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Quantification of Efferocytosis by Single-cell Fluorescence Microscopy
Published on: August 18, 2018
Mouse macrophages show different requirements for phosphatidylserine receptor Tim4 in efferocytosis
Yuichi Yanagihashi1, Katsumori Segawa1, Ryota Maeda2
1Laboratory of Biochemistry and Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.
Abstract:
Protein S (ProS) and growth arrest-specific 6 (Gas6) bind to phosphatidylserine (PtdSer) and induce efferocytosis upon binding TAM-family receptors (Tyro3, Axl, and Mer). Here, we produced mouse ProS, Gas6, and TAM-receptor extracellular region fused to IgG fragment crystallizable region in HEK293T cells. ProS and Gas6 bound Ca2+ dependently to PtdSer (Kd 20-40 nM), Mer, and Tyro3 (Kd 15-50 nM). Gas6 bound Axl strongly (Kd < 1.0 nM), but ProS did not bind Axl. Using NIH 3T3-based cell lines expressing a single TAM receptor, we showed that TAM-mediated efferocytosis was determined by the receptor-binding ability of ProS and Gas6. Tim4 is a membrane protein that strongly binds PtdSer. Tim4 alone did not support efferocytosis, but enhanced TAM-dependent efferocytosis. Resident peritoneal macrophages, Kupffer cells, and CD169+ skin macrophages required Tim4 for TAM-stimulated efferocytosis, whereas efferocytosis by thioglycollate-elicited peritoneal macrophages or primary cultured microglia was TAM dependent, but not Tim4 dependent. These results indicate that TAM and Tim4 collaborate for efficient efferocytosis in certain macrophage populations.
Insights
Protein S (ProS) and growth arrest-specific 6 (Gas6) facilitate efferocytosis by binding phosphatidylserine (PtdSer) and TAM receptors. Tim4 enhances this process in specific macrophages, revealing a collaborative mechanism for efficient efferocytosis.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Protein S (ProS) and growth arrest-specific 6 (Gas6) are ligands that bind phosphatidylserine (PtdSer).
- These ligands engage TAM-family receptors (Tyro3, Axl, Mer) to promote efferocytosis, the process of clearing apoptotic cells.
- Understanding the specific interactions and cellular requirements for TAM-mediated efferocytosis is crucial for immune regulation.
Purpose of the Study:
- To characterize the binding properties of mouse ProS and Gas6 to PtdSer and TAM receptors.
- To investigate the role of TAM receptors and Tim4 in mediating efferocytosis across different macrophage populations.
- To elucidate the collaborative mechanisms between TAM receptors and Tim4 in efferocytosis.
Main Methods:
- Production of recombinant mouse ProS, Gas6, and TAM receptor extracellular domains fused to an IgG Fc region using HEK293T cells.
- Binding assays to determine the affinity of ProS and Gas6 for PtdSer, Mer, Tyro3, and Axl.
- Functional assays using NIH 3T3 cell lines expressing single TAM receptors to assess efferocytosis.
- Analysis of efferocytosis in various primary macrophage populations (resident peritoneal, Kupffer, CD169+ skin, thioglycollate-elicited peritoneal, and microglia) in the presence or absence of Tim4.
Main Results:
- ProS and Gas6 bound PtdSer, Mer, and Tyro3 in a Ca2+-dependent manner with nanomolar affinity.
- Gas6 exhibited strong binding to Axl, while ProS did not bind Axl.
- TAM receptor-mediated efferocytosis efficiency correlated with the receptor-binding capabilities of ProS and Gas6.
- Tim4 enhanced TAM-dependent efferocytosis, with its requirement varying across different macrophage types; resident peritoneal macrophages, Kupffer cells, and CD169+ skin macrophages required Tim4, whereas thioglycollate-elicited peritoneal macrophages and microglia did not.
Conclusions:
- ProS and Gas6 display distinct binding profiles to TAM receptors, influencing their efferocytosis-inducing capacity.
- Tim4 acts as a crucial co-factor for TAM-mediated efferocytosis in specific macrophage populations, particularly those residing in tissues.
- These findings highlight a collaborative pathway involving TAM receptors and Tim4 for efficient efferocytosis, with cell-type specific regulation, impacting immune homeostasis and clearance processes.
