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Persistence of apoptotic cells without autoimmune disease or inflammation in CD14-/- mice.

Devitt A, Parker KG, Ogden CA, Oldreive C, Clay MF, Melville LA, Bellamy CO, Lacy-Hulbert A, Gangloff SC, Goyert SM, Gregory CD - J. Cell Biol. (2004)

Bottom Line: Significantly, CD14(-/-) macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process.However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14(-/-) macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells.We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Centre for Inflammation Research, University of Edinburgh, Edinburgh, Scotland, UK.

ABSTRACT
Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14(-/-) macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14(-/-) macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.

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Macrophage tethering of apoptotic cells by CD14. (A) Binding (in the absence of phagocytosis) of apoptotic BL cells (AC) by HMDMs assessed after coculture at 16–20°C for 60 min in the presence or absence of CD14 mAbs 61D3 or 63D3. Data shown are means ± SEM (n = 2). ANOVA: *, P < 0.05. (B) Binding of apoptotic BL cells (AC) by either mock-transfected or CD14-transfected COS-1 cells after coculture at 16–20°C for 60 min in the presence (gray bar) or absence (black bar) of CD14 mAb 61D3. (inset) Expression of CD14 by COS-1 cells as assessed by flow cytometry following immunofluorescence staining using mAb 63D3 and goat anti–mouse-FITC (shown in red) versus staining of mock transfectants (shown in blue). ANOVA: ***, P < 0.001. (C) Photomicrographs showing binding (but not phagocytosis) of apoptotic BL cells by COS cells. Binding is promoted by the expression of CD14 (arrow). (D) Binding of 10-d BMDMs from CD14+/+ (gray bar) and CD14−/− (black bar) mice with apoptotic BL cells assessed after 30-min coculture at 16–20°C. Data shown are means ± SEM (n = 3). ANOVA: ***, P < 0.001. (E) Binding of peritoneal macrophages from CD14+/+ (gray bar) and CD14−/− (black bar) mice with apoptotic BL cells assessed after 30-min coculture at 16–20°C. Data shown are means ± SEM (n = 3). ANOVA: ***, P < 0.001. (F) Binding of soluble recombinant human CD14 (sCD14) to apoptotic cells. BL cells undergoing spontaneous apoptosis were identified as “apoptotic” or “viable” according to light-scatter properties and confirmed by morphological analyses (Dive et al., 1992). Spontaneous apoptosis, a characteristic feature of group I BL cell lines (Gregory et al., 1991) was confirmed by morphological analysis as described previously (Devitt et al., 2003). Histograms of recombinant forms of CD14, sCD14-Fc, and sCD14-HIS binding to cells in these zones are shown. Control staining is shown in blue against staining with SCD14-Fc and SCD14-HIS in red.
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fig2: Macrophage tethering of apoptotic cells by CD14. (A) Binding (in the absence of phagocytosis) of apoptotic BL cells (AC) by HMDMs assessed after coculture at 16–20°C for 60 min in the presence or absence of CD14 mAbs 61D3 or 63D3. Data shown are means ± SEM (n = 2). ANOVA: *, P < 0.05. (B) Binding of apoptotic BL cells (AC) by either mock-transfected or CD14-transfected COS-1 cells after coculture at 16–20°C for 60 min in the presence (gray bar) or absence (black bar) of CD14 mAb 61D3. (inset) Expression of CD14 by COS-1 cells as assessed by flow cytometry following immunofluorescence staining using mAb 63D3 and goat anti–mouse-FITC (shown in red) versus staining of mock transfectants (shown in blue). ANOVA: ***, P < 0.001. (C) Photomicrographs showing binding (but not phagocytosis) of apoptotic BL cells by COS cells. Binding is promoted by the expression of CD14 (arrow). (D) Binding of 10-d BMDMs from CD14+/+ (gray bar) and CD14−/− (black bar) mice with apoptotic BL cells assessed after 30-min coculture at 16–20°C. Data shown are means ± SEM (n = 3). ANOVA: ***, P < 0.001. (E) Binding of peritoneal macrophages from CD14+/+ (gray bar) and CD14−/− (black bar) mice with apoptotic BL cells assessed after 30-min coculture at 16–20°C. Data shown are means ± SEM (n = 3). ANOVA: ***, P < 0.001. (F) Binding of soluble recombinant human CD14 (sCD14) to apoptotic cells. BL cells undergoing spontaneous apoptosis were identified as “apoptotic” or “viable” according to light-scatter properties and confirmed by morphological analyses (Dive et al., 1992). Spontaneous apoptosis, a characteristic feature of group I BL cell lines (Gregory et al., 1991) was confirmed by morphological analysis as described previously (Devitt et al., 2003). Histograms of recombinant forms of CD14, sCD14-Fc, and sCD14-HIS binding to cells in these zones are shown. Control staining is shown in blue against staining with SCD14-Fc and SCD14-HIS in red.

