are resistant to Fas-mediated apoptosis. However, Brown and Savill (4) recently reported that phagocytosis triggers macrophage release of FasL and induces apoptosis of bystander monocytes. More recently, Perlman et al. (20) reported that monocyte differentiation into macrophages was associated with upregulation of Fas-associated death domain-like IL-1beta -converting enzyme-inhibitory protein (Flip) and a decrease in Fas-mediated apoptosis.

In this study, we demonstrated for the first time that high levels of NO induced stimulation of sFasL release as well as upregulation of Fas expression in macrophages. In addition, fluorescence-activated cell sorting analysis revealed that neither cytokines nor NOC-12 increased the levels of cell surface FasL expression in macrophages (data not shown). Furthermore, macrophages isolated from Fas-lacking mice were resistant to NO-induced apoptosis. Taken together, these findings suggest that high levels of NO may sensitize macrophages to Fas-mediated apoptosis and that macrophages can serve as a source of sFasL, which may function in a paracrine pathway to mediate apoptosis in macrophages themselves.

Yaoita et al. (28) recently reported that a caspase inhibitor was effective in reducing myocardial ischemia-reperfusion injury in rats. More recently, Daemen et al. (6) also reported that administration of the anti-apoptotic agents insulin-like growth factor I and a caspase inactivator prevented the early onset of apoptosis, inflammation, and tissue injury, suggesting that inhibition of apoptosis by the caspase inhibitor may have beneficial effects. On the other hand, it has also been shown that airway inflammation is associated with an enhanced survival of inflammatory cells caused by reduced apoptosis (25, 27). In the present study, we demonstrated that inhibition of caspase activation augmented cytokine-induced peroxide generation from macrophages. These findings could raise the possibility that suppression of caspase activation may enhance proinflammatory products from macrophages and facilitate inflammation in certain diseases. Thus it might need to be considered carefully in the therapeutic potential of caspase inhibitors.

In conclusion, we demonstrated that high levels of NO released from cytokine-activated macrophages may sensitize macrophages themselves to Fas-mediated apoptosis. This may be a negative feedback loop serving to promote resolution of inflammation by accelerating deletion of macrophages by apoptosis. In addition, inhibition of macrophage apoptosis by caspase inhibitors may facilitate inflammation through enhancing reactive oxygen production.


    ACKNOWLEDGEMENTS

We thank Taeko Kaimoto for technical assistance and Tomoko Hironaka for secretarial assistance.


    FOOTNOTES

This work was supported by a grant from the Ministry of Education, Science, and Culture of Japan.

Address for reprint requests and other correspondence: K. Fukuo, Dept. of Geriatric Medicine, Osaka Univ. Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan (E-mail: fukuo{at}geriat.med.osaka-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 19 November 1999; accepted in final form 8 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Albina, JE, Cui S, Mateo RB, and Reichner JS. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol 150: 5080-5085, 1993[Abstract/Free Full Text].

2.   Bjorkerud, S, and Bjorkerud B. Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol 149: 367-380, 1996[Abstract].

3.   Boring, L, Gosling J, Cleary M, and Charo IF. Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature 394: 894-897, 1998[ISI][Medline].

4.   Brown, SB, and Savill J. Phagocytosis triggers macrophage release of Fas ligand and induces apoptosis of bystander leukocytes. J Immunol 162: 480-485, 1999[Abstract/Free Full Text].

5.   Burleigh, MC, Briggs AD, Lendon CL, Davies MJ, Born GVR, and Richardson PD. Collagen type I and III content, GAGs and mechanical strength of human atherosclerotic plaque caps: spanwise variations. Atherosclerosis 96: 71-81, 1992[ISI][Medline].

6.   Daemen, MARC, van't Veer C, Denecker G, Heemsekerk VH, Wolfs TGAM, Clauss M, Vandenabeele P, and Buurman WA. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin Invest 104: 541-549, 1999[Abstract/Free Full Text].

7.   Davies, MJ, Richardson PD, Woolf N, Katz DR, and Mann J. Risk of thrombosis in human atherosclerotic plaques: role of extracellularlipid, macrophage and smooth muscle cell content. Br Heart J 69: 377-381, 1993[Abstract].

8.   Gosling, J, Slaymaker S, Gu L, Tseng S, Zlot CH, Young SG, Rollins BJ, and Charo IF. MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B. J Clin Invest 103: 773-778, 1999[Abstract/Free Full Text].

