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Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids
Biochemical and Biophysical Research Communications 417 (2012) 1304–1309
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids Anna Nilsson a, Liselotte Vesterlund b, Per-Arne Oldenborg a,⇑ a b
Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, Umeå, Sweden Department of Biosciences and Nutrition at Novum, Karolinska Institute, Huddinge, Sweden
a r t i c l e
i n f o
Article history: Received 22 December 2011 Available online 3 January 2012 Keywords: Macrophages Efferocytosis Apoptotic cells Inflammation
a b s t r a c t 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/a2-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. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Recognition and ingestion of apoptotic cells have been shown to involve many phagocytic receptors, bridging molecules, and ‘‘eat-me’’ molecules on apoptotic cells [11]. The multifunctional scavenger receptor LDL receptor-related protein-1 (LRP1/CD91/ a2-macroglobulin receptor), homologous to the Caenorhabditis elegans protein CED-1, can also mediate macrophage phagocytosis of apoptotic cells [1,9]. We have previously reported that LRP1mediated macrophage phagocytosis of apoptotic cells is dependent on the interaction between calreticulin (CRT) on the apoptotic cell and LRP1 on the engulfing cell [1]. It was already known that a complex of CRT and LRP1 could function as a collectinreceptor on macrophages, to bind apoptotic cells opsonized with C1q, MBL, SP-A or SP-D [9,15]. However, CRT is also expressed on the surface of both viable and apoptotic cells, shows increased levels on the surface of apoptotic cells, and its binding to LRP1 on the macrophage can mediate apoptotic cell uptake [1]. In addition, despite the expression of CRT on the surface of viable cells, viable cells are normally not phagocytosed since CD47 on their surface can interact with the phagocytosis inhibitory receptor sig⇑ Corresponding author. Address: Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, SE-901 87 Umeå, Sweden. Fax: +46 90 786 66 96. E-mail address: [email protected] (P.-A. Oldenborg). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.137
nal regulatory protein alpha (SIRPa) on the macrophage [1,10]. However, on the surface of apoptotic cells, CD47 becomes clustered and does not induce an inhibitory SIRPa signal, despite its continuous ability to bind to SIRPa [1,8]. In addition, red blood cells (RBCs) express high levels of CRT on their surface, and viable unopsonized RBCs from CD47 / mice are phagocytosed by wildtype splenic macrophages, and to a lower extent also by resident peritoneal macrophages [1,8]. We found that a substantial part of this uptake mechanism is mediated by LRP1, which binds to CRT on the surface of RBCs [1]. Glucocorticoids (GCs) are powerful in treatment of a range of inflammatory conditions, so far explained by their ability to inhibit recruitment of inflammatory cells, and by down-regulation of production and responsiveness of cells to proinflammatory cytokines [12]. However, GCs were also shown to enhance macrophage uptake of apoptotic cells [2,5]. Autoantigens may be exposed or generated by apoptotic cells, and defective clearance of such cells is likely to be associated with spontaneous and/or persistent inflammatory responses. Thus, an important part of the anti-inflammatory effects of GCs is likely to enhance phagocytosis of apoptotic cells by macrophages. The molecular mechanisms behind this involve down-regulation of p130Cas, reduced accumulation of paxillin and pyk2 in focal contacts, and higher levels of active Rac [2]. It is not known if GC also affects macrophage expression of receptors involved in uptake of apoptotic cells. However, it has
A. Nilsson et al. / Biochemical and Biophysical Research Communications 417 (2012) 1304–1309
been shown that the prototypic GC dexamethasone can upregulate LRP1 expression in the hepatocarcinoma cell line HepG2 [4], and in rat microglial cells [6]. Therefore, we here investigated if the ability of GC to enhance uptake of apoptotic cells involved upregulation of LRP1 expression also in primary murine macrophages. 2. Materials and methods 2.1. Reagents Dexamethasone, RU486, and an apoptotic detection kit (Annexin V-FITC and propidium iodide) were from Sigma–Aldrich (St. Louis, MO). Anti-mouse CD16/CD32 mAb 2.4G2 and anti-LRP1 bchain mAb 11H4 were purified from hybridoma supernatants, using ammonium sulfate precipitation and protein G (mAb 2.4G2) or protein A (mAb 11H4) chromatography (Amersham Biosciences). FITC-conjugated anti-LRP1 (mouse IgG1) was from BioMac, Leipzig, Germany. A stock solution of RU486 (10 mM) was prepared in DMSO and stored at 20 °C. Fixation buffer and permeabilization wash buffer for intracellular staining were from BioLegend (San Diego, CA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum, and penicillin/streptomycin, were from Invitrogen, Stockholm, Sweden. RAP was a generous gift from Dr. Stefan Nilsson, Dept. of Medical Biosciences, Umeå University. 2.2. Mice Male and female Balb/c mice were from our own breeding colony. Animals were kept in accordance with local guidelines and maintained in a specific pathogen-free barrier facility. All experiments were approved by the local animal ethics committee.
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was analyzed by flow cytometry following staining with AnnexinV-FITC and propidium iodide. 2.6. Flow cytometry For flow cytometric analysis of macrophage LRP1 expression, macrophages were incubated with 5 lg/ml FITC-conjugated anti-LRP1 in PBS/1% BSA in the presence of 10 lg/ml mAb 2.4G2 (Fc-block) for 30 min on ice, washed, resuspended in PBS/1% BSA and analyzed by flow cytometry (FACS calibur, BD Biosciences, Mountain View, CA, USA) and CellQuest software (BD Biosciences, Mountain View, CA, USA). For intracellular LRP1 staining, macrophages were fixated in fixation buffer for 20 min at RT and washed three times in permeabilization buffer, followed by staining with FITC-conjugated anti-LRP1 or an isotype control antibody, and flow cytometric analysis. 2.7. Western blot analysis of LRP1 Day 6–8 BMM were lysed with buffer containing 10 mM Tris (pH 7.4), 250 mM sucrose, 2 mM EDTA, 1% Triton X-100, 5 lg/ml leupeptin, 20 lg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. BMM whole cell lysates were boiled in reducing SDS– PAGE sample buffer with 5% b-mercaptoethanol and separated on 8% SDS–PAGE, followed by transfer to nitrocellulose under standard conditions. Nonspecific binding was blocked by 3% BSA in TBST (2 mM Tris, 50 mM NaCl, 0.1% Tween 20 (pH 7.6)) followed by immunoblotting with anti-LRP1 mAb 11H4. The primary antibody was detected with peroxidase-conjugated goat anti-mouse IgG, and detection of signals was by chemiluminescence (ECL, Ammersham Biosciences). 2.8. Phagocytosis assay
2.3. Isolation and culture of resident peritoneal macrophages Resident peritoneal macrophages were isolated from 8– 12 weeks old Balb/c mice as previously described [1,8], and resuspended at 2 105/ml in complete DMEM. Macrophages were cultured on bacterial plastic at 37 °C and 5% CO2 in the presence or absence of dexamethasone and/or inhibitors as described in the figure legends. For analysis of macrophage LRP1 expression, cells were harvested by treatment with cold PBS/5 mM EDTA for 10 min. For use in phagocytosis experiments (described below), resident peritoneal macrophages in complete DMEM were plated on glass coverslips and incubated over night at 37 °C and 5% CO2, followed by incubation for 24 h in the presence or absence of 1 lM dexamethasone.
