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Mechanisms involved in macrophage phagocytosis of apoptotic cells
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Efficient removal of apoptotic cells is critical for development, tissue remodelling, maintenance of homeostasis, and response to injury. Phagocytosis of apoptotic cells is mediated by many phagocytic receptors, soluble bridging molecules, and pro-phagocytic ligands on the surface of apoptotic cells. Macrophage phagocytosis in general is controlled by stimulatory and inhibitory mechanisms. An example of the latter mechanism is that mediated by the cell surface glycoprotein CD47, which by binding to the inhibitory receptor Signal Regulatory Protein alpha (SIRPα) on macrophages, is known to inhibit phagocytosis of viable host cells. The studies of the present thesis aimed at investigating possible changes to CD47 on apoptotic cells, which could influence their elimination by macrophages.

The endoplasmatic protein calreticulin (CRT), in conjunction with Low density lipoprotein Receptorrelated Protein 1 (LRP1) on the phagocyte, can act as a receptor for collectin family members and mediate uptake of apoptotic cells. However, CRT itself was found to also be expressed on the surface of many viable cell types, and the CRT expression increased on apoptotic cells. By using antibodies to LRP1 or receptor‐associated protein (RAP), an antagonist blocking LRP1 ligand binding, we found that CRT on target cells could interact in trans with LRP1 on a phagocyte and stimulate phagocytosis. CD47 on the target cell inhibited LRP1‐mediated phagocytosis of viable cells (e.g. lymphocytes or erythtocytes), but not that of apoptotic cells. The inability of CD47 on apoptotic cells to inhibit LRP1‐ mediated phagocytosis could be explained in two ways: 1) Some apoptotic cell types (fibroblasts and neutrophils, but not Jurkat T cells) lost CD47 from the cell surface, or 2) CD47 is evenly distributed on the surface of viable cells, while it was redistributed into patches on apoptotic cells, segregated away from areas of the plasma membrane where the pro‐phagocytic ligands CRT and phoaphatidylserine (PS) were concentrated. Apoptotic murine thymocytes also showed a patched distribution of CD47, but no significant loss of the receptor. However, both PS‐independent and PS‐dependent macrophage phagocytosis of apoptotic CD47‐/‐ thymocytes was less efficient than uptake of apoptotic wild‐type (wt) thymocytes. This contradictory finding was explained by the fact that CD47 on apoptotic thymocytes did no longer inhibit phagocytosis, but rather mediated binding of the apoptotic cell to the macrophage. These effects could in part be dependent on the apoptotic cell type, since uptake of experimentally senescent PS+ wt or CD47‐/‐ erythrocytes by macrophage in vitro, or by dendritic cells (DC) in vivo, were the same. In vivo, PS+ erythrocytes were predominantly trapped by marginal zone macrophages and by CD8+ CD207+ DCs in the splenic marginal zone. DCs which had taken up PS+ erythrocytes showed a slight increase in expression levels of CD40, CD86 and MHC class II. These findings suggest that PS+ erythrocytes may be recognized by splenic macrophages and DCs in ways similar to that reported for apoptotic T cells. Uptake of senescent erythrocytes by DCs may serve as an important mechanism to maintain self‐tolerance to erythrocyte antigens, and defects in this function may facilitate development of AIHA.

Glucocorticoids are used to treat inflammatory conditions and can enhance macrophage uptake of apoptotic cells. We found that the glucocorticoid dexamethasone time‐ and dose‐dependently stimulated macrophage cell surface LRP1 expression. Dexamethasone‐stimulated macrophages also showed enhanced phagocytosis of apoptotic thymocytes and unopsonized viable CD47‐/‐ erythrocytes.

In summary, LRP1 can mediate phagocytosis of both viable and apoptotic cells by binding CRT on the target cell. Macrophage expression of LRP1 is increased by glucocorticoids, which could be one explanation for the anti‐inflammatory role of glucocorticoids. While CD47 on viable cells efficiently inhibits phagocytosis in macrophages, CD47 on apoptotic cells does not and can sometimes even promote their removal.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2009. , 50 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1307
Keyword [en]
macrophages, dendritic cells, phagocytosis, apoptotic cells, senescent erythrocytes, CD47, SIRPalpha, LRP1, calreticulin
National Category
Immunology
Research subject
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-29869ISBN: 978-91-7264-890-6 (print)OAI: oai:DiVA.org:umu-29869DiVA: diva2:278362
Public defence
2009-12-17, BiA201 Biologihuset, Umeå Universitet, Umeå, 00:00 (English)
Opponent
Supervisors
Available from: 2009-11-27 Created: 2009-11-25 Last updated: 2012-03-06Bibliographically approved
List of papers
1. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte
Open this publication in new window or tab >>Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte
Show others...
2005 (English)In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 123, no 2, 321-334 p.Article in journal (Refereed) Published
Abstract [en]

