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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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Persistence of apoptotic cells in tissues of normal CD14−/− mice. (A) Morphometric analysis of the frequency of ISEL+ nuclei present in the thymic cortex and medulla of CD14+/+ and CD14−/− mice. (B) Quantitative analyses of thymic sections showing the numbers of free and macrophage-associated apoptotic cells together with the ratios of free/macrophage-associated apoptotic cells for CD14+/+ and CD14−/− cortices. (C) Morphometric analyses of the distribution of macrophages (F4/80-reactive material) in the cortex and medulla of CD14+/+ and CD14−/− thymus. All thymus data shown are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.05; **, P < 0.01. (D) Morphometric analysis of the frequency of ISEL+ nuclei present in the splenic red and white pulp of CD14+/+ and CD14−/− mice. ANOVA: ***, P < 0.001. (E) Quantitative analysis of the numbers of free and apoptotic cells together with the ratios of free/macrophage-associated apoptotic cells for CD14+/+ and CD14−/− splenic red pulp. (F) Morphometric analyses of the distribution of F4/80+ macrophages in the cortex and medulla of CD14+/+ and CD14−/− spleen. All spleen data shown are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.05; ***, P < 0.001. (G) Persistence of apoptotic cells in non-lymphoid tissues of CD14−/− mice. Morphometric analyses of the frequency of ISEL+ nuclei present in the lung, liver, and large intestine (LI) of CD14+/+ and CD14−/− mice. Data are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.1; **, P < 0.01. (H) Photomicrographs showing histological detail of CD14+/+ versus CD14−/− thymus. Note preponderance of free apoptotic cells in CD14−/− thymus (arrows) and absence of inflammatory cell infiltrate.
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fig3: Persistence of apoptotic cells in tissues of normal CD14−/− mice. (A) Morphometric analysis of the frequency of ISEL+ nuclei present in the thymic cortex and medulla of CD14+/+ and CD14−/− mice. (B) Quantitative analyses of thymic sections showing the numbers of free and macrophage-associated apoptotic cells together with the ratios of free/macrophage-associated apoptotic cells for CD14+/+ and CD14−/− cortices. (C) Morphometric analyses of the distribution of macrophages (F4/80-reactive material) in the cortex and medulla of CD14+/+ and CD14−/− thymus. All thymus data shown are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.05; **, P < 0.01. (D) Morphometric analysis of the frequency of ISEL+ nuclei present in the splenic red and white pulp of CD14+/+ and CD14−/− mice. ANOVA: ***, P < 0.001. (E) Quantitative analysis of the numbers of free and apoptotic cells together with the ratios of free/macrophage-associated apoptotic cells for CD14+/+ and CD14−/− splenic red pulp. (F) Morphometric analyses of the distribution of F4/80+ macrophages in the cortex and medulla of CD14+/+ and CD14−/− spleen. All spleen data shown are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.05; ***, P < 0.001. (G) Persistence of apoptotic cells in non-lymphoid tissues of CD14−/− mice. Morphometric analyses of the frequency of ISEL+ nuclei present in the lung, liver, and large intestine (LI) of CD14+/+ and CD14−/− mice. Data are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.1; **, P < 0.01. (H) Photomicrographs showing histological detail of CD14+/+ versus CD14−/− thymus. Note preponderance of free apoptotic cells in CD14−/− thymus (arrows) and absence of inflammatory cell infiltrate.

Mentions: If CD14's role in the clearance process were redundant in vivo, the frequency of apoptotic cells in histological sections should be equivalent in tissues of CD14−/− and CD14+/+ mice. We initially analyzed the normal thymus of young adult animals because apoptosis normally occurs at high rate in this tissue and apoptotic thymocytes can be readily observed in situ (Surh and Sprent, 1994). Macroscopically, no differences were noted between CD14−/− and CD14+/+ thymi, and no overt microscopic differences in cellularity or tissue architecture were observed. However, quantitative analyses of apoptotic thymocytes identified by in situ end labeling (ISEL; Fig. 3 A) revealed increased frequencies of apoptotic events in both the cortex and medulla of the CD14−/− thymus compared with its CD14+/+ counterpart. Detailed morphological analyses of thymic cortex in toluidine blue–stained, resin-embedded sections allowed apoptotic cells to be defined as either “free” or “macrophage-associated” (Fig. 3 B). Quantitative analyses indicated that, although there were increased numbers both of free and of macrophage-associated apoptotic cells in the cortices of CD14−/− compared with CD14+/+ thymi, the ratio of free/macrophage-associated events was substantially higher in the CD14−/− animals (Fig. 3 B) despite equivalent distribution of macrophages throughout the thymus as assessed by F4/80 staining (Fig. 3 C). Similar results showing the presence of free apoptotic cells in thymus of CD14−/− and CD14+/+ mice were obtained after annexin V (AxV) and propidium iodide (PI) staining of dissociated thymi (see Fig. 5 A). Earlier studies have reported marginally lower levels of free apoptotic cells in the murine thymus than those reported here (Surh and Sprent, 1994; McIlroy et al., 2000), although it is important to note that different mouse strains and methodologies were used.