Phagocytosis experiments were performed as previously described [1,8]. Evaluation of phagocytosis was done in a blinded way, using light microscopy, and expressed as a phagocytosis percent (fraction of macrophages phagocytosing at least one target cell), and as a phagocytosis index ([number of phagocytosed targets/total number of macrophages] 100). There were duplicate cover-slips in each test-group and at least 200 macrophages were counted on each cover-slip. 2.9. Statistical analysis Statistical analysis was performed by using two-tailed Student’s t-test for paired samples (see legends to the figures).
2.4. Preparation of murine bone marrow-derived macrophages
3. Results
Bone marrow-derived macrophages (BMM) were derived from femurs of 8–12 weeks old Balb/c mice as previously described [8]. To study the effects of dexamethasone on LRP1 expression during macrophage differentiation, dexamethasone was added at 1 lM at days 0 or 3. On day 6, the macrophages were harvested and analyzed for cell surface LRP1 expression. For phagocytosis experiments, BMM cultured in the presence or absence of dexamethasone were harvested by treatment with cold PBS/5 mM EDTA for 10 min, washed, resuspended in complete DMEM, and adhered to glass cover slips at 37 °C and 5% CO2 for 60 min.
3.1. Murine macrophages express LRP1
2.5. Preparation of thymocytes and induction of apoptosis Thymuses from wt Balb/c mice were isolated as previously described [8]. To induce apoptosis, cells were incubated with 1 lM dexamethasone for 3 h at 37 °C. The fraction of apoptotic cells
We first confirmed that murine macrophages express LRP1. Western blot analysis of murine bone marrow-derived macrophage whole cell lysates, using a mAb against the LRP1 b-chain, detected a protein of about 90 kDa (Fig. 1A). In addition, flow cytometric analysis of resident peritoneal macrophages or bone marrow-derived macrophages, using a mAb directed against the LRP1 extracellular domain, showed that LRP1 was expressed on the cell surface in both types of macrophages (Fig. 1B, C). 3.2. Dexamethasone time- and dose-dependently stimulates macrophage cell surface LRP1 expression GCs (e.g. dexamethasone) can significantly enhance macrophage phagocytosis of apoptotic cells [2]. Based on our findings
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Fig. 1. LRP1 is expressed by murine macrophages and its expression can be enhanced by dexamethasone. (A) LRP1 was detected in western blot of BMM whole cell lysates, using the LRP1 b-chain mAb 11H4. Flow cytometric analysis of (B) BMM, or (C) resident peritoneal macrophages, confirmed cell surface expression of LRP1 (gray histogram = anti-LRP1; dashed line = isotype control). (D)Treatment of resident peritoneal macrophages with dexamethasone induced an overall increase in LRP1 expression, but also an increase in the LRP1hi peritoneal macrophage population shown in gate M1 (untreated (gray), 1 lM dexamethasone for 24 h (bold), isotype control (dotted). Dexamethasone treatment induced a (E) time-dependent and (F) dose-dependent upregulation of the LRP1hi population, as compared with untreated controls. Data are mean ± SEM for 4 separated experiments.
on the pivotal role of LRP1 in this process, we first investigated if dexamethasone could mediate upregulation of macrophage cell surface LRP1 expression. We found that freshly isolated resident peritoneal macrophages contained a subpopulation of about 5%, which expressed higher cell surface levels of LRP1 (Fig. 1C; gray histogram; gate M1). In addition to an overall increase in LRP1 expression during culture of peritoneal macrophages with 1 lM dexamethasone for 24 h, the population of macrophages which expressed high cell surface levels of LRP1 also increased to about 20– 30% of all cells (Fig. 1D; bold line; gate M1). Further analysis showed a time-dependent increase in the fraction of this LRP1hi macrophage population in the presence of 1 lM dexamethasone (Fig. 1E). Culture of the macrophages in the presence of increasing concentrations of dexamethasone for 24 h also showed that the effect of dexamethasone was dose-dependent, where dexamethasone already at 1–10 nM increased the fraction of LRP1hi macrophages (Fig. 1F). 3.3. The effect of dexamethasone on LRP1 expression is dependent on the macrophage differentiation level It has been suggested, that for GC to optimally stimulate apoptotic cell uptake by macrophages, GC has to be added early on during macrophage differentiation [5]. Thus, the fraction of resident peritoneal macrophages responsive to dexamethasone treatment in our system might be a population of younger macrophages or even monocytes, which recently migrated to the peritoneal cavity. To more in detail study if the macrophage differentiation level could affect dexamethasone-mediated LRP1 upregulation, we cultured non-adherent bone marrow precursor cells in the presence
of 15% L cell supernatant (as a source of M-CSF) for 6 days to induce macrophage differentiation. Dexamethasone at 0–1 lM was added on day 0 and macrophage cell surface LRP1 expression was quantified on day 6, using flow cytometry. The possibility that dexamethasone would affect the number of macrophages formed in these cultures was excluded by comparing the number of cells in each test group on day 6 before analysis of LRP1 expression (data not shown). These experiments showed that dexamethasone induced a dose-dependent upregulation of cell surface LRP1, with a statistically significant effect already at 10 nM dexamethasone (Fig. 2A). By adding 1 lM dexamethasone on day 0 or day 3 of culture, we found that the increase in LRP1 expression was reduced when dexamethasone was added to the cultures on day 3, as compared with addition of dexamethasone on day 0 (Fig. 2B). 3.4. Dexamethasone-stimulated LRP1 expression is mediated by the glucocorticoid receptor To study if the effect of dexamethasone was mediated via the glucocorticoid receptor (GCR), we next studied the ability of the GCR-inhibitor RU486 to inhibit dexamethasone-stimulated LRP1 expression in resident peritoneal macrophages. As shown in Fig. 3, RU486 at 0.1–10 lM could dose-dependently prevent the upregulation of LRP1 expression induced by 1 lM dexamethasone after 24 h of culture. RU486 on its own did not affect macrophage LRP1 expression (data not shown). Thus, the effects of dexamethasone in our system seemed to be mediated through the GCR. The known effects of the GC–GCR complex to induce gene expression would therefore indicate that the dexamethasone-mediated increase in macrophage LRP1 expression could in fact involve
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Fig. 2. Dexamethasone dose-dependently induced LRP1 expression during macrophage differentiation. (A) Non-adherent bone marrow precursors were cultured in the presence of 0–1000 nM dexamethasone for 6 days during macrophage differentiation. (B) Addition of dexamethasone at start of culture (day 0), or after 3 days of culture (day 3). All cultures were done in the presence of 15% L929-cell supernatant. Macrophage cell surface LRP1 expression was determined by flow cytometry on day 6. Data are mean ± SEM for four samples in each test group. ⁄P < 0.05, as compared with that in untreated control, using Student’s t-test for paired observations.
