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. 2013 Oct 15;8(10):e77615.
doi: 10.1371/journal.pone.0077615. eCollection 2013.

'Clustering' SIRPα into the plasma membrane lipid microdomains is required for activated monocytes and macrophages to mediate effective cell surface interactions with CD47

Binh Ha  1 , Zhiyuan Lv, Zhen Bian, Xiugen Zhang, Aarti Mishra, Yuan Liu
Affiliations

'Clustering' SIRPα into the plasma membrane lipid microdomains is required for activated monocytes and macrophages to mediate effective cell surface interactions with CD47

Binh Ha et al. PLoS One. .

Abstract

SIRPα, an ITIMs-containing signaling receptor, negatively regulates leukocyte responses through extracellular interactions with CD47. However, the dynamics of SIRPα-CD47 interactions on the cell surface and the governing mechanisms remain unclear. Here we report that while the purified SIRPα binds to CD47 and that SIRPα is expressed on monocytes and monocytic THP-1 or U937, these SIRPα are ineffective to mediate cell binding to immobilized CD47. However, cell binding to CD47 is significantly enhanced when monocytes transmigrating across endothelia, or being differentiated into macrophages. Cell surface labeling reveals SIRPα to be diffused on naïve monocytes but highly clustered on transmigrated monocytes and macrophages. Protein crosslink and equilibrium centrifugation confirm that SIRPα in the latter cells forms oligomerized complexes resulting in increased avidity for CD47 binding. Furthermore, formation of SIRPα complexes/clusters requires the plasma membrane 'lipid rafts' and the activity of Src family kinase during macrophage differentiation. These results together suggest that 'clustering' SIRPα into plasma membrane microdomains is essential for activated monocytes and macrophages to effectively interact with CD47 and initiate intracellular signaling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of SIRPα on monocytes.
Detection of SIRPα by WB in freshly isolated human PBMC and PMN using anti-SIRPα.ex (SIRPα.ex) and anti-SIRPα.ct (SIRPα.ct) antibodies. B) Immunofluorescence staining of PBMC. Cell surface SIRPα was labeled with anti-SIRPα.ex under non-permeable conditions. Monocytes (arrow heads) were defined by anti-human CD14 antibody, and T and B lymphocytes were defined with anti-Lck and anti-B220 antibodies, respectively. C) Determination of the distribution of SIRPα in PMN and monocytes by cell biotinylation. Cells were biotinylated with either NHS-biotin (cell permeable) or sulfo-NHS-biotin (non-cell permeable), followed by the IP of SIRPα from the lysates. Biotinylated and total SIRPα were blotted by streptavidin-HRP and anti-SIRPα.ct, respectively. The same amount of protein was used for IP as confirmed by the actin blots. D) FACS analysis to determine SIRPα in monocytes labeled under cell permeable and non-permeable conditions.
Figure 2
Figure 2. SIRPα in naïve monocytes do not mediate cell adhesion to immobilized CD47.
A) Schematic depiction of the cell adhesion assay. B) Adhesion of different cells to immobilized CD47-AP. Cell suspensions were incubated with CD47-AP or BSA (ctl.) for 30 min (25°C), followed by washing. C) Percentage of cell adhesion to CD47-AP versus BSA. D) Control adhesion performed using CHO cells with or without SIRPα transfection (CHO-mock and CHO-SIRPα, respectively), and in the presence or absence of inhibitory anti-SIRPα (SE5A5) and anti-CD47 (B6H12.2) mAbs (20 μg/ml for each). The data are expressed as the means ± SE and represent three independent experiments with triplicate wells per cell type. ***, P <0.001. E) FACS analyses of SIRPα on the cell surfaces of peripheral monocytes and CHO-SIRPα cells.
Figure 3
Figure 3. SIRPα in activated monocytes and macrophages mediate high avidity cell adhesion to immobilized CD47.
To obtain activated monocytes, freshly isolated PMBC were treated with 50 ng/ml MCP-1 (37°C, 1 h) and were also induced to transmigrate across HMEC-1 endothelial monolayers by MCP-1. To obtain macrophages, THP-1 cells were treated with PMA (50 nM) for 3-4 days. A) Cell adhesion assays. SE5A5 and SE7C2 are both inhibitory anti-SIRPα mAbs. B) Percentage of cell adhesion to CD47-AP versus BSA. ***, P <0.001. C) FACS analysis of SIRPα on the surface of naïve (PBMC) and activated monocytes, unstimulated and stimulated THP-1 using anti-SIRPα.ex antibody.
