The myeloid-associated Ig-like receptor family (CD300) consists of nine activating or inhibitory cell surface receptors preferentially expressed on myeloid cells and are encoded by the genes in a small cluster on mouse chromosome 11. One of the receptors, CD300LF (MAIR-V), has a long cytoplasmic tail containing two consensus ITIMs and an immunoreceptor tyrosine-based switching motif, suggesting that CD300LF regulates the activation of myeloid cells. However, the functional characteristics of this receptor are still incompletely understood. In this study, we demonstrate that cross-linking CD300LF with anti-CD300LF mAb induced cell death in peritoneal macrophages as well as in several transfectants expressing CD300LF. CD300LF-mediated cell death was dependent on the cytoplasmic region but did not require an ITIM or immunoreceptor tyrosine-based switching motif. Scanning electron microscopy revealed a loss of blebs from the surface of the dead cells mediated by CD300LF, a morphological feature similar to that observed in apoptotic cells. However, CD300LF-mediated cell death was not inhibited by a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, or autophagy inhibitors, 3-methyladenine or N-acetyl-l-cystein. Moreover, the splicing isoform of a transcription factor, X-box binding protein-1, which is produced in dead cells as a response to endoplasmic reticulum stress, was not detected. Together, these results indicate that CD300LF mediates caspase and endoplasmic reticulum stress-independent cell death by a novel mechanism.
Activating and inhibitory cell surface receptors play important roles in the regulation of immune responses (1, 2). Recent progress has demonstrated that many inhibitory receptors pair with activating, as well as inhibitory, isoforms (2, 3), both of whose genes are located in small clusters on a chromosome (4). The CMRF-35, recently assigned as CD300 (5), are a multigene family consisting of six genes on human chromosome 17 (6). The mouse counterparts of CD300 molecules, which were reported to be a myeloid-associated Ig-like receptor (MAIR)3 (7, 8, 9, 10)/CMRF-35-like molecule-1 (CLM) (11)/leukocyte mono-Ig-like receptor (LMIR) (12, 13)/dendritic cell (DC)-derived Ig receptor (DIgR) (14, 15), were encoded by nine genes on chromosome 11, syntenic region of human chromosome 17. CD300a (MAIR-I) and MAIR-II (7), also named as CLM8/LMIR1 and CLM4/LMIR2/DIgR1, respectively (14, 11, 12), constitute paired activating and inhibitory receptors. CD300a is preferentially expressed on myeloid cells, including macrophages, neutrophils, DCs, and bone marrow-derived cultured mast cells, and mediates an inhibitory signal through an ITIM in the cytoplasmic portion (7, 8). In contrast, MAIR-II, expressed on macrophages, associates with either ITAM-containing adapters DAP12 or FcRγ-chain and mediates an activating signal, resulting in inflammatory cytokine secretion from macrophages (7, 9).
We and others have recently reported that one of the genes in the mouse CD300 family, MAIR-IV, also named LMIR4 or CLM5 (13, 16), associates with the FcRγ-chain and mediates an activating signal in neutrophils (10). The Ig-like domain of MAIR-IV in the extracellular portion shares 91% amino acid identity with that of MAIR-V (CD300LF), also named LMIR3 or CLM1 (10, 13, 16). CD300LF has a long cytoplasmic tail containing two consensus ITIMs, suggesting that MAIR-IV and CD300LF constitute paired activating and inhibitory receptors. In fact, CD300LF associates with SHP-1, and expression of CD300LF in a macrophage cell line, RAW264, effectively inhibits in vitro osteoclast formation (11). Recently, Shi et al. (15) reported DIgR2 lacks 7 amino acids (PAIQVPI) in the stalk region of CD300LF, suggesting that DIgR2 is a splicing variant of CD300LF. Although DIgR2 was reported to mediate negative regulation of DC-induced Ag-specific T cell responses, whether CD300LF is also involved in the same function in DC remains undetermined.
In this study, we investigated the role of CD300LF in immune responses and describe that CD300LF-mediated signals induce caspase-independent cell death in myeloid cells.
Materials and Methods
Mice and cell lines
C57BL/6J mice were purchased from CLEA Japan. All mice used in this study were 8–10 wk of age and were maintained in our animal facility under specific pathogen-free conditions. All experiments were performed according to the guidelines of the animal ethics committee of the University of Tsukuba Animal Research Center. BW5147, RAW264, and P3U1 cells are murine T, macrophage, and myeloma cell lines, respectively. All these cell lines were purchased from the American Type Tissue Culture Collection.