LRP1 upregulation with increased LRP-dependent phagocytosis after dexamethasone treatment, we used dexamethasone-treated peritoneal macrophages and CD47 / RBCs in a phagocytosis assay. We found that treatment with 1 lM dexamethasone for 24 h increased the fraction of phagocytosing macrophages by 244.8 ± 13.2% (P < 0.001; data not shown), and increased the phagocytosis index by 297.8 ± 22.9% (P < 0.001; Fig. 4C). Receptor associated protein (RAP) is an LRP1-ligand able to block LRP1 binding to CRT [3], and we found that RAP significantly reduced the increased phagocytosis of CD47 / RBCs in dexamethasone-treated peritoneal macrophages (Fig. 4D). 4. Discussion Fig. 3. Dexamethasone-mediated enhancement of LRP1 expression is mediated through the glucocorticoid receptor. Resident peritoneal macrophages were incubated for 24 h in the presence or absence of 1 lM dexamethasone and the glucocorticoid receptor antagonist RU486, as indicated in the figure. Data are mean ± SEM for four separated experiments. ⁄P < 0.05, using Student’s t-test for paired observations.
increased transcription of the LRP1 gene. Therefore we investigated whether this LRP1 upregulation was due to induction of LRP1 synthesis or just recruitment of existing intracellular LRP1 to the surface. However, intracellular staining and flow cytometric analysis revealed that there was no change in the total LRP1 levels between untreated and dexamethasone-treated peritoneal macrophages at 24 h of treatment (data not shown). 3.5. Dexamethasone-treatment stimulates murine macrophage phagocytosis of apoptotic cells and viable CD47 / RBCs We next confirmed previously published data that dexamethasone treatment of macrophages should enhance their phagocytosis of apoptotic cells [7]. For this, BMM differentiated in the presence or absence of dexamethasone for 6 or 3 days were incubated with apoptotic murine thymocytes for 1 h. As expected, we found that the fraction of macrophages which had ingested at least one apoptotic target was increased by 192.6 ± 73.4% (P < 0.05; Fig. 4A) in macrophages incubated with dexamethasone for 6 days, and by 94.1 ± 51.0% when dexamethasone was added on day 3 (Fig. 4A). The phagocytosis index was affected by dexamethasone in a similar way, with a 352.1 ± 154.1% increase when dexamethasone was included for 6 days (P < 0.05; Fig. 4B). We have reported that uptake of CD47 / RBCs by resident peritoneal macrophage is mediated by the LRP1/CRT interaction [1]. To further link the observed
In the present study, we show that the glucocorticoid dexamethasone can induce a time and dose-dependent increase in cell surface LRP1 expression in murine macrophages. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU486. Moreover, dexamethasone treated macrophages showed a higher uptake of apoptotic thymocytes and viable unopsonized CD47 / RBCs, as compared with untreated macrophages. Our previous studies show that interaction of LRP1 on the phagocyte with CRT on a target cell, such as an apoptotic cell, can induce phagocytosis. These results therefore supports the hypothesis that the known enhancement of apoptotic cell elimination following dexamethasone treatment is, at least in part, due to an increased surface expression of LRP1 on the macrophage. Glucocorticoids, such as dexamethasone, have been shown to stimulate phagocytosis of apoptotic cells, and to modulate over 100 genes, but the exact mechanism behind the stimulatory effects on macrophage phagocytic capacity has remained elusive [7]. Increased phagocytosis as a result of dexamethasone treatment is most likely due to a number of different phenotypical changes in the macrophage. It was shown that dexamethasone could affect macrophage receptor expression, such as a slight increase in HLA-DR, FccRIII and CD163, and reduced expression levels of CD44, CD44v3, ICAM-1, CD36, and the b3 integrin subunit [2]. However, a changed expression pattern of these receptors could not explain an increased phagocytosis of apoptotic cells in dexamethasone treated macrophages [2]. Dexamethasone treatment could also down regulate expression of p130Cas, reduce the accumulation of paxillin and pyk2 in focal contacts, and induce higher levels of active Rac [2]. Thus, the effects of dexamethasone on apoptotic cell phagocytosis are likely multi-factorial. LRP1 is a multifunctional scavenger receptor known to recognize at least 30 different ligands [3], and LRP1 on the macrophage
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Fig. 4. Dexamethasone stimulates macrophage phagocytosis of apoptotic cells and CD47 / RBCs. (A) Increased fraction of macrophages phagocytosing apoptotic tymocytes, and (B) increased phagocytosis index, when 1 lM dexamethasone (Dex) was included during BMM differentiation. Phagocytosis data are expressed in percent of that in untreated controls. Data are mean ± SEM for three separate experiments. ⁄P < 0.05, as compared with that in untreated control, using Student’s t-test. (C) Increased phagocytosis of CD47 / RBCs by resident peritoneal macrophages treated with 1 lM dexamethasone for 24 h. (D) Dexamethasone-stimulated phagocytosis of CD47 / RBCs was sensitive to the LRP1-blocking ligand RAP. Data are mean ± SEM for four separate experiments. ⁄P < 0.05, using Student’s t-test for paired comparisons.