Mentions: To investigate tethering events independently of phagocytosis, macrophage–apoptotic cell interactions were conducted at 16–20°C, which permitted binding, but not phagocytic, events to occur (Fig. 2 and not depicted). Tethering of apoptotic cells to human monocyte-derived macrophages (HMDMs) was reduced significantly by mAb 61D3 but not by mAb 63D3 (Fig. 2 A). Enhanced tethering of apoptotic cells induced by transient expression of CD14 in COS transfectants (Fig. 2 C) was returned to background levels by 61D3 (Fig. 2 B). BMDMs (Fig. 2 D) and peritoneal macrophages (Fig. 2 E) from CD14−/− mice also bound apoptotic cells less effectively than their CD14+/+ counterparts. Finally, to determine whether or not CD14 is involved directly in the discrimination of viable and apoptotic cells, purified recombinant forms of CD14 were assessed for cell-binding ability. As shown in Fig. 2 F, recombinant CD14 bound apoptotic, but not viable, cells. The latter result raises the possibility that soluble CD14, which is present in high amounts in plasma and secretions might also be involved in apoptotic cell clearance. Together, these results demonstrate in both human and murine systems that CD14 can function independently of phagocytic events by tethering apoptotic cells to macrophages. Because of its relative specificity for apoptotic over viable cells, we conclude that CD14 also contributes to apoptotic cell recognition by macrophages.


Persistence of apoptotic cells without autoimmune disease or inflammation in CD14-/- mice.

Devitt A, Parker KG, Ogden CA, Oldreive C, Clay MF, Melville LA, Bellamy CO, Lacy-Hulbert A, Gangloff SC, Goyert SM, Gregory CD - J. Cell Biol. (2004)

