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LMIR5 extracellular domain activates myeloid cells through Toll-like receptor 4
Molecular Immunology 62 (2014) 169–177
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Short communication
LMIR5 extracellular domain activates myeloid cells through Toll-like receptor 4 Vongsavanh Phongsisay a,∗ , Ei’ichi Iizasa b , Hiromitsu Hara b , Sho Yamasaki a a b
Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan
a r t i c l e
i n f o
Article history: Received 5 March 2014 Received in revised form 16 May 2014 Accepted 9 June 2014 Keywords: LMIR5 CD300b TLR4 Inflammation
a b s t r a c t LMIR5/CD300b is an activating immunoglobulin-like receptor whose extracellular domain (LMIR5-Fc) is constitutively released from immune cells. The release of LMIR5-Fc is augmented upon stimulation with TLR agonists. LMIR5-Fc is reported to possess inflammatory activity and amplify LPS-induced lethal inflammation; however, its action mechanism has not been clarified. This study was aimed to identify receptors for LMIR5-Fc. Using NF-B reporter cells in human monocytes THP1, LMIR5-Fc was solely found to trigger NF-B activation among various signaling receptors examined. In addition, an injection of LMIR5-Fc into the mouse peritoneal resulted in a rapid production of inflammatory mediators and an amplification of LPS activity. Moreover, LMIR5-Fc-induced cytokine production was markedly reduced in TLR4-deficient mouse macrophages. Using TLR4 reporter cells, the LMIR5-Fc sample that contained a trace amount of endotoxin under the sensitivity of reporter cells triggered a potent NF-B activation. Furthermore, the inflammatory activity of LMIR5-Fc was completely lost by heating but unchanged by polymyxin B pretreatment. Using TLR4 fusion protein, TLR4 was found to interact specifically with LMIR5overexpressing cells. Therefore, LMIR5-Fc is new inflammatory mediator and endogenous ligand of TLR4. This study provides an insight into the positive feedback mechanism of inflammation through TLR4LMIR5-Fc axis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction LMIR5/CD300b is classified in the CD300 family of myeloid immunoglobulin receptors that are type I transmembrane proteins exhibiting a single IgV-like extracellular domain (Gasiorowski et al., 2013). Unusually, all CD300 members interact with each other, even with themselves, forming both homo- and heterodimers that differentially modulates signaling outcomes (Martinez-Barriocanal et al., 2010). LMIR5 is present on the cell membrane of neutrophils, peritoneal macrophages, and mast cells (Yamanishi et al., 2008, 2012). Upon stimulation with inflammatory stimuli, LMIR5 is dissociated from the membrane by a proteolytic cleavage of the membranous LMIR5 partly through matrix metalloproteinases (Yamanishi et al., 2012). LMIR5 mediates at least two different signaling pathways. Firstly, the membranous LMIR5 is mainly
∗ Corresponding author. Permanent address: Homsavanh School, Ban Hom, Hatsaifong, Vientiane, Laos.Tel.: +81 926424614/+856 20 55777636; fax: +81 926424614. E-mail addresses: [email protected], [email protected] (V. Phongsisay). http://dx.doi.org/10.1016/j.molimm.2014.06.012 0161-5890/© 2014 Elsevier Ltd. All rights reserved.
associated with the adaptor protein DAP12 under physiological condition (Yamanishi et al., 2008). DAP12 is well-known for its role in the delivery of activating signal by phosphorylation of ITAM motif at its cytoplasmic domain (Turnbull and Colonna, 2007). In LMIR5-transduced mast cells, cross-linking of anti-LMIR5 antibody induces phosphorylation of several signaling molecules (ERK, p38, Akt), resulting in cytokine/chemokine production, increased cell adhesion, histamine release, and increased cell survival. This phenotype is DAP12 and Syk-dependent (Yamanishi et al., 2008). Secondly, the fusion protein consisting of the LMIR5 extracellular domain and the human IgG Fc, LMIR5-Fc, induces cytokine production in mouse peritoneal cells (Yamanishi et al., 2012). To date, its action mechanism has not been clarified. Toll-like receptors are the pattern-recognition receptor that mediates the production of effective immunity against infectious agents. In humans and mice, there are at least 13 TLRs that differentially recognize microbial products (Akira and Takeda, 2004). For example, TLR2 recognizes zymosan and lipoteichoic acid. TLR4 recognizes LPS from Gram-negative bacteria and it co-operates with co-receptors MD-2 and CD14. Upon ligand stimulation, several signaling molecules are activated leading to NF-B activation, cytokine production, and cellular and humoral immune responses.