can induce phagocytosis by using CRT on the surface of a target cell as a ligand [1]. Herein we show that dexamethasone can induce an upregulation of macrophage LRP1 expression, which could be one explanation for an increased phagocytosis of apoptotic cells. In support of that hypothesis, we observed that addition of dexamethasone during culture of bone marrow-derived macrophages from hematopoietic bone marrow precursors resulted in a pronounced LRP1 upregulation. In vitro phagocytosis experiments with these bone marrow-derived macrophages showed an increased uptake of apoptotic thymocytes, indirectly indicating that the observed increase in apoptotic cell uptake could be due to increased levels of LRP1. Our previous studies showed that phagocytosis of unopsonized CD47 / RBCs by resident peritoneal macrophages is mediated by LRP1 [1]. Therefore, more direct evidence for a role of elevated LRP1 expression behind enhanced macrophage phagocytosis activity after dexamethasone treatment comes from our present findings of an increased uptake of unopsonized CD47 / RBCs, since this uptake was sensitive to inhibition by RAP, which blocks LRP1 binding to CRT [3]. In support of our present results, dexamethasone was also found to increase the expression of LRP1 in brain microglial cells [6]. SIRPa is a cell surface protein expressed by macrophages, known to inhibit phagocytosis upon interaction with CD47 on viable target host cells [10], and glucocorticoids were found to increase macrophage SIRPa expression [16]. Based on its role in phagocytosis inhibition, an increased SIRPa expression in macrophages would theoretically inhibit phagocytosis of apoptotic cells following glucocorticoid treatment. However, we and others have shown that CD47 on apoptotic cells does not inhibit, but rather stimulates phagocytosis, likely by facilitating tethering of apoptotic cells to the phagocyte [8,14].
Our data shows that the stimulatory effect of dexamethasone on macrophage LRP1 expression could be completely abolished by the GCR-antagonist RU486. The classical genomic mechanism of GC action involves passage through the plasma membrane and interaction with the cytosolic GCR (cGCR). This complex is then transported to the nucleus where it either induces a transactivation or transrepression of genes involved in the anti-inflammatory response [13]. There are also three suggested non-genomic mechanisms to explain some of the very rapid effects of GC. The first one is the binding of GC to the cGCR without entering the nucleus. Instead this receptor–ligand interaction induces the release of signaling molecules that are found in a complex with cGCR. The second proposed non-genomic mechanism involves non-specific interactions of GC with cellular membranes, changing their physiochemical properties and the activities of membrane-associated proteins. The third hypothesis is that of a specific interaction with a membrane-bound GCR (mGCR). This receptor is believed to be a variant of cGCR after differential splicing, promoter switching, or as a result of post-translational editing [13]. We did not observe a significant macrophage cell surface LRP1 upregulation in response to dexamethasone until about 12 h of incubation. Thus, this finding opens up the possibility of a genomic effect responsible for LRP1 upregulation in our experiments. However, although we found a slight increase in LRP1 mRNA in dexamethasone-treated macrophages (unpublished data), we could not detect an increase in the total amount of LRP1 at the protein level, as compared to untreated macrophages. This would imply that the dexamethasone effect on LRP1 expression is not due to the genomic mechanism, but rather one of the non-genomic mechanisms resulting in the transportation of pre-existing intracellular LRP1 to the plasma membrane.
A. Nilsson et al. / Biochemical and Biophysical Research Communications 417 (2012) 1304–1309
In conclusion, we found that the GC dexamethasone induces increased macrophage expression of LRP1, which could be one mechanism behind the phagocytosis-stimulatory effect reported for GC in terms of macrophage uptake of apoptotic cells. Acknowledgments Supported by grants from the Swedish Research Council (20104286), the Faculty of Medicine, Umeå University, and a Young Researcher Award from Umeå University (PAO). References [1] S.J. Gardai, K.A. McPhillips, S.C. Frasch, W.J. Janssen, A. Starefeldt, J.E. MurphyUllrich, D.L. Bratton, P.A. Oldenborg, M. Michalak, P.M. Henson, Cell-surface calreticulin initiates clearance of viable or apoptotic cells through transactivation of LRP on the phagocyte, Cell 123 (2005) 321–334. [2] K.M. Giles, K. Ross, A.G. Rossi, N.A. Hotchin, C. Haslett, I. Dransfield, Glucocorticoid augmentation of macrophage capacity for phagocytosis of apoptotic cells is associated with reduced p130Cas expression, loss of paxillin/ pyk2 phosphorylation, and high levels of active, Rac. J. Immunol. 167 (2001) 976–986. [3] J. Hertz, D.K. Strickland, LRP: a multifunctional scavenger and signaling receptor, J. Clin. Invest. 108 (2001) 779–784. [4] R.K. Kancha, M.M. Hussain, Up-regulation of the low density lipoprotein receptor-related protein by dexamethasone in HepG2 cells, Biochim. Biophys. Acta 1301 (1996) 213–220. [5] Y. liu, J.M. Cousin, J. Hughes, J. Van Damme, J.R. Seckl, C. Haslett, I. Dransfield, J. Savill, A.G. Rossi, Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes, J. Immunol. 162 (1999) 3639–3646. [6] M.P. Marzolo, B.R. von, G. Bu, N.C. Inestrosa, Expression of alpha(2)macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) in rat microglial cells, J. Neurosci. Res. 60 (2000) 401–411.