Abs and reagents
Control rat and mouse IgGs, anti-mouse CD11b (Mac1), CD11c, Ly6G (Gr-1), CD3, NK1.1 mAbs, and PE-conjugated annexin V were purchased from BD Biosciences; anti-Flag mAb were purchased from Sigma-Aldrich; and rabbit anti-rat IgG were purchased from Rockland. Anti-CD300LF (TX70 and TX73) and anti-Flag (TX74) mAbs were generated in our laboratory. In brief, 1 × 107 BW5147 transfectants expressing Flag-tagged CD300LF suspended in 500 μl PBS was emulsified with the same volume of CFA. A rat was s.c. injected in each footpad with 500 μl of the suspension of BW5147 transfectants on day 1 and 8. On day 11, popliteal lymph node cells were harvested and fused with the Sp2/0 myeloma cell line by a standard method. BW5147 transfectants expressing Flag-tagged CD300LF, CD300a, MAIR-II, or MAIR-IV were used for screening of specificity to CD300LF or Flag protein by flow cytometry. Wortmannin, LY294002, cytochalasin D, DTT, 3-methyladenine (3-MA), N-Acetyl-l-cystein (NAC), staurosphorine, etoposide, LPS, Earle’s balanced salts solution and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich. N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) was purchased from Calbiochem. Zymozan and CpG DNA (ODN1826) were purchased from Molecular Probes and Invivogen, respectively.
Establishment of transfectants expressing CD300LF
The CD300LF cDNA tagged with the Flag cDNA at the NH2 terminus was subcloned into the pMX retroviral vector (17). To generate site-directed CD300LF mutants at residues Y241, Y289, and Y325, we designed PCR primers containing a codon for F (TTT) instead of Y (TAT). BW5147, P3U1, and RAW264 cells stably expressing Flag-tagged CD300LF at the N terminus were established as described previously (18).
Induction and measurement of cell death
One million transfectants expressing CD300LF or thyoglycollate-elicited peritoneal macrophages (>80% purity, as determined by flow cytometry) were incubated in 1 ml of RPMI 1640 medium for 30 min in the presence of 2.5 μg premixed rat anti-CD300LF, anti-MAIR-I, anti-MAIR-IV, or anti-Flag mAbs with 4 μg rabbit anti-rat IgG. Cells were then washed with cold PBS twice and stained with annexin-V and propidium iodide (PI), as described (19). In brief, cells were resuspended in a binding solution (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) containing PE-conjugated annexin-V, at the dilutions recommended by the manufacturer (BD Biosciences). After 15 min, cells were resuspended in the binding solution containing PI and analyzed by flow cytometry. For inhibition of caspase activation, cells (106/ml) were pretreated with 50 μM pan-caspase inhibitor z-VAD-fmk for 24 h at 37°C, and then incubated as described above. For autophagy induction by amino acid starvation, cells were washed with PBS three times and incubated with 1 ml Earle’s balanced salt solution at 37°C for 4 h. For inhibition of autophagy, cells (106/ml) were pretreated with 10 μM of NAC or 10 mM of 3-MA for 12 h at 37°C, and then incubated as described above. Intracellular autophagic vacuoles were labeled with MDC, as described previously (20). In brief, cells were incubated with 0.05 mM MDC in PBS at 37°C for 10 min (21). After incubation, cells were washed four times with PBS and collected in 10 mM Tris-HCl (pH 8) containing 0.1% Triton X-100. Intracellular MDC was measured by fluorescence photometry (excitation wavelength 380 nm, emission filter 525 nm) in a Varioskan microplate reader from ThermoFisher Scientific. To normalize the measurements to the number of cells present in each well, a solution of ethidium bromide was added to a final concentration of 0.2 μM and the DNA fluorescence was measured (excitation wavelength 530 nm, emission filter 590 nm). The MDC incorporated was expressed as specific activity (arbitrary units).
Cells were incubated in a hypotonic buffer (0.1% sodium citrate, 0.1% Triton X-100) containing 50 μg/ml PI overnight at 4°C and analyzed by flow cytometry.