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In this study, we initially examined the immunological property of various receptor extracellular domains, including C-type lectin and immunoglobulin-like receptors. Anti-inflammatory activity was not observed for all the receptors exampled; indeed, LMIR5Fc was the only one capable of inducing inflammatory responses. Further study showed that LMIR5 extracellular domain is an endogenous activator of TLR4. 2. Materials and methods 2.1. Mice All mice are C57BL/6 background. Wild type mice were purchased from CLEA Japan. TLR2−/− and TLR4−/− mice were from Oriental Bioservice. FcR␥−/− and CARD9−/− mice were established as previously described (Park et al., 1998; Hara et al., 2007). MyD88−/− mice were purchased from Oriental Yeast. Mice were maintained in a filtrated-air laminar-flow enclosure and given standard laboratory food and water ad libitum. Animal protocols were approved by the committee of Ethics on Animal Experiment, Faculty of Medical Sciences, Kyushu University. 2.2. Reagents Polymyxin B-agarose (P1411), LPS (L4516), and zymosan (Z4250) were from Sigma. Pam3CSK4 (IMG-2201) was from IMGENEX. Antibody to LMIR5 (AF2580) was purchased from R&D. TSM buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM CaCl2 , 2 mM MgCl2 , pH 7.0) was prepared as previously described and used for all fusion protein experiments (van Sorge et al., 2009). Protein G SepharoseTM and PD MiniTrap G-25 columns containing SephadexTM G-25 medium were purchased from GE Healthcare. Amicon Ultracel-10K was from Millipore. ToxinSensorTM chromogenic LAL endotoxin assay kit (L00350) was purchased from Genscript. 2.3. Fusion proteins Fusion protein is consisted of the receptor extracellular domain and the human IgG Fc. For LMIR5-Fc fusion, a DNA fragment encoding for amino acids 65-177 of mouse LMIR5 (accession no. BC160263) was inserted via SfiI and SalI into pDisplayTM vector (Invitrogen). The human IgG1 Fc region was cloned into the vector via AccI. LMIR5-expressing vector was transfected into HEK 293 cells. FreeStyleTM 293 expression system (K9000-01, Invitrogen) was used to allow large-scale transfection of suspension 293 cells in defined, serum-free medium according to the manufacturer’s instruction. Production of other receptor-Fc fusion proteins was performed using the same method as mentioned. TLR4-Fc fusion protein was produced as previously described (Roger et al., 2009). All fusion proteins were diluted in TSM buffer. 2.4. Cells Cells were cultured in RPMI supplemented with 10% FCS, otherwise stated. Bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs) were induced for 7–10 days by M-CSF or GM-CSF, respectively. LMIR5-expressing cells were established by expressing the surface LMIR5-HA fusion in association with adaptor protein DAP12 in the NFAT-GFP-2B4 cells, as previously described (Yamasaki et al., 2008). LMIR4 reporter cells were established by expressing the surface LMIR4-HA fusion in association with FcR␥ in the NFAT-GFP-2B4 cells. For LMIR5 or LMIR4 reporter assay, 5 × 104 cells were incubated overnight with stimuli, which had been coated on the ELISA plate or cross-linked by using appropriate antibodies. A number of GFP-positive cells were
analysed by flow cytometry. NF-B reporter cells (TBRed) were established by expressing the fluorescent marker DsRed under the control of NF-B in the human monocytes THP1. For TBRed assay, cells were incubated overnight with 240 ng fusion protein or other stimuli. NF-B -positive cells were analyzed by flow cytometry. NKT cells RT-5 was obtained from Dr. Shimamura Michio. B3Z is CD8+ T cells. Microglia BV2 was provided by Dr. Makoto Tsuda and cultured in DMEM supplemented with 5% FCS. HEK-Blue mTLR4 (HB-4), HEK-Blue Null1-v (HB-N), and RAW-Blue were purchased from InvivoGen and cultured in DMEM supplemented with 10% FCS. TLR4 reporter cells were obtained by co-transfection of mTLR4 gene, MD-2/CD14 co-receptor genes, and secreted embryonic alkaline phosphatase (SEAP) reporter gene. The SEAP reporter gene was placed under the control of the IFN- minimal promoter fused to five NF-B and AP-1-binding sites. Activation of NF-B and AP-1 leads to the production of SEAP, which was measured at A630 using a detection medium QUANTI-Blue according to the manufacturer’s instruction (InvivoGen). 2.5. Cytokine assay BMDMs or BMDCs were incubated with stimuli for 24 h. Cytokines were measured using ELISA kits from BD biosciences. 2.6. Cell surface staining Cells were incubated with fusion protein in TSM-RPMI medium (50% TSM in 2% FCS/RPMI) followed by anti-human IgG Fc-PE in 2% FCS/RPMI. Cell staining was carried out on ice for 1 h, washed twice in 2% FCS/RPMI after each incubation, and analyzed by flow cytometry. 2.7. Assay of ligand-receptor interaction Nunc MaxiSorp 96-well plates were used throughout the experiments. Protein samples and LPS were coated in coating buffer (0.293% w/v of NaHCO3 and 0.053% w/v of Na2 CO3 , pH 9.6, in water) at 42 ◦ C overnight. The wells were blocked with 10% fresh FCS in TSM for 1 h and incubated with fusion protein with or without biotin conjugate in 10% FCS/TSM for 2 h. After washing with TSM buffer, anti-human IgG Fc-HRP conjugate or avidin-HRP was used for detection. 2.8. Statistics The statistical significance of differences was assessed using a two-tailed Mann–Whitney test. 3. Results and discussion 3.1. Inflammatory property of LMIR5-Fc To examine if the receptor extracellular domains possess inflammatory or anti-inflammatory property, NF-B reporter cells TBRed were stimulated with various fusion proteins in the presence or absence of LPS or zymosan. C-type lectin receptors (CLEC2, CLEC5a, CLEC9a, DCAR, DECTIN1, DECTIN2, DCSIGN-short, DCSIGNlong, MGL1, MINCLE, SIGNR1, and SIGNR3) and immunoglobulin family (TREM1, TREM2, TREM3, LMIR2, LMIR4, LMIR5, LMIR7, LMIR8, SIRP␣, and SIRP-1) were included. DCSIGN-short and DCSIGN-long are human receptors while the others are mouse receptors. Anti-inflammatory activity was not observed for all the receptor fusions examined. Interestingly, NF-B activation was only induced by LMIR5-Fcin a dose dependent manner. Addition of LMIR5-Fc did not obviously affect the NF-B activation induced by LPS or zymosan. The possibility that the fused human IgG Fc was
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Fig. 1. NF-B activation by LMIR5-Fc in human monocytes. (A) 5 × 104 TBRed cells were incubated overnight with 240 ng/well (1.2 g/ml) fusion proteins in the presence or absence of 10 ng/ml LPS. A number of NF-B-positive cells were analyzed by flow cytometry. (B) TBRed cells were incubated with various concentrations of LMIR5-Fc in the presence or absence of 10 ng/ml LPS or 20 g/ml zymosan. (C) 240 ng fusion protein and 10 ng/ml LPS samples were heated at 100 ◦ C for 40 min and subjected to TBRed assay as previously mentioned. Data are representative of two independent experiments and reported as mean ± SEM.
