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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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Spontaneous monocyte apoptosis is regulated by Fas–FasL. (A) Monocytes and macrophages express surface Fas and FasL. Cultures were blocked in human serum, stained with FITC-labeled anti-Fas, anti-CD3, anti–TCR-γ/δ, or isotype control. Additional cells were stained with anti-FasL (C-20), anti-FasL (4H9), or normal rabbit IgG, then with FITC-labeled goat anti–rabbit or rabbit anti–hamster antibody. All cultures were then analyzed by flow cytometry. Control Igs are in blue; the experimental antibodies (anti-Fas, anti-FasL, anti–TCR-γ/δ, or anti-CD3) are in red. (B) Neutralizing anti-FasL antibody protects monocytes cultured in serum from spontaneous apoptosis. Immediately after isolation, triplicate cultures of monocytes were treated with neutralizing anti-FasL (10 μg/ml), control IgG (10 μg/ml), zVAD.fmk (20 μM), TNF-α (10 ng/ml), or control medium (Mock) for 24 h. Cultures were fixed in 70% ETOH, incubated in PI (50 μg/ml), and analyzed by flow cytometry. Values represent the mean ± SE of three independent experiments. Anti-FasL and control IgG–treated cultures, and zVAD.fmk-, TNF-α–, and mock-treated cultures were compared for statistical significance by unpaired two-tailed Student's t test. *P < 0.0001 for anti-FasL, P < 0.01 for zVAD.fmk, and P < 0.02 for TNF-α. (C) Whole cell extracts (25 μg) were prepared from peripheral blood monocytes and macrophages which had been cultured in 20% FBS/RPMI/1 μg/ml polymyxin B, and isolated at 0, 1, 2, and 7 d. Extracts were subjected to SDS-PAGE on 12.5% polyacrylamide gels. Gels were transferred to Immobilon P for immunoblot analysis with anti-caspases 8 and 3 antibodies. The anti-caspase 8 and 3 immunoblots are representative of three individuals.
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Figure 2: Spontaneous monocyte apoptosis is regulated by Fas–FasL. (A) Monocytes and macrophages express surface Fas and FasL. Cultures were blocked in human serum, stained with FITC-labeled anti-Fas, anti-CD3, anti–TCR-γ/δ, or isotype control. Additional cells were stained with anti-FasL (C-20), anti-FasL (4H9), or normal rabbit IgG, then with FITC-labeled goat anti–rabbit or rabbit anti–hamster antibody. All cultures were then analyzed by flow cytometry. Control Igs are in blue; the experimental antibodies (anti-Fas, anti-FasL, anti–TCR-γ/δ, or anti-CD3) are in red. (B) Neutralizing anti-FasL antibody protects monocytes cultured in serum from spontaneous apoptosis. Immediately after isolation, triplicate cultures of monocytes were treated with neutralizing anti-FasL (10 μg/ml), control IgG (10 μg/ml), zVAD.fmk (20 μM), TNF-α (10 ng/ml), or control medium (Mock) for 24 h. Cultures were fixed in 70% ETOH, incubated in PI (50 μg/ml), and analyzed by flow cytometry. Values represent the mean ± SE of three independent experiments. Anti-FasL and control IgG–treated cultures, and zVAD.fmk-, TNF-α–, and mock-treated cultures were compared for statistical significance by unpaired two-tailed Student's t test. *P < 0.0001 for anti-FasL, P < 0.01 for zVAD.fmk, and P < 0.02 for TNF-α. (C) Whole cell extracts (25 μg) were prepared from peripheral blood monocytes and macrophages which had been cultured in 20% FBS/RPMI/1 μg/ml polymyxin B, and isolated at 0, 1, 2, and 7 d. Extracts were subjected to SDS-PAGE on 12.5% polyacrylamide gels. Gels were transferred to Immobilon P for immunoblot analysis with anti-caspases 8 and 3 antibodies. The anti-caspase 8 and 3 immunoblots are representative of three individuals.