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[7] A. McColl, S. Bournazos, S. Franz, M. Perretti, B.P. Morgan, C. Haslett, I. Dransfield, Glucocorticoids induce protein S-dependent phagocytosis of apoptotic neutrophils by human macrophages, J. Immunol. 183 (2009) 2167–2175. [8] A. Nilsson, P.A. Oldenborg, CD47 promotes both phosphatidylserineindependent and phosphatidylserine-dependent phagocytosis of apoptotic murine thymocytes by non-activated macrophages, Biochem. Biophys. Res. Commun. 387 (2009) 58–63. [9] C.A. Ogden, A. deCathelineau, P.R. Hoffmann, D. Bratton, B. Ghebrehiwet, V.A. Fadok, P.M. Henson, C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells., J. Exp. Med. 194 (2001) 781–795. [10] P.-A. Oldenborg, A. Zheleznyak, Y.-F. Fang, C.F. Lagenaur, H.D. Gresham, F.P. Lindberg, Role of CD47 as a marker of self on red blood cells, Science 288 (2000) 2051–2054. [11] J. Savill, I. Dransfield, C. Gregory, C. Haslett, A blast from the past: clearance of apoptotic cells regulates immune responses, Nat. Rev. Immunol 2 (2002) 965– 975. [12] R.P. Schleimer, An overview of glucocorticoid anti-inflammatory actions, Eur. J. Clin. Pharmacol. 45 (Suppl 1) (1993) S3–S7. [13] C. Stahn, M. Lowenberg, D.W. Hommes, F. Buttgereit, Molecular mechanisms of glucocorticoid action and selective glucocorticoid receptor agonists, Mol. Cell Endocrinol. 275 (2007) 71–78. [14] K. Tada, M. Tanaka, R. Hanayama, K. Miwa, A. Shinohara, A. Iwamatsu, S. Nagata, Tethering of apoptotic cells to phagocytes through binding of CD47 to Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1, J. Immunol. 171 (2003) 5718–5726. [15] R.W. Vandivier, C.A. Ogden, V.A. Fadok, P.R. Hoffmann, K.K. Brown, M. Botto, M.J. Walport, J.H. Fisher, P.M. Henson, K.E. Greene, Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex, J. Immunol. 169 (2002) 3978–3986. [16] X. Wang, Y. Li, X. Zhu, Y. Wang, F. Diao, J. Lu, Signal regulatory protein alpha1 is involved in the inhibitory effect of glucocorticoid receptor on the proliferation of murine macrophage RAW264.7 cell and mouse peritoneal macrophage, J. Mol. Endocrinol. 41 (2008) 393–403.
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids Anna Nilsson a, Liselotte Vesterlund b, Per-Arne Oldenborg a,⇑ a b
Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, Umeå, Sweden Department of Biosciences and Nutrition at Novum, Karolinska Institute, Huddinge, Sweden
a r t i c l e
i n f o
Article history: Received 22 December 2011 Available online 3 January 2012 Keywords: Macrophages Efferocytosis Apoptotic cells Inflammation
a b s t r a c t 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/a2-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. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Recognition and ingestion of apoptotic cells have been shown to involve many phagocytic receptors, bridging molecules, and ‘‘eat-me’’ molecules on apoptotic cells [11]. The multifunctional scavenger receptor LDL receptor-related protein-1 (LRP1/CD91/ a2-macroglobulin receptor), homologous to the Caenorhabditis elegans protein CED-1, can also mediate macrophage phagocytosis of apoptotic cells [1,9]. We have previously reported that LRP1mediated macrophage phagocytosis of apoptotic cells is dependent on the interaction between calreticulin (CRT) on the apoptotic cell and LRP1 on the engulfing cell [1]. It was already known that a complex of CRT and LRP1 could function as a collectinreceptor on macrophages, to bind apoptotic cells opsonized with C1q, MBL, SP-A or SP-D [9,15]. However, CRT is also expressed on the surface of both viable and apoptotic cells, shows increased levels on the surface of apoptotic cells, and its binding to LRP1 on the macrophage can mediate apoptotic cell uptake [1]. In addition, despite the expression of CRT on the surface of viable cells, viable cells are normally not phagocytosed since CD47 on their surface can interact with the phagocytosis inhibitory receptor sig⇑ Corresponding author. Address: Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, SE-901 87 Umeå, Sweden. Fax: +46 90 786 66 96. E-mail address: [email protected] (P.-A. Oldenborg). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.137
nal regulatory protein alpha (SIRPa) on the macrophage [1,10]. However, on the surface of apoptotic cells, CD47 becomes clustered and does not induce an inhibitory SIRPa signal, despite its continuous ability to bind to SIRPa [1,8]. In addition, red blood cells (RBCs) express high levels of CRT on their surface, and viable unopsonized RBCs from CD47 / mice are phagocytosed by wildtype splenic macrophages, and to a lower extent also by resident peritoneal macrophages [1,8]. We found that a substantial part of this uptake mechanism is mediated by LRP1, which binds to CRT on the surface of RBCs [1]. Glucocorticoids (GCs) are powerful in treatment of a range of inflammatory conditions, so far explained by their ability to inhibit recruitment of inflammatory cells, and by down-regulation of production and responsiveness of cells to proinflammatory cytokines [12]. However, GCs were also shown to enhance macrophage uptake of apoptotic cells [2,5]. Autoantigens may be exposed or generated by apoptotic cells, and defective clearance of such cells is likely to be associated with spontaneous and/or persistent inflammatory responses. Thus, an important part of the anti-inflammatory effects of GCs is likely to enhance phagocytosis of apoptotic cells by macrophages. The molecular mechanisms behind this involve down-regulation of p130Cas, reduced accumulation of paxillin and pyk2 in focal contacts, and higher levels of active Rac [2]. It is not known if GC also affects macrophage expression of receptors involved in uptake of apoptotic cells. However, it has
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been shown that the prototypic GC dexamethasone can upregulate LRP1 expression in the hepatocarcinoma cell line HepG2 [4], and in rat microglial cells [6]. Therefore, we here investigated if the ability of GC to enhance uptake of apoptotic cells involved upregulation of LRP1 expression also in primary murine macrophages. 2. Materials and methods 2.1. Reagents Dexamethasone, RU486, and an apoptotic detection kit (Annexin V-FITC and propidium iodide) were from Sigma–Aldrich (St. Louis, MO). Anti-mouse CD16/CD32 mAb 2.4G2 and anti-LRP1 bchain mAb 11H4 were purified from hybridoma supernatants, using ammonium sulfate precipitation and protein G (mAb 2.4G2) or protein A (mAb 11H4) chromatography (Amersham Biosciences). FITC-conjugated anti-LRP1 (mouse IgG1) was from BioMac, Leipzig, Germany. A stock solution of RU486 (10 mM) was prepared in DMSO and stored at 20 °C. Fixation buffer and permeabilization wash buffer for intracellular staining were from BioLegend (San Diego, CA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum, and penicillin/streptomycin, were from Invitrogen, Stockholm, Sweden. RAP was a generous gift from Dr. Stefan Nilsson, Dept. of Medical Biosciences, Umeå University. 2.2. Mice Male and female Balb/c mice were from our own breeding colony. Animals were kept in accordance with local guidelines and maintained in a specific pathogen-free barrier facility. All experiments were approved by the local animal ethics committee.