responsible for NF-B activation could be ruled out because the human IgG Fc and the other fusion proteins neither induced NFB activation nor affected LPS activity (Fig. 1A and B). Importantly, heated LMIR5-Fc was not able to trigger NF-B activation whereas LPS sample with or without heating showed a similar inflammatory activity (Fig. 1C). To demonstrate the inflammatory activity of LMIR5-Fc in vivo, mice were given with 20 g LMIR5-Fc or 4 g LPS per mouse by i.p. injection. All mice were sacrificed at 3 or 5 h after injection and blood samples were collected for cytokine assays. IL-6, IL12-p40, and MCP-1 were rapidly increased at the early time point and significantly decreased at 5 h. The similar expression pattern of these cytokines was observed for LPS administration but LPS induced a higher cytokine production. Although LPS-induced NF-B activation in TBRed cells was unchanged in the presence of LMIR5-Fc in vitro experiment, LMIR5-Fc statistically increased LPS-induced production of IL-12p40, MCP-1, and IFN-␥ at 3 h. In contrast to LPS, LMIR5-Fc was unable to induce IFN-␥ production (Fig. 2A–D). IL-10 production was not induced by LPS or LMIR5-Fc at both time points (data not shown). Collectively, these results showed the unique inflammatory property of LMIR5 extracellular domain. T cell Ig domain and mucin domain protein 1 (TIM1) is recently identified as an endogenous ligand for LMIR5 (Yamanishi et al., 2010). TIM1 is predominantly expressed on B rather than T cells. It is the costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation (Ding et al., 2011). TIM-1 is expressed by a large majority of IL-10-expressing regulatory B cells that can be induced through TIM1 ligation to promote tolerance (Ding et al., 2011). A TIM1-LMIR5 interaction results in cytokine production and is pivotal in neutrophil accumulation in the mouse model of kidney injury (Yamanishi et al., 2010). However, the same group reports the controversial finding
that LMIR5-Fc could induce cytokine production in the peritoneal cells in which TIM1 expression is undetectable (Yamanishi et al., 2012). One explanation is that other receptors are responsible for the inflammatory activity of LMIR5-Fc. 3.2. Requirement of TLR4 in the inflammatory activity of LMIR5-Fc Immunoreceptor tyrosine-based activation motifs (ITAMs) and toll-like receptors play a crucial role in antigen receptor signalings. MyD88 is the adaptor protein essential for TLR signaling (Akira and Takeda, 2004). ITAM receptors are associated with several adaptor proteins, including FcR␥, whose downstream signaling event is regulated by the adaptor protein CARD9 (Hara et al., 2007). To identify signaling pathways involved in the inflammatory activity of LMIR5, BMDMs from mutant mice lacking these adaptors were stimulated with LMIR5-Fc. MyD88−/− , CARD9−/− , and FcR␥−/− mice were included in this study. Production of IL-6 and IL-12p40 was examined. LMIR5-Fc-induced cytokine production was observed in WT BMDMs but it was substantially lost in the absence of MyD88 and slightly reduced or unchanged in the absence of CARD9 and FcR␥. Notably, IL-12p40 production was reduced in FcR␥−/− BMDMs, suggesting the involvement of FcR␥-associated receptor(s). However, this observation was specific for IL-12p40 as LMIR5-Fc-induced IL6 production was not different between WT and FcR␥−/− BMDMs (Fig. 3A and B). These results suggested that MyD88-associated receptors might be the key players responsible for the inflammatory activity of LMIR5-Fc. To address if TLR2 was the action target of LMIR5-Fc, WT and TLR2−/− BMDMs were stimulated with LMIR5-Fc. Cytokines IL-6 and IL-12p40 were measured from the cell-culture supernatant. Zymosan stimulation was used as a positive control for TLR2
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Fig. 2. Production of inflammatory mediators upon intra-peritoneal injection of LMIR5-Fc. ((A)–(D)) cytokines were measured from the blood samples at 3 or 5 h after i.p. injection of LMIR5-Fc (20 g/mouse) and/or LPS (4 g/mouse). Three mice were included for each group. Data are representative of six experimental replicates of one experiment (* , P < 0.05). Data are represented as mean ± SEM.
