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FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis.

Perlman H, Pagliari LJ, Georganas C, Mano T, Walsh K, Pope RM - J. Exp. Med. (1999)

Bottom Line: FLICE (Fas-associated death domain-like interleukin 1beta-converting enzyme)-inhibitory protein (Flip), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis.Here, we show that monocyte differentiation into macrophages was associated with upregulation of Flip and a decrease in Fas-mediated apoptosis.Addition of an antagonistic Fas ligand antibody to Flip antisense-treated macrophages rescued cultures from apoptosis, demonstrating that endogenous Flip blocked Fas-induced cell death.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Northwestern University Medical School, Chicago, Illinois 60611, USA.

ABSTRACT
Macrophages differentiated from circulating peripheral blood monocytes are essential for host immune responses and have been implicated in the pathogenesis of rheumatoid arthritis and atherosclerosis. In contrast to monocytes, macrophages are resistant to Fas-induced cell death by an unknown mechanism. FLICE (Fas-associated death domain-like interleukin 1beta-converting enzyme)-inhibitory protein (Flip), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Here, we show that monocyte differentiation into macrophages was associated with upregulation of Flip and a decrease in Fas-mediated apoptosis. Overexpression of Flip protected monocytes from Fas-mediated apoptosis, whereas acute Flip inhibition in macrophages induced apoptosis. Addition of an antagonistic Fas ligand antibody to Flip antisense-treated macrophages rescued cultures from apoptosis, demonstrating that endogenous Flip blocked Fas-induced cell death. Thus, the expression of Flip in macrophages conferred resistance to Fas-mediated apoptosis, which may contribute to the development of inflammatory disease.

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Macrophage resistance to Fas-mediated apoptosis is associated with Flip expression. (A) Macrophages are resistant to Fas-induced apoptosis. Triplicate cultures of macrophages differentiated for 7 d in 20% FBS/polymixin B were treated with activating anti-Fas mAb (250 ng/ml), control Ig, or TNF-α (10 ng/ml) for 1 d, fixed in 70% ETOH, and incubated in PI (50 μg/ml) for flow cytometry analysis to determine hypodiploid DNA content. Parallel cultures were incubated with Rh123 (0.1 μg/ml) for 30 min before analysis by flow cytometry. The results represent a mean of three experiments. (B) Flip is upregulated in macrophages. Whole cell extracts (25 μg) were prepared from peripheral blood monocytes and macrophages cultured in 20% FBS/RPMI/1 μg/ml polymyxin B and isolated at the indicated times. Extracts were subjected to SDS-PAGE on 12.5% polyacrylamide gels. Gels were transferred to Immobilon P for immunoblot analysis with anti-Flip. The anti-Flip immunoblot is a representative of 10 individuals. (C) Flip transcript levels are upregulated during monocyte to macrophage differentiation. RT-PCR analysis was performed on total RNA, and the 1,470-bp FlipL, 620-bp FlipS, and 838-bp β-actin amplified fragments were fractionated on 1.0% agarose gel and visualized by ethidium bromide staining.
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Figure 3: Macrophage resistance to Fas-mediated apoptosis is associated with Flip expression. (A) Macrophages are resistant to Fas-induced apoptosis. Triplicate cultures of macrophages differentiated for 7 d in 20% FBS/polymixin B were treated with activating anti-Fas mAb (250 ng/ml), control Ig, or TNF-α (10 ng/ml) for 1 d, fixed in 70% ETOH, and incubated in PI (50 μg/ml) for flow cytometry analysis to determine hypodiploid DNA content. Parallel cultures were incubated with Rh123 (0.1 μg/ml) for 30 min before analysis by flow cytometry. The results represent a mean of three experiments. (B) Flip is upregulated in macrophages. Whole cell extracts (25 μg) were prepared from peripheral blood monocytes and macrophages cultured in 20% FBS/RPMI/1 μg/ml polymyxin B and isolated at the indicated times. Extracts were subjected to SDS-PAGE on 12.5% polyacrylamide gels. Gels were transferred to Immobilon P for immunoblot analysis with anti-Flip. The anti-Flip immunoblot is a representative of 10 individuals. (C) Flip transcript levels are upregulated during monocyte to macrophage differentiation. RT-PCR analysis was performed on total RNA, and the 1,470-bp FlipL, 620-bp FlipS, and 838-bp β-actin amplified fragments were fractionated on 1.0% agarose gel and visualized by ethidium bromide staining.