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)

Persistence of apoptotic cells in tissues of normal CD14−/− mice. (A) Morphometric analysis of the frequency of ISEL+ nuclei present in the thymic cortex and medulla of CD14+/+ and CD14−/− mice. (B) Quantitative analyses of thymic sections showing the numbers of free and macrophage-associated apoptotic cells together with the ratios of free/macrophage-associated apoptotic cells for CD14+/+ and CD14−/− cortices. (C) Morphometric analyses of the distribution of macrophages (F4/80-reactive material) in the cortex and medulla of CD14+/+ and CD14−/− thymus. All thymus data shown are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.05; **, P < 0.01. (D) Morphometric analysis of the frequency of ISEL+ nuclei present in the splenic red and white pulp of CD14+/+ and CD14−/− mice. ANOVA: ***, P < 0.001. (E) Quantitative analysis of the numbers of free and apoptotic cells together with the ratios of free/macrophage-associated apoptotic cells for CD14+/+ and CD14−/− splenic red pulp. (F) Morphometric analyses of the distribution of F4/80+ macrophages in the cortex and medulla of CD14+/+ and CD14−/− spleen. All spleen data shown are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.05; ***, P < 0.001. (G) Persistence of apoptotic cells in non-lymphoid tissues of CD14−/− mice. Morphometric analyses of the frequency of ISEL+ nuclei present in the lung, liver, and large intestine (LI) of CD14+/+ and CD14−/− mice. Data are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.1; **, P < 0.01. (H) Photomicrographs showing histological detail of CD14+/+ versus CD14−/− thymus. Note preponderance of free apoptotic cells in CD14−/− thymus (arrows) and absence of inflammatory cell infiltrate.
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fig3: Persistence of apoptotic cells in tissues of normal CD14−/− mice. (A) Morphometric analysis of the frequency of ISEL+ nuclei present in the thymic cortex and medulla of CD14+/+ and CD14−/− mice. (B) Quantitative analyses of thymic sections showing the numbers of free and macrophage-associated apoptotic cells together with the ratios of free/macrophage-associated apoptotic cells for CD14+/+ and CD14−/− cortices. (C) Morphometric analyses of the distribution of macrophages (F4/80-reactive material) in the cortex and medulla of CD14+/+ and CD14−/− thymus. All thymus data shown are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.05; **, P < 0.01. (D) Morphometric analysis of the frequency of ISEL+ nuclei present in the splenic red and white pulp of CD14+/+ and CD14−/− mice. ANOVA: ***, P < 0.001. (E) Quantitative analysis of the numbers of free and apoptotic cells together with the ratios of free/macrophage-associated apoptotic cells for CD14+/+ and CD14−/− splenic red pulp. (F) Morphometric analyses of the distribution of F4/80+ macrophages in the cortex and medulla of CD14+/+ and CD14−/− spleen. All spleen data shown are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.05; ***, P < 0.001. (G) Persistence of apoptotic cells in non-lymphoid tissues of CD14−/− mice. Morphometric analyses of the frequency of ISEL+ nuclei present in the lung, liver, and large intestine (LI) of CD14+/+ and CD14−/− mice. Data are means ± SEM (n = 3 animals in each case). ANOVA: *, P < 0.1; **, P < 0.01. (H) Photomicrographs showing histological detail of CD14+/+ versus CD14−/− thymus. Note preponderance of free apoptotic cells in CD14−/− thymus (arrows) and absence of inflammatory cell infiltrate.
Mentions: If CD14's role in the clearance process were redundant in vivo, the frequency of apoptotic cells in histological sections should be equivalent in tissues of CD14−/− and CD14+/+ mice. We initially analyzed the normal thymus of young adult animals because apoptosis normally occurs at high rate in this tissue and apoptotic thymocytes can be readily observed in situ (Surh and Sprent, 1994). Macroscopically, no differences were noted between CD14−/− and CD14+/+ thymi, and no overt microscopic differences in cellularity or tissue architecture were observed. However, quantitative analyses of apoptotic thymocytes identified by in situ end labeling (ISEL; Fig. 3 A) revealed increased frequencies of apoptotic events in both the cortex and medulla of the CD14−/− thymus compared with its CD14+/+ counterpart. Detailed morphological analyses of thymic cortex in toluidine blue–stained, resin-embedded sections allowed apoptotic cells to be defined as either “free” or “macrophage-associated” (Fig. 3 B). Quantitative analyses indicated that, although there were increased numbers both of free and of macrophage-associated apoptotic cells in the cortices of CD14−/− compared with CD14+/+ thymi, the ratio of free/macrophage-associated events was substantially higher in the CD14−/− animals (Fig. 3 B) despite equivalent distribution of macrophages throughout the thymus as assessed by F4/80 staining (Fig. 3 C). Similar results showing the presence of free apoptotic cells in thymus of CD14−/− and CD14+/+ mice were obtained after annexin V (AxV) and propidium iodide (PI) staining of dissociated thymi (see Fig. 5 A). Earlier studies have reported marginally lower levels of free apoptotic cells in the murine thymus than those reported here (Surh and Sprent, 1994; McIlroy et al., 2000), although it is important to note that different mouse strains and methodologies were used.

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