signaling. Zymosan-induced IL-6 production was observed in WT BMDMs and significantly reduced in TLR2−/− BMDMs. In contrast, LMIR5-Fc stimulation resulted in IL-6 production in both WT and TLR2−/− at the similar level. IL-12p40 production was also unchanged regardless of the expression of TLR2. Unexpectedly, IL12p40 production was not reduced in TLR2−/− BMDMs in response to zymosan stimulation when compared to WT BMDMs (Fig. 3C and D). Possibly, the expression of IL-12p40 is regulated by several immune receptors associated with Myd88 and FcR␥ as previously shown in Fig. 3A. These results suggested that LMIR5-Fc might act on other TLRs. To examine whether LMIR5-Fc acted on TLR4, WT and TLR4−/− BMDMs were stimulated with LMIR5-Fc or LPS. Pam3 as a ligand for TLR1 and TLR2 was also included. Production of IL-6 and IL-12p40 was examined. As expected, cytokine production was completely lost in TLR4−/− BMDMs in response to LPS, but not Pam3. LMIR5Fc stimulation resulted in a lower cytokine production in TLR4−/− BMDMs when compared to WT BMDMs. Heated LMIR5-Fc failed to induce cytokine production while heating did not affect the inflammatory activity of LPS (Fig. 3E and F). To further ensure TLR4-mediated inflammatory activity of LMIR5-Fc, TLR4 reporter cells (HB-4) were stimulated with LMIR5Fc or LPS. The control reporter cells without TLR4 (HB-N) was included for a comparative analysis. Both HB-4 and HB-N are originally derived from HEK 293 cells. LPS stimulation in HB-4 resulted in the activation of NF-B leading to the production of reporter gene product SEAP. LMIR5-Fc stimulation induced NF-B activation in HB-4. As expected, HB-N showed unresponsiveness to LPS or LMIR5-Fc stimulation (Fig. 4A). It was noteworthy that the lowest amount of Escherichia coli endotoxin standard required for
NF-B activation in HB-4 cells was 0.055 EU/ml. LMIR5-Fc used in this experiment contained 0.031 EU/ml endotoxin per 100 ng LMIR5-Fc. LMIR5-Fc at 15 and 60 ng still triggered a potent NFB activation. Indeed, LMIR5-Fc used in this study derived from the same lot of preparation in which endotoxin was detected at 0.075 ± 0.106 EU/ml per 240 ng LMIR5-Fc as previously mentioned. The detected endotoxin in 240 ng LMIR5-Fc, which was the highest amount used in this study, did not possess any inflammatory activity. NF-B activation was not induced by heated LMIR5-Fc. Heating did not affect the inflammatory activity of LPS (Fig. 4B). These results suggested that TLR4-mediated NF-B activation in HB-4 was due to the activity of LMIR5-Fc, not contaminating endotoxin. 3.3. A direct interaction between TLR4 and LMIR5 To demonstrate a direct interaction between LMIR5 and TLR4, we initially examined the binding capacity of LMIR5-Fc to HB-4 by cell surface staining. LMIR5-Fc-binding cells were detected using anti-human IgG Fc-PE conjugate. HB-N cells without TLR4 were used as a negative control. Unexpectedly, LMIR5-Fc interacted with all the cells examined at the same level regardless of the expression of TLR4 (data not shown). Alternative approaches are needed to demonstrate the LMIR5-TLR4 binding. TLR4 is composed of N-terminal, central, and C-terminal domains. MD-2 binds to the concave surface of the N-terminal and central TLR4 domains (Roger et al., 2009). To alternatively demonstrate the LMIR5-TLR4 interaction, we first generated a soluble recombinant chimeric protein composed of the N-terminal half of the mouse TLR4 ectodomain (amino acids 1-334) fused to the Fc
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Fig. 3. Requirement of MyD88 and TLR4 in the inflammatory activity of LMIR5-Fc. ((A)–(F)) BMDMs from wild type (WT) and gene-deficient mice (−/− ) were stimulated with 1.2 g/ml fusion protein, 100 ng/ml Pam3, 50 g/ml zymosan, or 10 ng/ml LPS for 24 h. Cytokines were measured using ELISA kits. In some experiments, heating was performed at 100 ◦ C for 40 min. Data are representative of two independent experiments and represented as mean ± SEM (* , P < 0.05).
domain of human IgG1 (TLR4-Fc). The recombinant TLR4-Fc protein was produced in HEK 293 cells to ensure posttranscriptional modifications as previously described (Roger et al., 2009). In the presence of serum as a source of soluble MD-2, LPS was shown to bind to TLR4-Fc as also demonstrated previously (data not shown) (Roger et al., 2009). Next, we examined the binding capacity of TLR4-Fc to the lymphocyte 2B4 cells, which express the surface LMIR5 in association with its adaptor protein DAP12. As expected, TLR4-Fc interacted with LMIR5-positive cells, but not the control cells without LMIR5 (Fig. 4C). We next examined if TLR4-Fc was able to induce signal transduction through LMIR5 using LMIR5 reporter cells 2B4. This reporter expresses a surface LMIR5-HA fusion in association with DAP12 and a fluorescent maker GFP under the control of transcriptional factor NFAT. Cross-linking of anti-HA antibody resulted in NFAT activation, which could be determined by a number of GFP-positive cells. However, cross-linking of TLR4-Fc did not trigger NFAT activation (Fig. 4D). Because TLR4-Fc interacted with both LMIR5 and LPS, we asked whether LPS could inhibit TLR4-LMIR5 interaction. Addition of LPS did not affect the binding capacity of TLR4-Fc to LMIR5-expressing 2B4 cells (Fig. 4E) or coated LMIR5 on ELISA plate (Fig. 4F). However, TLR4 could inhibit the inflammatory activity of LPS and LMIR5-Fc in a dose dependent manner (Fig. 4G and H). These results suggested that the binding sites on TLR4 might be different for LMIR5-Fc and LPS. Binding capacity of LMIR5-Fc was examined for TBRed, NKT cells, CD8+ T cells B3Z, RAW-Blue cells, microglia cells BV2, lymphocyte 2B4, epithelial cells HEK-293, BMDMs, and BMDCs. LMIR5 interacted with most cells examined, except for RAWBlue and 2B4 (Fig. 5A). Attempts have been made to explore the