Mentions: The ability of agonistic Fas antibody and TNF-α to induce apoptosis in macrophages was examined. Macrophages were resistant to spontaneous and agonistic Fas antibody–induced apoptosis (Fig. 3 A) as indicated by normal DNA content and mitochondrial membrane integrity, suggesting that a potent inhibitor of the death receptor–mediated pathway, not present in monocytes, may be upregulated in macrophages. As a control, agonistic Fas antibody induced apoptosis in Jurkat T cells and U937 cells (not shown). In certain cell types, Flip overexpression has been shown to directly inhibit Fas-mediated apoptosis 29, indicating that Flip may function to promote survival during monocyte to macrophage differentiation. Previous investigations demonstrated that by day 3 in culture, monocytes begin to differentiate into macrophages based on morphology, cell surface markers including vitronectin (CD51 [32]), transferrin receptor (CD71; not shown), and cytolytic activity 46. Immunoblot analyses performed on isolated peripheral blood monocytes harvested at 0, 1, 2, 3, 7, and 14 d revealed that Flip upregulation was associated with macrophage differentiation and reduced apoptosis, beginning on day 3 (Fig. 3 B). FlipL was detected at 7 and 14 d of macrophage differentiation, whereas FlipS was highly expressed 3 d after isolation (Fig. 3 B). The ratio of FlipL to FlipS on days 3, 7, and 14 varied between individuals. Although low levels of Flip were detected before day 3 in some individuals, Flip upregulation at 3, 7, and 14 d was observed in every individual examined (n = 10). These data demonstrate that the decreased spontaneous apoptosis seen during and after macrophage differentiation is associated with Flip upregulation.


FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis.

Perlman H, Pagliari LJ, Georganas C, Mano T, Walsh K, Pope RM - J. Exp. Med. (1999)