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was analyzed by flow cytometry following staining with AnnexinV-FITC and propidium iodide. 2.6. Flow cytometry For flow cytometric analysis of macrophage LRP1 expression, macrophages were incubated with 5 lg/ml FITC-conjugated anti-LRP1 in PBS/1% BSA in the presence of 10 lg/ml mAb 2.4G2 (Fc-block) for 30 min on ice, washed, resuspended in PBS/1% BSA and analyzed by flow cytometry (FACS calibur, BD Biosciences, Mountain View, CA, USA) and CellQuest software (BD Biosciences, Mountain View, CA, USA). For intracellular LRP1 staining, macrophages were fixated in fixation buffer for 20 min at RT and washed three times in permeabilization buffer, followed by staining with FITC-conjugated anti-LRP1 or an isotype control antibody, and flow cytometric analysis. 2.7. Western blot analysis of LRP1 Day 6–8 BMM were lysed with buffer containing 10 mM Tris (pH 7.4), 250 mM sucrose, 2 mM EDTA, 1% Triton X-100, 5 lg/ml leupeptin, 20 lg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. BMM whole cell lysates were boiled in reducing SDS– PAGE sample buffer with 5% b-mercaptoethanol and separated on 8% SDS–PAGE, followed by transfer to nitrocellulose under standard conditions. Nonspecific binding was blocked by 3% BSA in TBST (2 mM Tris, 50 mM NaCl, 0.1% Tween 20 (pH 7.6)) followed by immunoblotting with anti-LRP1 mAb 11H4. The primary antibody was detected with peroxidase-conjugated goat anti-mouse IgG, and detection of signals was by chemiluminescence (ECL, Ammersham Biosciences). 2.8. Phagocytosis assay
2.3. Isolation and culture of resident peritoneal macrophages Resident peritoneal macrophages were isolated from 8– 12 weeks old Balb/c mice as previously described [1,8], and resuspended at 2 105/ml in complete DMEM. Macrophages were cultured on bacterial plastic at 37 °C and 5% CO2 in the presence or absence of dexamethasone and/or inhibitors as described in the figure legends. For analysis of macrophage LRP1 expression, cells were harvested by treatment with cold PBS/5 mM EDTA for 10 min. For use in phagocytosis experiments (described below), resident peritoneal macrophages in complete DMEM were plated on glass coverslips and incubated over night at 37 °C and 5% CO2, followed by incubation for 24 h in the presence or absence of 1 lM dexamethasone.
Phagocytosis experiments were performed as previously described [1,8]. Evaluation of phagocytosis was done in a blinded way, using light microscopy, and expressed as a phagocytosis percent (fraction of macrophages phagocytosing at least one target cell), and as a phagocytosis index ([number of phagocytosed targets/total number of macrophages] 100). There were duplicate cover-slips in each test-group and at least 200 macrophages were counted on each cover-slip. 2.9. Statistical analysis Statistical analysis was performed by using two-tailed Student’s t-test for paired samples (see legends to the figures).
2.4. Preparation of murine bone marrow-derived macrophages
3. Results
Bone marrow-derived macrophages (BMM) were derived from femurs of 8–12 weeks old Balb/c mice as previously described [8]. To study the effects of dexamethasone on LRP1 expression during macrophage differentiation, dexamethasone was added at 1 lM at days 0 or 3. On day 6, the macrophages were harvested and analyzed for cell surface LRP1 expression. For phagocytosis experiments, BMM cultured in the presence or absence of dexamethasone were harvested by treatment with cold PBS/5 mM EDTA for 10 min, washed, resuspended in complete DMEM, and adhered to glass cover slips at 37 °C and 5% CO2 for 60 min.
3.1. Murine macrophages express LRP1
2.5. Preparation of thymocytes and induction of apoptosis Thymuses from wt Balb/c mice were isolated as previously described [8]. To induce apoptosis, cells were incubated with 1 lM dexamethasone for 3 h at 37 °C. The fraction of apoptotic cells
We first confirmed that murine macrophages express LRP1. Western blot analysis of murine bone marrow-derived macrophage whole cell lysates, using a mAb against the LRP1 b-chain, detected a protein of about 90 kDa (Fig. 1A). In addition, flow cytometric analysis of resident peritoneal macrophages or bone marrow-derived macrophages, using a mAb directed against the LRP1 extracellular domain, showed that LRP1 was expressed on the cell surface in both types of macrophages (Fig. 1B, C). 3.2. Dexamethasone time- and dose-dependently stimulates macrophage cell surface LRP1 expression GCs (e.g. dexamethasone) can significantly enhance macrophage phagocytosis of apoptotic cells [2]. Based on our findings
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Fig. 1. LRP1 is expressed by murine macrophages and its expression can be enhanced by dexamethasone. (A) LRP1 was detected in western blot of BMM whole cell lysates, using the LRP1 b-chain mAb 11H4. Flow cytometric analysis of (B) BMM, or (C) resident peritoneal macrophages, confirmed cell surface expression of LRP1 (gray histogram = anti-LRP1; dashed line = isotype control). (D)Treatment of resident peritoneal macrophages with dexamethasone induced an overall increase in LRP1 expression, but also an increase in the LRP1hi peritoneal macrophage population shown in gate M1 (untreated (gray), 1 lM dexamethasone for 24 h (bold), isotype control (dotted). Dexamethasone treatment induced a (E) time-dependent and (F) dose-dependent upregulation of the LRP1hi population, as compared with untreated controls. Data are mean ± SEM for 4 separated experiments.