biological significance of LMIR5-Fc bindings to these cells, including BMDCs. Although maturation marker CD80 was not up-regulated by LMIR5-Fc stimulation, LMIR5-Fc was able to induce IL-6 and IL12p40 secretion from BMDCs (Fig. 5B–D). This capacity was not due to endotoxin contamination as heated LMIR5-Fc failed to induce cytokine production (Fig. 5C and D). Not only microbial products but also self-components are able to activate TLR4 signaling. LPS from Gram-negative bacteria has long been known as an exogenous activator of TLR4 (Akira and Takeda, 2004). TLR4 is recently reported to recognize Shiga toxins and mediates the lethal cardiac toxicity of anthrax (Brigotti et al., 2013; Kandadi et al., 2012). Pertussis toxin-induced activation of several signaling molecules is nearly abolished in the absence of TLR4 (Kerfoot et al., 2004). Endogenous activators of TLR4 include S100 proteins, HMGB1, histones, cold-inducible RNA-binding protein, and heat shock proteins (Vogl et al., 2007; Xu et al., 2009, 2011; Yang et al., 2010; Qiang et al., 2013; Quintana and Cohen, 2005). The release of these endogenous activators contributes to septic death (Vogl et al., 2007; Xu et al., 2009; Qiang et al., 2013). In this study, the LMIR5 extracellular domain is identified as another endogenous activator of TLR4. It will be interesting to examine the involvement of other toll-like receptors in addition to TLR2 and TLR4. A single receptor LMIR5 is capable of inducing different activation signals through its extracellular and intracellular domains. For extracellular domain, this study finds that LMIR5 delivers the activation signal through a TLR4-MyD88-NF-B pathway. In addition, FcR␥-associated receptors may be involved in LMIR5-induced IL-12p40 production (see, Fig. 3A). For intracellular domain, the membranous LMIR5 is able to deliver the activation signal through DAP10 and DAP12 in LMIR5-transducing mast cells upon LMIR5specific antibody cross-linking (Yamanishi et al., 2008). However,
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Fig. 4. Interaction between TLR4 and LMIR5. (A) 5 × 104 HB-4 and HB-N were incubated overnight with 10 ng/ml LPS (+), reagent (−), and various concentrations of LMIR5Fc. Reporter cell assay was measured at A630. A630 > 0.3 is considered as positive. Data are representative of at least five independent experiments and represented as mean ± SEM. (B) 240 ng fusion protein and 10 ng/ml LPS samples were heated at 100 ◦ C for 40 min and subjected to HB-4 assay. (C) TLR4-Fc was examined for its binding capacity to LMIR5-expressing cells 2B4. Black histogram is for the control cells without LMIR5. Empty histogram is for LMIR5-expressing 2B4. Data are representative of two independent experiments. The most obvious results are shown. (D) TLR4-Fc, hIgG Fc, medium (negative) or anti-HA antibody (positive) was cross-linked to LMIR5 reporter cells DAP12 + LMIR5+ or control cells DAP12+ for assay of NFAT-GFP activation. (E) TLR4-Fc was examined for its binding capacity to LMIR5 or LMIR4-expressing 2B4 in the presence or absence of 10 g LPS low and 30 g LPS high. (F) TLR4-Fc-biotin conjugate was premixed with or without 10 g LPS for 30 min. The mixture or reagent (negative) was added to ELISA wells containing of coated LMIR5-Fc or LPS. ((G)–(H)) HB-4 was incubated overnight with LPS or LMIR5-Fc in the presence or absence of TLR4-Fc. Data are represented as mean ± SEM (* , P < 0.05).
natural ligands for the membranous LMIR5 have not been identified. Furthermore, the 29-aa cytoplasmic tail of LMIR5 contains the tyrosine-based motif that becomes a docking site for Grb2 upon c-Fyn phosphorylation (Martinez-Barriocanal and Sayos, 2006). 3.4. Examination of NF-B-activating contaminants in LMIR5-Fc samples To exclude the possibility that contaminants were responsible for the inflammatory activity of LMIR5-Fc, the following experiments were performed. Firstly, LMIR5-Fc samples were fractionated using a gel filtration and 14 fractions were collected. The presence of LMIR5-Fc was examined by incubating each fraction with HB-4 cells. Binding of LMIR5-Fc to HB-4 was detected using anti-hIgG Fc-PE conjugate and analyzed by flow cytometry. Seven fractions (4–10) were positive for LMIR5-Fc and
consistently responsible for NF-B activation. As expected, other factions were negative for both LMIR5-Fc and NF-B activation (Fig. 6A and B). Secondly, LMIR5-Fc samples were purified using a protein G. Protein G-binding components were eluted and loaded on the gel filtration. Eighteen fractions were collected for HB-4 and TBRed assays. Ten fractions (3–12) induced NF-B activation in both reporter cells. The other factions were negative. Notably, factions 2 and 13 were weakly positive for HB-4 but negative for TBRed. This inconsistent result might be due to the fact that HB-4 is more sensitive than TBRed (Fig. 6C and D). It was noted that endotoxin was detected at 0.100 ± 0.005 EU/ml for 5 l fraction 7 (Fig. 6A and B) and 0.111 ± 0.017 EU/ml for 5 l fraction 7 (Fig. 6C and D). The same amount of LMIR5-Fc fraction (5 l) was used for TBRed and HB-4 assays. The contribution of contaminated endotoxin could be excluded because this level of endotoxin did not possess inflammatory
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Fig. 5. Binding capacity of LMIR5-Fc to various cells. (A) Binding of LMIR5-Fc was examined for various cell lines, BMDMs, and BMDCs. Black histogram is for hIgG binding. Empty histogram is for LMIR5-Fc binding. (B) BMDCs were stimulated with 240 ng fusion protein or 10 ng/ml LPS for 48 h. Black and empty histograms are unstimulated and stimulated cells, respectively. ((C) and (D)) BMDCs were stimulated with 240 ng fusion protein or 10 ng/ml LPS for 24 h for assay of cytokine production. Heat denaturation was performed at 100 ◦ C for 40 min. Data are represented as mean ± SEM (* , P < 0.05).