Macrophage tethering of apoptotic cells by CD14. (A) Binding (in the absence of phagocytosis) of apoptotic BL cells (AC) by HMDMs assessed after coculture at 16–20°C for 60 min in the presence or absence of CD14 mAbs 61D3 or 63D3. Data shown are means ± SEM (n = 2). ANOVA: *, P < 0.05. (B) Binding of apoptotic BL cells (AC) by either mock-transfected or CD14-transfected COS-1 cells after coculture at 16–20°C for 60 min in the presence (gray bar) or absence (black bar) of CD14 mAb 61D3. (inset) Expression of CD14 by COS-1 cells as assessed by flow cytometry following immunofluorescence staining using mAb 63D3 and goat anti–mouse-FITC (shown in red) versus staining of mock transfectants (shown in blue). ANOVA: ***, P < 0.001. (C) Photomicrographs showing binding (but not phagocytosis) of apoptotic BL cells by COS cells. Binding is promoted by the expression of CD14 (arrow). (D) Binding of 10-d BMDMs from CD14+/+ (gray bar) and CD14−/− (black bar) mice with apoptotic BL cells assessed after 30-min coculture at 16–20°C. Data shown are means ± SEM (n = 3). ANOVA: ***, P < 0.001. (E) Binding of peritoneal macrophages from CD14+/+ (gray bar) and CD14−/− (black bar) mice with apoptotic BL cells assessed after 30-min coculture at 16–20°C. Data shown are means ± SEM (n = 3). ANOVA: ***, P < 0.001. (F) Binding of soluble recombinant human CD14 (sCD14) to apoptotic cells. BL cells undergoing spontaneous apoptosis were identified as “apoptotic” or “viable” according to light-scatter properties and confirmed by morphological analyses (Dive et al., 1992). Spontaneous apoptosis, a characteristic feature of group I BL cell lines (Gregory et al., 1991) was confirmed by morphological analysis as described previously (Devitt et al., 2003). Histograms of recombinant forms of CD14, sCD14-Fc, and sCD14-HIS binding to cells in these zones are shown. Control staining is shown in blue against staining with SCD14-Fc and SCD14-HIS in red.
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fig2: Macrophage tethering of apoptotic cells by CD14. (A) Binding (in the absence of phagocytosis) of apoptotic BL cells (AC) by HMDMs assessed after coculture at 16–20°C for 60 min in the presence or absence of CD14 mAbs 61D3 or 63D3. Data shown are means ± SEM (n = 2). ANOVA: *, P < 0.05. (B) Binding of apoptotic BL cells (AC) by either mock-transfected or CD14-transfected COS-1 cells after coculture at 16–20°C for 60 min in the presence (gray bar) or absence (black bar) of CD14 mAb 61D3. (inset) Expression of CD14 by COS-1 cells as assessed by flow cytometry following immunofluorescence staining using mAb 63D3 and goat anti–mouse-FITC (shown in red) versus staining of mock transfectants (shown in blue). ANOVA: ***, P < 0.001. (C) Photomicrographs showing binding (but not phagocytosis) of apoptotic BL cells by COS cells. Binding is promoted by the expression of CD14 (arrow). (D) Binding of 10-d BMDMs from CD14+/+ (gray bar) and CD14−/− (black bar) mice with apoptotic BL cells assessed after 30-min coculture at 16–20°C. Data shown are means ± SEM (n = 3). ANOVA: ***, P < 0.001. (E) Binding of peritoneal macrophages from CD14+/+ (gray bar) and CD14−/− (black bar) mice with apoptotic BL cells assessed after 30-min coculture at 16–20°C. Data shown are means ± SEM (n = 3). ANOVA: ***, P < 0.001. (F) Binding of soluble recombinant human CD14 (sCD14) to apoptotic cells. BL cells undergoing spontaneous apoptosis were identified as “apoptotic” or “viable” according to light-scatter properties and confirmed by morphological analyses (Dive et al., 1992). Spontaneous apoptosis, a characteristic feature of group I BL cell lines (Gregory et al., 1991) was confirmed by morphological analysis as described previously (Devitt et al., 2003). Histograms of recombinant forms of CD14, sCD14-Fc, and sCD14-HIS binding to cells in these zones are shown. Control staining is shown in blue against staining with SCD14-Fc and SCD14-HIS in red.
Mentions: To investigate tethering events independently of phagocytosis, macrophage–apoptotic cell interactions were conducted at 16–20°C, which permitted binding, but not phagocytic, events to occur (Fig. 2 and not depicted). Tethering of apoptotic cells to human monocyte-derived macrophages (HMDMs) was reduced significantly by mAb 61D3 but not by mAb 63D3 (Fig. 2 A). Enhanced tethering of apoptotic cells induced by transient expression of CD14 in COS transfectants (Fig. 2 C) was returned to background levels by 61D3 (Fig. 2 B). BMDMs (Fig. 2 D) and peritoneal macrophages (Fig. 2 E) from CD14−/− mice also bound apoptotic cells less effectively than their CD14+/+ counterparts. Finally, to determine whether or not CD14 is involved directly in the discrimination of viable and apoptotic cells, purified recombinant forms of CD14 were assessed for cell-binding ability. As shown in Fig. 2 F, recombinant CD14 bound apoptotic, but not viable, cells. The latter result raises the possibility that soluble CD14, which is present in high amounts in plasma and secretions might also be involved in apoptotic cell clearance. Together, these results demonstrate in both human and murine systems that CD14 can function independently of phagocytic events by tethering apoptotic cells to macrophages. Because of its relative specificity for apoptotic over viable cells, we conclude that CD14 also contributes to apoptotic cell recognition by macrophages.

Bottom Line: Significantly, CD14(-/-) macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process.However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14(-/-) macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells.We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Centre for Inflammation Research, University of Edinburgh, Edinburgh, Scotland, UK.

ABSTRACT
Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14(-/-) macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14(-/-) macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.

Show MeSH
Related in: MedlinePlus