Figure 4
Figure 4. Purified SIRPα binding to CD47.
SIRPα, affinity purified from naïve and activated monocytes and unstimulated and stimulated THP-1, was tested for direct binding to CD47-AP. A) Affinity purification of SIRPα. SIRPα in different cell lysates was absorbed by anti-SIRPα.ex-conjugated Sepharose and eluted at pH 4.0 in the presence of 1% OG. B) Binding of purified SIRPα from various sources to CD47-AP. Purified SIRPα was immobilized in 96-well plates following a 1:10 dilution of OG. Binding to CD47 was tested by incubation with CD47-AP (2 μg/ml, 30 min), followed by washing and detection of AP activity. The figure inset shows similar amounts of immobilized SIRPα in the wells as detected by WB (anti-SIRPα.ct). C) Binding of SIRPα purified from PBMC to CD47-AP in the presence of inhibitory anti-SIRPα (SE5A5) and anti-CD47 (B6H12.2) mAbs, or SIRPα.ex Fc. Purified SIRPα samples were absorbed onto 96-well plates (lower panel) or nitrocellulose membrane (upper panel). Following blocking with BSA and incubation with CD47-AP, the binding was assessed by AP activity.
Figure 5
Figure 5. SIRPα on activated monocytes and macrophages forms clusters/complexes.
A) Assessing SIRPα on naïve PBMC, transmigrated monocytes, PMA-induced THP-1 and murine macrophages by immunofluorescence staining using anti-SIRPα.ex and mAb P84, an antibody specific for the murine SIRPα extracellular domain. B) Staining of SIRPα and CD47 on PMA-stimulated THP-1 using anti-SIRPα.ex and mAb B6H12.2. C) DTSSP crosslinking to assess protein complexes consisting SIRPα. Different cells were treated with DTSSP (25°C, 30 min) prior to lysis in detergent-containing buffer. Lysate samples were analyzed by SDS-PAGE and WB under non-reducing or reducing (+DTT) conditions. The latter reverses the DTSSP crosslinking. Note: murine SIRPα appears to be 110-120 kD, larger than the human counterpart. Dot blot detection of SIRPα was performed by directly absorbing cell lysates onto nitrocellulose, prior to antibody blotting. D) Equilibrium centrifugation analysis of SIRPα complexes. After DTSSP crosslinking, cell lysates were applied to ultracentrifugation in sucrose density gradients. Following fractionation, SIRPα localization was analyzed by WB and compared to the sedimentation of a reference protein marker.
Figure 6
Figure 6. Clustering of SIRPα on cell surfaces requires cholesterol-rich lipid rafts.
PMA-stimulated THP-1 cells were treated with lipid raft/cholesterol perturbation/modification agents, including MβCD (10 mg/ml), triparanol (5 µM), cholesterol oxidase (CO) (1 unit/ml) and filipin (0.5 µg/ml), prior to A) assessment of SIRPα on the cell surface by immunofluorescence staining, B) analysis of SIRPα localization in lipid rafts by co-staining for raft marker GM1 by cholera toxin B subunit (CT-B), C) assay of SIRPα-mediated cell adhesion to immobilized CD47-AP. **, P <0.01. ***, P <0.001. The effect of the actin filament disruption agent cytochalasin D on lipid rafts and SIRPα distribution was also tested (shown in C).
Figure 7
Figure 7. Role of the Src family tyrosine kinase in SIRPα clustering.
THP-1 cells were induced to differentiate into macrophages by treating with PMA for 5 days. From day 3, various pharmacological inhibitors were added into the cell culture, including the Src tyrosine kinase inhibitors genistein (100 µg/ml) and PP1 (35 µM), the genistein analog daidzein (non-inhibitory, 100 µg/ml), a mixture of MAP kinase inhibitors (MAP combo) SB203580 and PD98059 (20 µM each), the PI3 kinase inhibitor LY294002 (25 µM), the Btk inhibitor LFM-A13 (100 µM), the JAK inhibitor (JAK inhibitor I, 100 nM). A) Immunofluorescence staining of SIRPα on the THP-1 surface by anti-SIRPα.ex. B) Assay of SIRPα-mediated cell adhesion to immobilized CD47-AP. ***, P <0.001. C) A model of SIRPα distribution and complex formation on monocytes and macrophages.
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