XBP-1 splicing was assessed using RT-PCR with primers that spanned the splice site in the XBP-1 mRNA: forward, ACGAGAGAAAACTCATGGC; reverse, ACAGGGTCCAACTTGTCCAG. PCR products were sequenced to confirm their identities.
Statistical analyses were performed using the unpaired Student’s t test.
Generation of mAbs against CD300LF
To determine the functional characteristics of CD300LF, we generated two mAbs (TX70 and TX73) against CD300LF and a mAb against Flag protein by immunizing a rat with a mouse T cell line, BW5147, transfected with Flag-tagged CD300LF. To confirm the specificity of these mAbs to CD300LF, we also established BW5147 transfectants stably expressing CD300LF, CD300a, or MAIR-IV tagged at the N terminus with Flag. TX70 and TX73 mAbs specifically stained the transfectants expressing CD300LF, but not those expressing CD300a or MAIR-IV, indicating that these mAbs are specific to CD300LF (Fig. 1 A).
Analyses by flow cytometry using TX70 mAb showed that CD300LF was expressed on macrophages, but not on B cells, T cells, or granulocytes, derived from the spleen and peritoneal cavity (Fig. 1 B). Thyoglycollate-elicited peritoneal macrophages expressed an amount of CD300LF comparable to peritoneal macrophages from naive mice. In vitro stimulation of peritoneal macrophages with the ligands for toll-like receptors, such as LPS (1∼100 μg/ml), zymozan (1∼100 μg/ml), or CpG DNA (100 nM∼1 μM), did not affect the expression of CD300LF on peritoneal macrophages (data not shown). We confirmed similar results by using TX73 anti-CD300LF mAb (data not shown).
CD300LF mediates cell death
We found that CD300LF contains a consensus immunoreceptor tyrosine-based switching motif (ITSM) as well as two consensus ITIMs in the cytoplasmic tail. To examine whether CD300LF-mediated signals affect cell activation or proliferation, BW5147 transfectant expressing CD300LF was stimulated with anti-CD300LF (TX70) in the presence of the secondary anti-rat IgG Ab for 30 min. As demonstrated in Fig. 2,A, cross-linking CD300LF with the Ab significantly induced cell death, as analyzed by flow cytometry for binding with annexin-V and/or PI, compared with the transfectant stimulated with the IC of a control rat IgG with the secondary anti-rat IgG Ab. In contrast, cross-linking MAIR-I or MAIR-IV on BW5147 transfectants with anti-MAIR-I or anti-MAIR-IV mAbs, respectively, did not induce cell death (Fig. 2,B). Because the BW5147 cell is a T cell line and did not express the receptors for IgG (FcγRs) (data not shown), FcγRs were excluded to be involved in the effect of anti-CD300LF and the secondary Ab. Cross-linking CD300LF with anti-CD300LF mAb also induced cell death in CD300LF-transfected myeloma cell line P3U1 and macrophage cell line RAW264 (Fig. 2,C). Moreover, cell death was also induced by TX73 anti-CD300LF and anti-Flag mAbs (Fig. 2,D). Taken together, these results suggested that the cell death was specifically mediated by CD300LF. Finally, we examined whether CD300LF-mediated cell death also occurred in primary cells. As demonstrated in Fig. 2 E, cross-linking CD300LF with TX70 anti-CD300LF mAb significantly induced cell death in peritoneal macrophages, suggesting that CD300LF plays an important role in survival of peritoneal macrophages.
CD300LF-mediated cell death was independent of ITIM or ITSM
To investigate the molecular mechanism of CD300LF-mediated cell death, we first examined whether cross-linking CD300LF induces cell death at 4°C. We observed that culture of the BW5147 transfectant at 4°C abrogated cell death mediated by stimulation with anti-CD300LF mAb (Fig. 3,A), suggesting that a CD300LF-mediated signal is involved in cell death. To examine whether ITIM or ITSM in the cytoplasmic region of CD300LF is involved in the cell death, we established BW5147 transfectants stably expressing mutant CD300LF, in which the cytoplasmic region was deleted (Δcyto) or the tyrosines (Y) at residue 241, 289, and 325 were replaced with phenylalanines (F) (Y-F 241, 289, 325) (Fig. 3,B). These transfectants expressed an amount of CD300LF comparable to that of the transfectant expressing WT CD300LF (Fig. 3,C). We found that cross-linking the mutant CD300LF lacking the cytoplasmic region (Δcyto) with TX70 mAb did not induce cell death (Fig. 3,D), indicating that the cytoplasmic region of CD300LF is responsible for the cell death. However, cross-linking Y-F (241, 289, 325) mutant CD300LF still induced cell death comparably to wild type (WT) CD300LF (Fig. 3 D). These results indicated that the cytoplasmic region other than ITIMs or ITSM played an essential role in induction of cell death.