Apoptotic-cell removal is critical for development, tissue homeostasis, and resolution of inflammation. Although many candidate systems exist, only phosphatidylserine has been identified as a general recognition ligand on apoptotic cells. We demonstrate here that calreticulin acts as a second general recognition ligand by binding and activating LDL-receptor-related protein (LRP) on the engulfing cell. Since surface calreticulin is also found on viable cells, a mechanism preventing inadvertent uptake was sought. Disruption of interactions between CD47 (integrin-associated protein) on the target cell and SIRPalpha (SHPS-1), a heavily glycosylated transmembrane protein on the engulfing cell, permitted uptake of viable cells in a calreticulin/LRP-dependent manner. On apoptotic cells, CD47 was altered and/or lost and no longer activated SIRPalpha. These changes on the apoptotic cell create an environment where "don't eat me" signals are rendered inactive and "eat me" signals, including calreticulin and phosphatidylserine, congregate together and signal for removal.

Keyword
Animals, Antibodies; Monoclonal/metabolism, Antigens; CD47/metabolism, Apoptosis/genetics/*immunology, Calreticulin/*metabolism, Cell Line, Embryo, Fibroblasts/cytology/metabolism, Heterozygote, Humans, Jurkat Cells, Macrophages/immunology, Mice, Mice; Knockout, Microscopy; Video, Models; Biological, Neutrophils/cytology/metabolism, Phagocytes/*immunology, Phagocytosis/genetics/immunology, Receptors; Immunologic/genetics/*metabolism, Trans-Activation (Genetics)
Identifiers
urn:nbn:se:umu:diva-12284 (URN)10.1016/j.cell.2005.08.032 (DOI)16239148 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2010-06-24Bibliographically approved
2. Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids
Open this publication in new window or tab >>Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids
2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, no 4, 1304-1309 p.Article in journal (Refereed) Published
Abstract [en]

Macrophage phagocytosis of apoptotic cells, or unopsonized viable CD47(-/-) red blood cells, can be mediated by the interaction between calreticulin (CRT) on the target cell and LDL receptor-related protein-1 (LRP1/CD91/alpha 2-macroglobulin receptor) on the macrophage. Glucocorticoids (GC) are powerful in treatment of a range of inflammatory conditions, and were shown to enhance macrophage uptake of apoptotic cells. Here we investigated if the ability of GC to promote macrophage uptake of apoptotic cells could in part be mediated by an upregulation of macrophage LRP1 expression. Using both resident peritoneal and bone marrow-derived macrophages, we found that the GC dexamethasone could dose- and time-dependently increase macrophage LRP1 expression. The GC receptor-inhibitor RU486 could dose-dependently prevent LRP1 upregulation. Dexamethasone-treated macrophages did also show enhanced phagocytosis of apoptotic thymocytes as well as unopsonized viable CD47(-/-) red blood cells, which was sensitive to inhibition by the LRP1-agonist RAP. In conclusion, these data suggest that GC-stimulated macrophage uptake of apoptotic cells may involve an upregulation of macrophage LRP1 expression and enhanced LRP1-mediated phagocytosis. (C) 2012 Elsevier Inc. All rights reserved.