activity as previously shown. Thirdly, coated anti-human IgG Fc antibody was used to cross-link LMIR5-Fc to HB-4 cells. Nonspecific or unbound components were washed away before adding HB-4. LMIR4-Fc was used as a negative control. Cross-linking of LMIR5-Fc, but not LMIR4-Fc, could induce NF-B activation
and this was not observed in the wells without antibodies (Fig. 6E). Finally, LMIR5-Fc or LPS samples were incubated with polymyxin B-agarose conjugate (PMB) for 2 h in order to remove LPS. After centrifugation, the supernatant was collected and concentrated before incubation with HB-4. As expected, 5 ng/ml LPS
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Fig. 6. Examination of NF-B-inducing contaminants in LMIR5-Fc samples. ((A) and (B)) LMIR5-Fc samples were loaded on PD MiniTrap G-25 columns and 14 fractions (1–14) were collected. Each fraction was examined for the presence of LMIR5-Fc by HB-4 binding assay and the NF-B activation by HB-4 reporter cell assay. ((C) and (D)) LMIR5-Fc samples were purified using a protein G. Protein G-binding components were eluted and fractioned using a gel filtration as previously mentioned. Eighteen factions were collected and subjected to HB-4 and TBRed assays. (E) Anti-human IgG Fc antibody was coated in PBS on 96-well plate at 4 ◦ C overnight, washed twice with PBS, incubated with fusion protein for 2 h, and washed with PBS, consecutively. HB-4 reporter cell assay was performed. (F) LMIR5-Fc samples were mixed with polymyxin B (PMB) for 2 h at room temperature and the supernatant was concentrated using Ultracel-10 K before addition of HB4. Data are representative of two independent experiments and reported as mean ± SEM.
was totally captured by PMB. The supernatant from PMB-treated LPS samples was unable to induce NF-B activation in HB-4. In contrast to LPS, LMIR5-Fc samples with or without PMB treatment showed a similar inflammatory activity (Fig. 6F). Notably, a new LMIR5-Fcsample was reproduced in HEK 293 and examined for its inflammatory property, which was reproducible in HB-4 (data not shown). Collectively, the contaminants were not the cause of NF-B activation. To our knowledge, the immune receptors that are responsible for the inflammatory activity of the LMIR5 extracellular domain
have not been identified. In this study, we provide the evidence that supports the existence of LMIR5-TLR4 signaling pathway as follows: (i) LMIR5-Fc-induced inflammation is substantially lost in the absence of TLR4, (ii) LMIR5-Fc could induce NF-B activation in TLR4 reporter cells, (iii) a direct interaction between TLR4-Fc and LMIR5-expressing cells could be demonstrated, and (iv) NF-Bactivating contaminants are undetectable from LMIR5-Fc samples. Activation of TLR4 by the LMIR5 extracellular domain may play an important role in the amplification of inflammatory responses. Indeed, the contribution of LMIR5 in sepsis is recently
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demonstrated. LMIR5 deficiency profoundly reduces a systemic cytokine production and a septic mortality in LPS-administered mice (Yamanishi et al., 2012). Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death is decreased by LMIR5-Fc administration (Yamanishi et al., 2012). In this study, LMIR5 extracellular domain amplifies LPS-induced inflammation in vivo and it constitutively activates myeloid cells through TLR4-MyD88-NF-B signaling pathway. Thus, identification of endogenous activator of TLR4 not only advances our understanding of additional inflammatory mediators but will also help in the development of new therapeutic strategies; for example, anti-LMIR5 neutralizing antibodies for treatment of sepsis. A recent study has demonstrated that fusion protein containing antigen of interest fused to the extra domain A from fibronectin, which is an endogenous TLR4 ligand, is highly immunogenic and induces a potent immune responses against the fused antigen (Arribillaga et al., 2013). Indeed, TLR4 activation is critical for the development of potent humoral and cellular responses and it mediates vaccine-induced protective immunity (Lartigue et al., 2009; Higgins et al., 2006; Hutchens et al., 2008; Hwang et al., 2009; Loser et al., 2010). We report here that LMIR5-Fc interacts with several cell types, including antigen-presenting cells, and induces cytokine production from dendritic cells. Therefore, the finding from here may open a new way to use the LMIR5 extracellular domain-based vaccine delivery for vaccination against infectious diseases and cancer. Acknowledgments This work was supported by grant-in-aid for scientific research from Japan Society for the Promotion of Science (JSPS, 11F01107 to S.Y.) and the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government (24390256 to H.H.). V.P. is the recipient of a JSPS fellowship under the FY2011-2013 program of JSPS Postdoctoral Fellowship for Foreign Researcher. References Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511. Arribillaga, L., Durantez, M., Lozano, T., Rudilla, F., Rehberger, F., Casares, N., Villanueva, L., Martinez, M., Gorraiz, M., Borras-Cuesta, F., Sarobe, P., Prieto, J., Lasarte, J.J., 2013. A fusion protein between streptavidin and the endogenous TLR4 ligand EDA targets biotinylated antigens to dendritic cells and induces T cell responses in vivo. BioMed Res. Int. 2013, 864720. Brigotti, M., Carnicelli, D., Arfilli, V., Tamassia, N., Borsetti, F., Fabbri, E., Tazzari, P.L., Ricci, F., Pagliaro, P., Spisni, E., Cassatella, M.A., 2013. Identification of TLR4 as the receptor that recognizes Shiga toxins in human neutrophils. J. Immunol. 191, 4748–4758. Ding, Q., Yeung, M., Camirand, G., Zeng, Q., Akiba, H., Yagita, H., Chalasani, G., Sayegh, M.H., Najafian, N., Rothstein, D.M., 2011. Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice. J. Clin. Invest. 121, 3645–3656. Gasiorowski, R.E., Ju, X., Hart, D.N., Clark, G.J., 2013. CD300 molecule regulation of human dendritic cell functions. Immunol. Lett. 149, 93–100. Hara, H., Ishihara, C., Takeuchi, A., Imanishi, T., Xue, L., Morris, S.W., Inui, M., Takai, T., Shibuya, A., Saijo, S., Iwakura, Y., Ohno, N., Koseki, H., Yoshida, H., Penninger, J.M., Saito, T., 2007. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat. Immunol. 8, 619–629. Higgins, S.C., Jarnicki, A.G., Lavelle, E.C., Mills, K.H., 2006. TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells. J. Immunol. 177, 7980–7989. Hutchens, M.A., Luker, K.E., Sonstein, J., Nunez, G., Curtis, J.L., Luker, G.D., 2008. Protective effect of Toll-like receptor 4 in pulmonary vaccinia infection. PLoS Pathog. 4, e1000153.