CD300LF has a PI3K binding motif (Y276xxM) in the cytoplasmic region. To investigate whether the cell death is mediated by PI3K, we pretreated the BW5147 transfectant with the PI3K inhibitors, wortmannin (1 μM) or LY294002 (1 μM), and stimulated the transfectant with TX70 anti-CD300LF mAb in the presence of the PI3K inhibitors. However, we did not observe any effect of the PI3 kinase inhibitors on the cell death (data not shown), suggesting that CD300LF-mediated cell death was also independent of PI3K.
CD300LF-mediated cell death was independent of caspases
To examine whether caspase activation is implicated in CD300LF-mediated cell death, BW5147 transfectant-expressing CD300LF was stimulated with TX70 anti-CD300LF in the presence or absence of a broad-spectrum caspase inhibitor, z-VAD-fmk. Although z-VAD-fmk effectively suppressed caspases-dependent cell death induced by etoposide (40 μM) (Fig. 4,A), CD300LF-mediated cell death was not affected by z-VAD-fmk, suggesting that it is independent of caspases. Caspase activation induces cell death with nuclear features, such as chromatin condensation and DNA fragmentation. Flow cytometric analysis by staining of nucleic acids with PI showed no significant DNA fragmentation in the BW5147 transfectant stimulated with TX70 anti-CD300LF (Fig. 4 B). Moreover, we failed to demonstrate internucleosomal DNA laddering (data not shown). Taken together, these results indicated that CD300LF-mediated cell death was independent of caspases.
CD300LF-mediated cell death was independent of endoplasmic reticulum (ER) stress
ER stress caused by misfolded proteins or cytotoxic drugs induces programmed cell death (22), which is not prevented by caspase inhibitors. Activation of unfolded protein response causes IRE1-dependent splicing of a transcription factor X-box binding protein-1 (XBP-1) mRNA (23). To test whether ER stress is involved in CD300LF-mediated cell death, we examined XBP-1 splicing in BW5147 transfectant-expressing CD300LF. Although the splicing isoform of XBP-1 (XBP-1s) was detected in the transfectant after treatment with DTT, an ER stress inducer, it was not detected in the transfectant stimulated with TX70 anti-CD300LF mAb (Fig. 5). These results indicated that CD300LF-mediated cell death was independent of ER stress.
CD300LF-mediated cell death was independent of autophagy
Autophagic programmed cell death associated with autophagosomes is another type of cell death (24, 25). To examine whether CD300LF mediates autophagic programmed cell death, BW5147 transfectant-expressing WT CD300LF was pretreated with autophagy inhibitors 3-MA or NAC (26, 27) and then stimulated with TX70 anti-CD300LF mAb. Although these inhibitors effectively suppressed the autophagic programmed cell death induced by staurosporine, they did not affect CD300LF-mediated cell death (Fig. 6,A). Moreover, while BW5147 transfectant-expressing WT CD300LF was effectively stained with MDC, a specific autophagolysosome marker (20), after autophagy induction by amino acid starvation, MDC did not stain the transfectant stimulated with TX70 anti-CD300LF mAb (Fig. 6 B). Together, these results indicate that CD300LF-mediated cell death was independent of autophagy.
Morphology of CD300LF-mediated cell death
To further characterize the CD300LF-mediated cell death, morphological analyses were performed by electron microscope (EM). As demonstrated in Fig. 7, scanning EM demonstrated that BW5147 transfectant-expressing WT CD300LF that had been stimulated with TX70 anti-CD300LF mAb showed cell shrinkage and aggregation. Notably, we observed lack of fine blebs on the cell surface of dead BW5147 transfectant expressing WT, but not mutant (Δcyto), CD300LF. The similar morphological feature was also observed in apoptotic BW5147 transfectant induced by UV, but not in necrotic BW5147 transfectant induced by heat shock at 56°C. However, transmitted EM showed no obvious morphological difference in the cytoplasm and nuclei between BW5147 transfectants expressing WT and mutant (Δcyto) CD300LF (data not shown).