Mentions: Monocytes cultured in serum undergo spontaneous apoptosis that may be mediated by the Fas–FasL pathway. Flow cytometric analyses of surface Fas and FasL revealed that Fas was present on essentially all the cells, although it was more strongly expressed on monocytes compared with macrophages (Fig. 2 A). FasL was also detected on the surface of both monocytes and macrophages, though more FasL was expressed on macrophages (Fig. 2 A). To confirm that the detection of Fas and FasL was specific, flow cytometric analysis using FITC-conjugated anti-CD3 and anti–TCR-γ/δ antibodies demonstrated comparable staining to the isotype control in both monocytes and macrophages (Fig. 2 A). To define the mechanism(s) responsible for monocyte apoptosis, inhibitory FasL antibodies and the general caspase inhibitor zVAD.fmk were compared with TNF-α, which was previously shown to inhibit monocyte apoptosis 253031. Addition of either C-20 (82% inhibition) or 4H9 (41% inhibition, not shown) neutralizing anti-FasL antibodies to isolated monocytes in 20% serum significantly (P < 0.02) inhibited spontaneous apoptosis compared with IgG-treated or medium control–treated (mock) monocytes (Fig. 2 B). Similarly, TNF-α inhibited apoptosis by 62% (P < 0.02) while the general caspase inhibitor zVAD.fmk partially blocked spontaneous apoptosis (41%, P < 0.01) in 1-d (Fig. 2 B) and 2-d (not shown) monocyte cultures. The contribution of caspase activation to Fas-mediated apoptosis in monocytes was further examined by immunoblot analyses. Reduced detection of the procaspases 8 and 3 was observed in extracts from 0 and 1-d monocytes (Fig. 2 C) compared with 7-d macrophages. The intermediate active form of caspase 3 (p24 45) was detected in extracts of monocytes on days 0 and 1, but not those from day 7 macrophages. As a control, Fas-agonistic antibody–treated Jurkat T cells also displayed the intermediate active caspase 3 (not shown). These data indicate that the spontaneous monocyte apoptosis that occurs in serum was attributed to Fas–FasL interaction and caspase activation.


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)

Spontaneous monocyte apoptosis is regulated by Fas–FasL. (A) Monocytes and macrophages express surface Fas and FasL. Cultures were blocked in human serum, stained with FITC-labeled anti-Fas, anti-CD3, anti–TCR-γ/δ, or isotype control. Additional cells were stained with anti-FasL (C-20), anti-FasL (4H9), or normal rabbit IgG, then with FITC-labeled goat anti–rabbit or rabbit anti–hamster antibody. All cultures were then analyzed by flow cytometry. Control Igs are in blue; the experimental antibodies (anti-Fas, anti-FasL, anti–TCR-γ/δ, or anti-CD3) are in red. (B) Neutralizing anti-FasL antibody protects monocytes cultured in serum from spontaneous apoptosis. Immediately after isolation, triplicate cultures of monocytes were treated with neutralizing anti-FasL (10 μg/ml), control IgG (10 μg/ml), zVAD.fmk (20 μM), TNF-α (10 ng/ml), or control medium (Mock) for 24 h. Cultures were fixed in 70% ETOH, incubated in PI (50 μg/ml), and analyzed by flow cytometry. Values represent the mean ± SE of three independent experiments. Anti-FasL and control IgG–treated cultures, and zVAD.fmk-, TNF-α–, and mock-treated cultures were compared for statistical significance by unpaired two-tailed Student's t test. *P < 0.0001 for anti-FasL, P < 0.01 for zVAD.fmk, and P < 0.02 for TNF-α. (C) Whole cell extracts (25 μg) were prepared from peripheral blood monocytes and macrophages which had been cultured in 20% FBS/RPMI/1 μg/ml polymyxin B, and isolated at 0, 1, 2, and 7 d. Extracts were subjected to SDS-PAGE on 12.5% polyacrylamide gels. Gels were transferred to Immobilon P for immunoblot analysis with anti-caspases 8 and 3 antibodies. The anti-caspase 8 and 3 immunoblots are representative of three individuals.