on the pivotal role of LRP1 in this process, we first investigated if dexamethasone could mediate upregulation of macrophage cell surface LRP1 expression. We found that freshly isolated resident peritoneal macrophages contained a subpopulation of about 5%, which expressed higher cell surface levels of LRP1 (Fig. 1C; gray histogram; gate M1). In addition to an overall increase in LRP1 expression during culture of peritoneal macrophages with 1 lM dexamethasone for 24 h, the population of macrophages which expressed high cell surface levels of LRP1 also increased to about 20– 30% of all cells (Fig. 1D; bold line; gate M1). Further analysis showed a time-dependent increase in the fraction of this LRP1hi macrophage population in the presence of 1 lM dexamethasone (Fig. 1E). Culture of the macrophages in the presence of increasing concentrations of dexamethasone for 24 h also showed that the effect of dexamethasone was dose-dependent, where dexamethasone already at 1–10 nM increased the fraction of LRP1hi macrophages (Fig. 1F). 3.3. The effect of dexamethasone on LRP1 expression is dependent on the macrophage differentiation level It has been suggested, that for GC to optimally stimulate apoptotic cell uptake by macrophages, GC has to be added early on during macrophage differentiation [5]. Thus, the fraction of resident peritoneal macrophages responsive to dexamethasone treatment in our system might be a population of younger macrophages or even monocytes, which recently migrated to the peritoneal cavity. To more in detail study if the macrophage differentiation level could affect dexamethasone-mediated LRP1 upregulation, we cultured non-adherent bone marrow precursor cells in the presence
of 15% L cell supernatant (as a source of M-CSF) for 6 days to induce macrophage differentiation. Dexamethasone at 0–1 lM was added on day 0 and macrophage cell surface LRP1 expression was quantified on day 6, using flow cytometry. The possibility that dexamethasone would affect the number of macrophages formed in these cultures was excluded by comparing the number of cells in each test group on day 6 before analysis of LRP1 expression (data not shown). These experiments showed that dexamethasone induced a dose-dependent upregulation of cell surface LRP1, with a statistically significant effect already at 10 nM dexamethasone (Fig. 2A). By adding 1 lM dexamethasone on day 0 or day 3 of culture, we found that the increase in LRP1 expression was reduced when dexamethasone was added to the cultures on day 3, as compared with addition of dexamethasone on day 0 (Fig. 2B). 3.4. Dexamethasone-stimulated LRP1 expression is mediated by the glucocorticoid receptor To study if the effect of dexamethasone was mediated via the glucocorticoid receptor (GCR), we next studied the ability of the GCR-inhibitor RU486 to inhibit dexamethasone-stimulated LRP1 expression in resident peritoneal macrophages. As shown in Fig. 3, RU486 at 0.1–10 lM could dose-dependently prevent the upregulation of LRP1 expression induced by 1 lM dexamethasone after 24 h of culture. RU486 on its own did not affect macrophage LRP1 expression (data not shown). Thus, the effects of dexamethasone in our system seemed to be mediated through the GCR. The known effects of the GC–GCR complex to induce gene expression would therefore indicate that the dexamethasone-mediated increase in macrophage LRP1 expression could in fact involve
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Fig. 2. Dexamethasone dose-dependently induced LRP1 expression during macrophage differentiation. (A) Non-adherent bone marrow precursors were cultured in the presence of 0–1000 nM dexamethasone for 6 days during macrophage differentiation. (B) Addition of dexamethasone at start of culture (day 0), or after 3 days of culture (day 3). All cultures were done in the presence of 15% L929-cell supernatant. Macrophage cell surface LRP1 expression was determined by flow cytometry on day 6. Data are mean ± SEM for four samples in each test group. ⁄P < 0.05, as compared with that in untreated control, using Student’s t-test for paired observations.
LRP1 upregulation with increased LRP-dependent phagocytosis after dexamethasone treatment, we used dexamethasone-treated peritoneal macrophages and CD47 / RBCs in a phagocytosis assay. We found that treatment with 1 lM dexamethasone for 24 h increased the fraction of phagocytosing macrophages by 244.8 ± 13.2% (P < 0.001; data not shown), and increased the phagocytosis index by 297.8 ± 22.9% (P < 0.001; Fig. 4C). Receptor associated protein (RAP) is an LRP1-ligand able to block LRP1 binding to CRT [3], and we found that RAP significantly reduced the increased phagocytosis of CD47 / RBCs in dexamethasone-treated peritoneal macrophages (Fig. 4D). 4. Discussion Fig. 3. Dexamethasone-mediated enhancement of LRP1 expression is mediated through the glucocorticoid receptor. Resident peritoneal macrophages were incubated for 24 h in the presence or absence of 1 lM dexamethasone and the glucocorticoid receptor antagonist RU486, as indicated in the figure. Data are mean ± SEM for four separated experiments. ⁄P < 0.05, using Student’s t-test for paired observations.
increased transcription of the LRP1 gene. Therefore we investigated whether this LRP1 upregulation was due to induction of LRP1 synthesis or just recruitment of existing intracellular LRP1 to the surface. However, intracellular staining and flow cytometric analysis revealed that there was no change in the total LRP1 levels between untreated and dexamethasone-treated peritoneal macrophages at 24 h of treatment (data not shown). 3.5. Dexamethasone-treatment stimulates murine macrophage phagocytosis of apoptotic cells and viable CD47 / RBCs We next confirmed previously published data that dexamethasone treatment of macrophages should enhance their phagocytosis of apoptotic cells [7]. For this, BMM differentiated in the presence or absence of dexamethasone for 6 or 3 days were incubated with apoptotic murine thymocytes for 1 h. As expected, we found that the fraction of macrophages which had ingested at least one apoptotic target was increased by 192.6 ± 73.4% (P < 0.05; Fig. 4A) in macrophages incubated with dexamethasone for 6 days, and by 94.1 ± 51.0% when dexamethasone was added on day 3 (Fig. 4A). The phagocytosis index was affected by dexamethasone in a similar way, with a 352.1 ± 154.1% increase when dexamethasone was included for 6 days (P < 0.05; Fig. 4B). We have reported that uptake of CD47 / RBCs by resident peritoneal macrophage is mediated by the LRP1/CRT interaction [1]. To further link the observed
In the present study, we show that the glucocorticoid dexamethasone can induce a time and dose-dependent increase in cell surface LRP1 expression in murine macrophages. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU486. Moreover, dexamethasone treated macrophages showed a higher uptake of apoptotic thymocytes and viable unopsonized CD47 / RBCs, as compared with untreated macrophages. Our previous studies show that interaction of LRP1 on the phagocyte with CRT on a target cell, such as an apoptotic cell, can induce phagocytosis. These results therefore supports the hypothesis that the known enhancement of apoptotic cell elimination following dexamethasone treatment is, at least in part, due to an increased surface expression of LRP1 on the macrophage. Glucocorticoids, such as dexamethasone, have been shown to stimulate phagocytosis of apoptotic cells, and to modulate over 100 genes, but the exact mechanism behind the stimulatory effects on macrophage phagocytic capacity has remained elusive [7]. Increased phagocytosis as a result of dexamethasone treatment is most likely due to a number of different phenotypical changes in the macrophage. It was shown that dexamethasone could affect macrophage receptor expression, such as a slight increase in HLA-DR, FccRIII and CD163, and reduced expression levels of CD44, CD44v3, ICAM-1, CD36, and the b3 integrin subunit [2]. However, a changed expression pattern of these receptors could not explain an increased phagocytosis of apoptotic cells in dexamethasone treated macrophages [2]. Dexamethasone treatment could also down regulate expression of p130Cas, reduce the accumulation of paxillin and pyk2 in focal contacts, and induce higher levels of active Rac [2]. Thus, the effects of dexamethasone on apoptotic cell phagocytosis are likely multi-factorial. LRP1 is a multifunctional scavenger receptor known to recognize at least 30 different ligands [3], and LRP1 on the macrophage
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Fig. 4. Dexamethasone stimulates macrophage phagocytosis of apoptotic cells and CD47 / RBCs. (A) Increased fraction of macrophages phagocytosing apoptotic tymocytes, and (B) increased phagocytosis index, when 1 lM dexamethasone (Dex) was included during BMM differentiation. Phagocytosis data are expressed in percent of that in untreated controls. Data are mean ± SEM for three separate experiments. ⁄P < 0.05, as compared with that in untreated control, using Student’s t-test. (C) Increased phagocytosis of CD47 / RBCs by resident peritoneal macrophages treated with 1 lM dexamethasone for 24 h. (D) Dexamethasone-stimulated phagocytosis of CD47 / RBCs was sensitive to the LRP1-blocking ligand RAP. Data are mean ± SEM for four separate experiments. ⁄P < 0.05, using Student’s t-test for paired comparisons.