This is the first report to demonstrate that cross-linking CD300LF induced cell death in primary peritoneal macrophages as well as in several transfectants expressing CD300LF. We showed that CD300LF-mediated cell death was dependent on the cytoplasmic region. However, the tyrosines in the ITIM or ITSM were not required for the cell death. CD300LF has another tyrosine at residue 276 in the cytoplasmic region, which may be involved in PI3K binding. We showed that this tyrosine may also not be responsible for the cell death, because PI3K inhibitors, wortmannin or LY294002, did not affect cell death mediated by CD300LF. At present, which domain in the cytoplasmic region of CD300LF is responsible for cell death remains undetermined.
Although the morphological feature of the dead cells mediated by CD300LF revealed by SEM was similar to that of apoptotic cells, we showed that the cell death was independent of caspases activation, a major signaling event in programmed cell death in many developmental and physiological settings (28, 29). Recent evidence has demonstrated that programmed cell death can occur in the absence of caspases (30). Several cell surface receptors have been reported to mediate caspase-independent programmed cell death in immune cells. For example, sialic acid-binding Ig-like lectin (Siglec)-9, which is expressed on neutrophils and monocytes, mediates in part caspase-independent cell death in neutrophils (26). Morphological analyses of Siglec-9-mediated cell death showed autophagosomes in the cytoplasm (26), suggesting that cross-linking Siglec-9 induced autophagic programmed cell death. In fact, an autophagy inhibitor NAC partially inhibited Siglec-9-mediated cell death (26). CD47 also mediates caspase-independent cell death in chronic lymphocytic leukemia cells (31, 32), T cells (33), monocytes, and DC (34). Although the molecular mechanisms of the caspase-independent cell death in these cell types are still undetermined, CD47-mediated cell death was suppressed by cytochalasin D, an inhibitor of actin polymerization, suggesting that cytoskeleton reorganization may be involved in CD47-mediated, caspase-independent cell death (32). In contrast, CD300LF-mediated cell death was not inhibited in the presence of NAC, 3-MA, or cytochalasin D (data not shown), indicating that the mechanism of CD300LF-mediated, caspase-independent cell death is different from that induced by Siglec-9 or CD47.
Because ER stress also induces caspase-independent cell death (22), we examined the splicing variant of XBP-1 produced by ER stress in dead cells mediated by CD300LF. However, we did not detect any splicing variant of XBP-1, indicating that CD300LF-mediated cell death was not caused by ER stress. Future studies are required to clarify the molecular mechanism of CD300LF-mediated cell death.
Among the nine molecules in the mouse CD300 (MAIR) family, CD300a (MAIR-I) and CD300LF have ITIMs in the cytoplasmic region that mediates inhibitory signals. We previously demonstrated that coligation of CD300a (MAIR-I) and FcεRI inhibited IgE-mediated degranulation from bone marrow-derived cultured mast cells and a rat basophil cell line, RBL-2H3, transfected with CD300LF (7, 8). In contrast, the inhibitory function of CD300LF in immune responses remains unclear. In this study, we demonstrated that CD300LF mediated cell death was induced in peritoneal macrophages as well as transfectants expressing CD300LF. Regulation of survival of immune cells, including macrophages, should be fundamentally important for immune regulation. Identification and characterization of the ligand for CD300LF is essential to clarify the in vivo role of CD300LF-mediated cell death as well as its inhibitory function in immune responses.
We thank Yurika Soeda for secretarial assistance. We also thank Flaminia Miyamasu for editorial assistance.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This research was supported in part by grants provided by the Ministry of Education, Science, and Culture of Japan.
Abbreviations used in this paper: MAIR, myeloid-associated Ig-like receptor; CLM, CMRF-35-like molecule-1; LMIR, leukocyte mono-Ig-like receptor; DC, dendritic cell; DIgR, DC-derived Ig receptor; 3-MA, 3-methyladenine; NAC, N-Acetyl-l-cystein; MDC, monodansylcadaverine; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; PI, propidium iodide; ITSM, immunoreceptor tyrosine-based switching motif; WT, wild type; ER, endoplasmic reticulum; XBP-1, X-box binding protein-1; EM, electron microscope; Siglec, sialic acid-binding Ig-like lectin.