Place, publisher, year, edition, pages
San Diego: Elsevier, 2012
Keyword
Macrophages, Efferocytosis, Apoptotic cells, Inflammation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-52856 (URN)10.1016/j.bbrc.2011.12.137 (DOI)000300196100034 ()
Available from: 2012-03-06 Created: 2012-03-05 Last updated: 2017-12-07Bibliographically approved
3. CD47 promotes both phosphatidylserine-independent and phosphatidylserine-dependent phagocytosis of apoptotic murine thymocytes by non-activated macrophages
Open this publication in new window or tab >>CD47 promotes both phosphatidylserine-independent and phosphatidylserine-dependent phagocytosis of apoptotic murine thymocytes by non-activated macrophages
2009 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 387, no 1, 58-63 p.Article in journal (Refereed) Published
Abstract [en]

The ubiquitously expressed cell surface glycoprotein CD47 on host cells can inhibit phagocytosis of unopsonized or opsonized viable host target cells. Here we studied the role of target cell CD47 in macrophage uptake of viable or apoptotic murine thymocytes. As expected, IgG-opsonized viable CD47-/- thymocytes were taken up more efficiently than equally opsonized Wt thymocytes. However IgG-opsonized apoptotic thymocytes from Wt and CD47-/- mice were taken up equally. Although uptake of apoptotic thymocytes by non-activated bone marrow-derived macrophages was phosphatidylserine (PS)-independent, while uptake by non-activated resident peritoneal macrophages was PS-dependent, both macrophage populations showed a reduced uptake of non-opsonized apoptotic CD47-/- thymocytes, as compared with the uptake of apoptotic Wt thymocytes. This difference was only seen with non-activated macrophages, and not with β-1,3-glucan-activated macrophages. CD47 promoted binding of thymocytes to macrophages, which did not require F-actin polymerization. CD47 became clustered on apoptotic thymocytes, both colocalized with or separated from, clustered PS and cholesterol-rich GM-1 domains. Thus, CD47 does not inhibit, but rather support, both PS-independent and PS-dependent uptake of apoptotic cells in the murine system. This mechanism only comes into play in non-activated macrophages.

Place, publisher, year, edition, pages
Elsevier, 2009
Keyword
Macrophages, Apoptosis, T cells, Phagocytosis
National Category
Immunology
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-29853 (URN)10.1016/j.bbrc.2009.06.121 (DOI)
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2013-11-27Bibliographically approved
4. Role of CD47 in regulating macrophage and dendritic cell uptake of calcium-induced experimentally senescent erythrocytes in vitro and in vivo
Open this publication in new window or tab >>Role of CD47 in regulating macrophage and dendritic cell uptake of calcium-induced experimentally senescent erythrocytes in vitro and in vivo
(English)Manuscript (preprint) (Other academic)
Abstract [en]

During senescence, erythrocytes are rapidly eliminated by phagocytes in the spleen and liver,and the interplay between macrophages or dendritic cells (DCs) with lymphocytes in the spleen could be of importance to regulate self-tolerance and to prevent development of autoimmune hemolytic anemia (AIHA).The cell surface glycoprotein CD47 on the surface of host target cells can inhibit phagocytosis of normal circulating blood cells by binding to signal regulatory protein alpha (SIRPα) on macrophages and DCs. Here we investigated the clearance of Ca2+-ionophore-treated experimentally senescent erythrocytes (Ca2+-RBCs) in vivo, and if CD47 on Ca2+-RBCs regulated their uptake by macrophages in vitro or their clearance in vivo. Ca2+-RBCs exposed phosphatidylserine (PS) on their surface and were rapidly phagocytosed by macrophages in vitro. This uptake was dependent on heat-stable serum factors, but was not inhibited by CD47 on the erythrocytes. Following intravenous injection, a large fraction of PKH26-labeled Ca2+-RBCs were trapped by splenic marginal zone macrophages and DCs within 1 hour. In addition, at 20 hours after injection fluorescence from the injected cells could also be detected within the T cell area of the splenic white pulp and associated with both CD8+ and CD8- DCs. We found that DCs in wild-type (Wt) recipient mice showed equal uptake of transfused Wt and CD47-/- Ca2+-RBCs. DCs which had taken up Ca2+-RBCs showed a slight increase in expression levels of CD40, CD86 and MHC class II. In recipients of CD47-/- Ca2+-RBCs, DCs negative for RBC-uptake showed strongly increased expression of CD86, as compared with that in recipients of Wt Ca2+-RBCs. These findings suggest that PS+ erythrocytes may be recognized by splenic macrophages and DCs in ways similar to that reported for nucleated apoptotic cells. Uptake of senescent erythrocytes by DCs may serve as an important mechanism to maintain self-tolerance to erythrocyte antigens, and defects in this function may facilitate development of AIHA.

National Category
Immunology
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-29861 (URN)
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2010-06-24Bibliographically approved

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