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Short communication
LMIR5 extracellular domain activates myeloid cells through Toll-like receptor 4 Vongsavanh Phongsisay a,∗ , Ei’ichi Iizasa b , Hiromitsu Hara b , Sho Yamasaki a a b
Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan
a r t i c l e
i n f o
Article history: Received 5 March 2014 Received in revised form 16 May 2014 Accepted 9 June 2014 Keywords: LMIR5 CD300b TLR4 Inflammation
a b s t r a c t LMIR5/CD300b is an activating immunoglobulin-like receptor whose extracellular domain (LMIR5-Fc) is constitutively released from immune cells. The release of LMIR5-Fc is augmented upon stimulation with TLR agonists. LMIR5-Fc is reported to possess inflammatory activity and amplify LPS-induced lethal inflammation; however, its action mechanism has not been clarified. This study was aimed to identify receptors for LMIR5-Fc. Using NF-B reporter cells in human monocytes THP1, LMIR5-Fc was solely found to trigger NF-B activation among various signaling receptors examined. In addition, an injection of LMIR5-Fc into the mouse peritoneal resulted in a rapid production of inflammatory mediators and an amplification of LPS activity. Moreover, LMIR5-Fc-induced cytokine production was markedly reduced in TLR4-deficient mouse macrophages. Using TLR4 reporter cells, the LMIR5-Fc sample that contained a trace amount of endotoxin under the sensitivity of reporter cells triggered a potent NF-B activation. Furthermore, the inflammatory activity of LMIR5-Fc was completely lost by heating but unchanged by polymyxin B pretreatment. Using TLR4 fusion protein, TLR4 was found to interact specifically with LMIR5overexpressing cells. Therefore, LMIR5-Fc is new inflammatory mediator and endogenous ligand of TLR4. This study provides an insight into the positive feedback mechanism of inflammation through TLR4LMIR5-Fc axis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction LMIR5/CD300b is classified in the CD300 family of myeloid immunoglobulin receptors that are type I transmembrane proteins exhibiting a single IgV-like extracellular domain (Gasiorowski et al., 2013). Unusually, all CD300 members interact with each other, even with themselves, forming both homo- and heterodimers that differentially modulates signaling outcomes (Martinez-Barriocanal et al., 2010). LMIR5 is present on the cell membrane of neutrophils, peritoneal macrophages, and mast cells (Yamanishi et al., 2008, 2012). Upon stimulation with inflammatory stimuli, LMIR5 is dissociated from the membrane by a proteolytic cleavage of the membranous LMIR5 partly through matrix metalloproteinases (Yamanishi et al., 2012). LMIR5 mediates at least two different signaling pathways. Firstly, the membranous LMIR5 is mainly
∗ Corresponding author. Permanent address: Homsavanh School, Ban Hom, Hatsaifong, Vientiane, Laos.Tel.: +81 926424614/+856 20 55777636; fax: +81 926424614. E-mail addresses: [email protected], [email protected] (V. Phongsisay). http://dx.doi.org/10.1016/j.molimm.2014.06.012 0161-5890/© 2014 Elsevier Ltd. All rights reserved.
associated with the adaptor protein DAP12 under physiological condition (Yamanishi et al., 2008). DAP12 is well-known for its role in the delivery of activating signal by phosphorylation of ITAM motif at its cytoplasmic domain (Turnbull and Colonna, 2007). In LMIR5-transduced mast cells, cross-linking of anti-LMIR5 antibody induces phosphorylation of several signaling molecules (ERK, p38, Akt), resulting in cytokine/chemokine production, increased cell adhesion, histamine release, and increased cell survival. This phenotype is DAP12 and Syk-dependent (Yamanishi et al., 2008). Secondly, the fusion protein consisting of the LMIR5 extracellular domain and the human IgG Fc, LMIR5-Fc, induces cytokine production in mouse peritoneal cells (Yamanishi et al., 2012). To date, its action mechanism has not been clarified. Toll-like receptors are the pattern-recognition receptor that mediates the production of effective immunity against infectious agents. In humans and mice, there are at least 13 TLRs that differentially recognize microbial products (Akira and Takeda, 2004). For example, TLR2 recognizes zymosan and lipoteichoic acid. TLR4 recognizes LPS from Gram-negative bacteria and it co-operates with co-receptors MD-2 and CD14. Upon ligand stimulation, several signaling molecules are activated leading to NF-B activation, cytokine production, and cellular and humoral immune responses.