9.   Heck, DE, Bisaccia E, Armus S, and Laskin JD. Production of hydrogen peroxide by cutaneous T-cell lymphoma following photopheresis with psoralens and ultraviolet light. Cancer Chemother Pharmacol 28: 344-350, 1991[ISI][Medline].

10.   Iwai, K, Miyawaki T, Takizawa T, Konno A, Ohta K, Yachie A, Seki H, and Taniguchi N. Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils. Blood 84: 1201-1208, 1994[Abstract/Free Full Text].

11.   Kiener, PA, Davis PM, Starling GC, Mehlin C, Klebanoff SJ, Ledbetter JA, and Liles WC. Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages. J Exp Med 185: 1511-1516, 1997[Abstract/Free Full Text].

12.   Krammer, PH, Dhein J, Walczak H, Behrmann I, Mariani S, Matiba B, Fath M, Daniel PT, Knipping E, Westendorp MO, Stricker K, Baumler C, Hellbardt S, Germer M, Peter ME, and Debatin K-M. The role of APO-1-mediated apoptosis in the immune system. Immunol Rev 142: 175-191, 1994[ISI][Medline].

13.   Mangan, DF, and Wahl SM. Differential regulation of human monocyte programmed cell death (apoptosis) by chemotactic factors and proinflammatory cytokines. J Immunol 147: 3408-3412, 1991[Abstract/Free Full Text].

14.   Mannick, JB, Asano K, Izumi K, Kieff E, and Stamler JS. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 79: 1137-1146, 1994[ISI][Medline].

15.   Mannick, JB, Miao XQ, and Stamler JS. Nitric oxide inhibits Fas-induced apoptosis. J Biol Chem 272: 24125-24128, 1997[Abstract/Free Full Text].

16.   Moreno, PR, Bernardi VH, Lopez-Cuellar J, Murcia AM, Palacios IF, Gold HK, Mehran R, Sharma SK, Nemerson Y, Fuster V, and Fallon JT. Macrophages, smooth muscle cells, and tissue factor in unstable angina. Implications for cell-mediated thrombogenicity in acute coronary syndromes. Circulation 94: 3090-3097, 1996[Abstract/Free Full Text].

17.   Moreno, PR, Falk E, Palacios IF, Newell JB, Fuster V, and Fallon JT. Macrophage infiltration in acute coronary syndromes. Implications for plaque rupture. Circulation 90: 775-778, 1994[Abstract].

18.   Nagata, S. Apoptosis by death factor. Cell 88: 355-365, 1997[ISI][Medline].

19.   Nagata, S, and Golstein P. The Fas death factor. Science 267: 1449-1456, 1995[ISI][Medline].

20.   Perlman, H, Pagliari LJ, Georganas C, Mano T, Walsh K, and Pope RM. FLICE-inhibitory protein expression during macrophage differentiation confers resistance to Fas-mediated apoptosis. J Exp Med 190: 1679-1688, 1999[Abstract/Free Full Text].

21.   Rojas, M, Barrera LF, Puzo G, and Garcia LF. Differential induction of apoptosis by virulent mycobacterium tuberculosis in resistant and susceptible murine macrophages. Role of nitric oxide and mycobacterial products. J Immunol 159: 1352-1361, 1997[Abstract].

22.   Ross, R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801-809, 1993[ISI][Medline].

23.   Sarih, M, Souvannavong V, and Adam A. Nitric oxide synthase induces macrophage death by apoptosis. Biochem Biophys Res Commun 191: 503-508, 1993[ISI][Medline].

24.   Shah, PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Maihac A, and Villareal-Levy G. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques. Potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation 92: 1565-1569, 1995[ISI][Medline].

25.   Tsuyuki, S, Bertrand C, Erard F, Trifilieff A, Tsuyuki J, Wesp M, Anderson GP, and Coyle AJ. Activation of the Fas receptor on lung eosinophils leads to apoptosis and the resolution of eosinophilic inflammation of the airways. J Clin Invest 96: 2924-2931, 1995[ISI][Medline].

26.   Van der Wal, AC, Becker AE, van der Loos CM, and Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of dominant plaque morphology. Circulation 89: 36-45, 1994[Abstract].

27.   Vignola, AM, Chanez P, Chiappara G, Siena L, Merendino A, Reina C, Gagliardo R, Profita M, Bousquet J, and Bonsignore G. Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis. J Allergy Clin Immunol 103: 563-573, 1999[ISI][Medline].

28.   Yaoita, H, Ogawa K, Maehara K, and Maruyama Y. Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation 97: 276-281, 1998[Abstract/Free Full Text].


Am J Physiol Cell Physiol 279(2):C504-C509
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society