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Figure 2: Spontaneous monocyte apoptosis is regulated by Fas–FasL. (A) Monocytes and macrophages express surface Fas and FasL. Cultures were blocked in human serum, stained with FITC-labeled anti-Fas, anti-CD3, anti–TCR-γ/δ, or isotype control. Additional cells were stained with anti-FasL (C-20), anti-FasL (4H9), or normal rabbit IgG, then with FITC-labeled goat anti–rabbit or rabbit anti–hamster antibody. All cultures were then analyzed by flow cytometry. Control Igs are in blue; the experimental antibodies (anti-Fas, anti-FasL, anti–TCR-γ/δ, or anti-CD3) are in red. (B) Neutralizing anti-FasL antibody protects monocytes cultured in serum from spontaneous apoptosis. Immediately after isolation, triplicate cultures of monocytes were treated with neutralizing anti-FasL (10 μg/ml), control IgG (10 μg/ml), zVAD.fmk (20 μM), TNF-α (10 ng/ml), or control medium (Mock) for 24 h. Cultures were fixed in 70% ETOH, incubated in PI (50 μg/ml), and analyzed by flow cytometry. Values represent the mean ± SE of three independent experiments. Anti-FasL and control IgG–treated cultures, and zVAD.fmk-, TNF-α–, and mock-treated cultures were compared for statistical significance by unpaired two-tailed Student's t test. *P < 0.0001 for anti-FasL, P < 0.01 for zVAD.fmk, and P < 0.02 for TNF-α. (C) Whole cell extracts (25 μg) were prepared from peripheral blood monocytes and macrophages which had been cultured in 20% FBS/RPMI/1 μg/ml polymyxin B, and isolated at 0, 1, 2, and 7 d. Extracts were subjected to SDS-PAGE on 12.5% polyacrylamide gels. Gels were transferred to Immobilon P for immunoblot analysis with anti-caspases 8 and 3 antibodies. The anti-caspase 8 and 3 immunoblots are representative of three individuals.
Mentions: Monocytes cultured in serum undergo spontaneous apoptosis that may be mediated by the Fas–FasL pathway. Flow cytometric analyses of surface Fas and FasL revealed that Fas was present on essentially all the cells, although it was more strongly expressed on monocytes compared with macrophages (Fig. 2 A). FasL was also detected on the surface of both monocytes and macrophages, though more FasL was expressed on macrophages (Fig. 2 A). To confirm that the detection of Fas and FasL was specific, flow cytometric analysis using FITC-conjugated anti-CD3 and anti–TCR-γ/δ antibodies demonstrated comparable staining to the isotype control in both monocytes and macrophages (Fig. 2 A). To define the mechanism(s) responsible for monocyte apoptosis, inhibitory FasL antibodies and the general caspase inhibitor zVAD.fmk were compared with TNF-α, which was previously shown to inhibit monocyte apoptosis 253031. Addition of either C-20 (82% inhibition) or 4H9 (41% inhibition, not shown) neutralizing anti-FasL antibodies to isolated monocytes in 20% serum significantly (P < 0.02) inhibited spontaneous apoptosis compared with IgG-treated or medium control–treated (mock) monocytes (Fig. 2 B). Similarly, TNF-α inhibited apoptosis by 62% (P < 0.02) while the general caspase inhibitor zVAD.fmk partially blocked spontaneous apoptosis (41%, P < 0.01) in 1-d (Fig. 2 B) and 2-d (not shown) monocyte cultures. The contribution of caspase activation to Fas-mediated apoptosis in monocytes was further examined by immunoblot analyses. Reduced detection of the procaspases 8 and 3 was observed in extracts from 0 and 1-d monocytes (Fig. 2 C) compared with 7-d macrophages. The intermediate active form of caspase 3 (p24 45) was detected in extracts of monocytes on days 0 and 1, but not those from day 7 macrophages. As a control, Fas-agonistic antibody–treated Jurkat T cells also displayed the intermediate active caspase 3 (not shown). These data indicate that the spontaneous monocyte apoptosis that occurs in serum was attributed to Fas–FasL interaction and caspase activation.

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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Related in: MedlinePlus