can induce phagocytosis by using CRT on the surface of a target cell as a ligand [1]. Herein we show that dexamethasone can induce an upregulation of macrophage LRP1 expression, which could be one explanation for an increased phagocytosis of apoptotic cells. In support of that hypothesis, we observed that addition of dexamethasone during culture of bone marrow-derived macrophages from hematopoietic bone marrow precursors resulted in a pronounced LRP1 upregulation. In vitro phagocytosis experiments with these bone marrow-derived macrophages showed an increased uptake of apoptotic thymocytes, indirectly indicating that the observed increase in apoptotic cell uptake could be due to increased levels of LRP1. Our previous studies showed that phagocytosis of unopsonized CD47 / RBCs by resident peritoneal macrophages is mediated by LRP1 [1]. Therefore, more direct evidence for a role of elevated LRP1 expression behind enhanced macrophage phagocytosis activity after dexamethasone treatment comes from our present findings of an increased uptake of unopsonized CD47 / RBCs, since this uptake was sensitive to inhibition by RAP, which blocks LRP1 binding to CRT [3]. In support of our present results, dexamethasone was also found to increase the expression of LRP1 in brain microglial cells [6]. SIRPa is a cell surface protein expressed by macrophages, known to inhibit phagocytosis upon interaction with CD47 on viable target host cells [10], and glucocorticoids were found to increase macrophage SIRPa expression [16]. Based on its role in phagocytosis inhibition, an increased SIRPa expression in macrophages would theoretically inhibit phagocytosis of apoptotic cells following glucocorticoid treatment. However, we and others have shown that CD47 on apoptotic cells does not inhibit, but rather stimulates phagocytosis, likely by facilitating tethering of apoptotic cells to the phagocyte [8,14].
Our data shows that the stimulatory effect of dexamethasone on macrophage LRP1 expression could be completely abolished by the GCR-antagonist RU486. The classical genomic mechanism of GC action involves passage through the plasma membrane and interaction with the cytosolic GCR (cGCR). This complex is then transported to the nucleus where it either induces a transactivation or transrepression of genes involved in the anti-inflammatory response [13]. There are also three suggested non-genomic mechanisms to explain some of the very rapid effects of GC. The first one is the binding of GC to the cGCR without entering the nucleus. Instead this receptor–ligand interaction induces the release of signaling molecules that are found in a complex with cGCR. The second proposed non-genomic mechanism involves non-specific interactions of GC with cellular membranes, changing their physiochemical properties and the activities of membrane-associated proteins. The third hypothesis is that of a specific interaction with a membrane-bound GCR (mGCR). This receptor is believed to be a variant of cGCR after differential splicing, promoter switching, or as a result of post-translational editing [13]. We did not observe a significant macrophage cell surface LRP1 upregulation in response to dexamethasone until about 12 h of incubation. Thus, this finding opens up the possibility of a genomic effect responsible for LRP1 upregulation in our experiments. However, although we found a slight increase in LRP1 mRNA in dexamethasone-treated macrophages (unpublished data), we could not detect an increase in the total amount of LRP1 at the protein level, as compared to untreated macrophages. This would imply that the dexamethasone effect on LRP1 expression is not due to the genomic mechanism, but rather one of the non-genomic mechanisms resulting in the transportation of pre-existing intracellular LRP1 to the plasma membrane.
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In conclusion, we found that the GC dexamethasone induces increased macrophage expression of LRP1, which could be one mechanism behind the phagocytosis-stimulatory effect reported for GC in terms of macrophage uptake of apoptotic cells. Acknowledgments Supported by grants from the Swedish Research Council (20104286), the Faculty of Medicine, Umeå University, and a Young Researcher Award from Umeå University (PAO). References [1] S.J. Gardai, K.A. McPhillips, S.C. Frasch, W.J. Janssen, A. Starefeldt, J.E. MurphyUllrich, D.L. Bratton, P.A. Oldenborg, M. Michalak, P.M. Henson, Cell-surface calreticulin initiates clearance of viable or apoptotic cells through transactivation of LRP on the phagocyte, Cell 123 (2005) 321–334. [2] K.M. Giles, K. Ross, A.G. Rossi, N.A. Hotchin, C. Haslett, I. Dransfield, Glucocorticoid augmentation of macrophage capacity for phagocytosis of apoptotic cells is associated with reduced p130Cas expression, loss of paxillin/ pyk2 phosphorylation, and high levels of active, Rac. J. Immunol. 167 (2001) 976–986. [3] J. Hertz, D.K. Strickland, LRP: a multifunctional scavenger and signaling receptor, J. Clin. Invest. 108 (2001) 779–784. [4] R.K. Kancha, M.M. Hussain, Up-regulation of the low density lipoprotein receptor-related protein by dexamethasone in HepG2 cells, Biochim. Biophys. Acta 1301 (1996) 213–220. [5] Y. liu, J.M. Cousin, J. Hughes, J. Van Damme, J.R. Seckl, C. Haslett, I. Dransfield, J. Savill, A.G. Rossi, Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes, J. Immunol. 162 (1999) 3639–3646. [6] M.P. Marzolo, B.R. von, G. Bu, N.C. Inestrosa, Expression of alpha(2)macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) in rat microglial cells, J. Neurosci. Res. 60 (2000) 401–411.
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