Macrophage resistance to Fas-mediated apoptosis is associated with Flip expression. (A) Macrophages are resistant to Fas-induced apoptosis. Triplicate cultures of macrophages differentiated for 7 d in 20% FBS/polymixin B were treated with activating anti-Fas mAb (250 ng/ml), control Ig, or TNF-α (10 ng/ml) for 1 d, fixed in 70% ETOH, and incubated in PI (50 μg/ml) for flow cytometry analysis to determine hypodiploid DNA content. Parallel cultures were incubated with Rh123 (0.1 μg/ml) for 30 min before analysis by flow cytometry. The results represent a mean of three experiments. (B) Flip is upregulated in macrophages. Whole cell extracts (25 μg) were prepared from peripheral blood monocytes and macrophages cultured in 20% FBS/RPMI/1 μg/ml polymyxin B and isolated at the indicated times. Extracts were subjected to SDS-PAGE on 12.5% polyacrylamide gels. Gels were transferred to Immobilon P for immunoblot analysis with anti-Flip. The anti-Flip immunoblot is a representative of 10 individuals. (C) Flip transcript levels are upregulated during monocyte to macrophage differentiation. RT-PCR analysis was performed on total RNA, and the 1,470-bp FlipL, 620-bp FlipS, and 838-bp β-actin amplified fragments were fractionated on 1.0% agarose gel and visualized by ethidium bromide staining.
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Figure 3: Macrophage resistance to Fas-mediated apoptosis is associated with Flip expression. (A) Macrophages are resistant to Fas-induced apoptosis. Triplicate cultures of macrophages differentiated for 7 d in 20% FBS/polymixin B were treated with activating anti-Fas mAb (250 ng/ml), control Ig, or TNF-α (10 ng/ml) for 1 d, fixed in 70% ETOH, and incubated in PI (50 μg/ml) for flow cytometry analysis to determine hypodiploid DNA content. Parallel cultures were incubated with Rh123 (0.1 μg/ml) for 30 min before analysis by flow cytometry. The results represent a mean of three experiments. (B) Flip is upregulated in macrophages. Whole cell extracts (25 μg) were prepared from peripheral blood monocytes and macrophages cultured in 20% FBS/RPMI/1 μg/ml polymyxin B and isolated at the indicated times. Extracts were subjected to SDS-PAGE on 12.5% polyacrylamide gels. Gels were transferred to Immobilon P for immunoblot analysis with anti-Flip. The anti-Flip immunoblot is a representative of 10 individuals. (C) Flip transcript levels are upregulated during monocyte to macrophage differentiation. RT-PCR analysis was performed on total RNA, and the 1,470-bp FlipL, 620-bp FlipS, and 838-bp β-actin amplified fragments were fractionated on 1.0% agarose gel and visualized by ethidium bromide staining.
Mentions: The ability of agonistic Fas antibody and TNF-α to induce apoptosis in macrophages was examined. Macrophages were resistant to spontaneous and agonistic Fas antibody–induced apoptosis (Fig. 3 A) as indicated by normal DNA content and mitochondrial membrane integrity, suggesting that a potent inhibitor of the death receptor–mediated pathway, not present in monocytes, may be upregulated in macrophages. As a control, agonistic Fas antibody induced apoptosis in Jurkat T cells and U937 cells (not shown). In certain cell types, Flip overexpression has been shown to directly inhibit Fas-mediated apoptosis 29, indicating that Flip may function to promote survival during monocyte to macrophage differentiation. Previous investigations demonstrated that by day 3 in culture, monocytes begin to differentiate into macrophages based on morphology, cell surface markers including vitronectin (CD51 [32]), transferrin receptor (CD71; not shown), and cytolytic activity 46. Immunoblot analyses performed on isolated peripheral blood monocytes harvested at 0, 1, 2, 3, 7, and 14 d revealed that Flip upregulation was associated with macrophage differentiation and reduced apoptosis, beginning on day 3 (Fig. 3 B). FlipL was detected at 7 and 14 d of macrophage differentiation, whereas FlipS was highly expressed 3 d after isolation (Fig. 3 B). The ratio of FlipL to FlipS on days 3, 7, and 14 varied between individuals. Although low levels of Flip were detected before day 3 in some individuals, Flip upregulation at 3, 7, and 14 d was observed in every individual examined (n = 10). These data demonstrate that the decreased spontaneous apoptosis seen during and after macrophage differentiation is associated with Flip upregulation.

Bottom Line: FLICE (Fas-associated death domain-like interleukin 1beta-converting enzyme)-inhibitory protein (Flip), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis.Here, we show that monocyte differentiation into macrophages was associated with upregulation of Flip and a decrease in Fas-mediated apoptosis.Addition of an antagonistic Fas ligand antibody to Flip antisense-treated macrophages rescued cultures from apoptosis, demonstrating that endogenous Flip blocked Fas-induced cell death.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Northwestern University Medical School, Chicago, Illinois 60611, USA.

ABSTRACT
Macrophages differentiated from circulating peripheral blood monocytes are essential for host immune responses and have been implicated in the pathogenesis of rheumatoid arthritis and atherosclerosis. In contrast to monocytes, macrophages are resistant to Fas-induced cell death by an unknown mechanism. FLICE (Fas-associated death domain-like interleukin 1beta-converting enzyme)-inhibitory protein (Flip), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Here, we show that monocyte differentiation into macrophages was associated with upregulation of Flip and a decrease in Fas-mediated apoptosis. Overexpression of Flip protected monocytes from Fas-mediated apoptosis, whereas acute Flip inhibition in macrophages induced apoptosis. Addition of an antagonistic Fas ligand antibody to Flip antisense-treated macrophages rescued cultures from apoptosis, demonstrating that endogenous Flip blocked Fas-induced cell death. Thus, the expression of Flip in macrophages conferred resistance to Fas-mediated apoptosis, which may contribute to the development of inflammatory disease.

Show MeSH
Related in: MedlinePlus