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In this study, we initially examined the immunological property of various receptor extracellular domains, including C-type lectin and immunoglobulin-like receptors. Anti-inflammatory activity was not observed for all the receptors exampled; indeed, LMIR5Fc was the only one capable of inducing inflammatory responses. Further study showed that LMIR5 extracellular domain is an endogenous activator of TLR4. 2. Materials and methods 2.1. Mice All mice are C57BL/6 background. Wild type mice were purchased from CLEA Japan. TLR2−/− and TLR4−/− mice were from Oriental Bioservice. FcR␥−/− and CARD9−/− mice were established as previously described (Park et al., 1998; Hara et al., 2007). MyD88−/− mice were purchased from Oriental Yeast. Mice were maintained in a filtrated-air laminar-flow enclosure and given standard laboratory food and water ad libitum. Animal protocols were approved by the committee of Ethics on Animal Experiment, Faculty of Medical Sciences, Kyushu University. 2.2. Reagents Polymyxin B-agarose (P1411), LPS (L4516), and zymosan (Z4250) were from Sigma. Pam3CSK4 (IMG-2201) was from IMGENEX. Antibody to LMIR5 (AF2580) was purchased from R&D. TSM buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM CaCl2 , 2 mM MgCl2 , pH 7.0) was prepared as previously described and used for all fusion protein experiments (van Sorge et al., 2009). Protein G SepharoseTM and PD MiniTrap G-25 columns containing SephadexTM G-25 medium were purchased from GE Healthcare. Amicon Ultracel-10K was from Millipore. ToxinSensorTM chromogenic LAL endotoxin assay kit (L00350) was purchased from Genscript. 2.3. Fusion proteins Fusion protein is consisted of the receptor extracellular domain and the human IgG Fc. For LMIR5-Fc fusion, a DNA fragment encoding for amino acids 65-177 of mouse LMIR5 (accession no. BC160263) was inserted via SfiI and SalI into pDisplayTM vector (Invitrogen). The human IgG1 Fc region was cloned into the vector via AccI. LMIR5-expressing vector was transfected into HEK 293 cells. FreeStyleTM 293 expression system (K9000-01, Invitrogen) was used to allow large-scale transfection of suspension 293 cells in defined, serum-free medium according to the manufacturer’s instruction. Production of other receptor-Fc fusion proteins was performed using the same method as mentioned. TLR4-Fc fusion protein was produced as previously described (Roger et al., 2009). All fusion proteins were diluted in TSM buffer. 2.4. Cells Cells were cultured in RPMI supplemented with 10% FCS, otherwise stated. Bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs) were induced for 7–10 days by M-CSF or GM-CSF, respectively. LMIR5-expressing cells were established by expressing the surface LMIR5-HA fusion in association with adaptor protein DAP12 in the NFAT-GFP-2B4 cells, as previously described (Yamasaki et al., 2008). LMIR4 reporter cells were established by expressing the surface LMIR4-HA fusion in association with FcR␥ in the NFAT-GFP-2B4 cells. For LMIR5 or LMIR4 reporter assay, 5 × 104 cells were incubated overnight with stimuli, which had been coated on the ELISA plate or cross-linked by using appropriate antibodies. A number of GFP-positive cells were
analysed by flow cytometry. NF-B reporter cells (TBRed) were established by expressing the fluorescent marker DsRed under the control of NF-B in the human monocytes THP1. For TBRed assay, cells were incubated overnight with 240 ng fusion protein or other stimuli. NF-B -positive cells were analyzed by flow cytometry. NKT cells RT-5 was obtained from Dr. Shimamura Michio. B3Z is CD8+ T cells. Microglia BV2 was provided by Dr. Makoto Tsuda and cultured in DMEM supplemented with 5% FCS. HEK-Blue mTLR4 (HB-4), HEK-Blue Null1-v (HB-N), and RAW-Blue were purchased from InvivoGen and cultured in DMEM supplemented with 10% FCS. TLR4 reporter cells were obtained by co-transfection of mTLR4 gene, MD-2/CD14 co-receptor genes, and secreted embryonic alkaline phosphatase (SEAP) reporter gene. The SEAP reporter gene was placed under the control of the IFN- minimal promoter fused to five NF-B and AP-1-binding sites. Activation of NF-B and AP-1 leads to the production of SEAP, which was measured at A630 using a detection medium QUANTI-Blue according to the manufacturer’s instruction (InvivoGen). 2.5. Cytokine assay BMDMs or BMDCs were incubated with stimuli for 24 h. Cytokines were measured using ELISA kits from BD biosciences. 2.6. Cell surface staining Cells were incubated with fusion protein in TSM-RPMI medium (50% TSM in 2% FCS/RPMI) followed by anti-human IgG Fc-PE in 2% FCS/RPMI. Cell staining was carried out on ice for 1 h, washed twice in 2% FCS/RPMI after each incubation, and analyzed by flow cytometry. 2.7. Assay of ligand-receptor interaction Nunc MaxiSorp 96-well plates were used throughout the experiments. Protein samples and LPS were coated in coating buffer (0.293% w/v of NaHCO3 and 0.053% w/v of Na2 CO3 , pH 9.6, in water) at 42 ◦ C overnight. The wells were blocked with 10% fresh FCS in TSM for 1 h and incubated with fusion protein with or without biotin conjugate in 10% FCS/TSM for 2 h. After washing with TSM buffer, anti-human IgG Fc-HRP conjugate or avidin-HRP was used for detection. 2.8. Statistics The statistical significance of differences was assessed using a two-tailed Mann–Whitney test. 3. Results and discussion 3.1. Inflammatory property of LMIR5-Fc To examine if the receptor extracellular domains possess inflammatory or anti-inflammatory property, NF-B reporter cells TBRed were stimulated with various fusion proteins in the presence or absence of LPS or zymosan. C-type lectin receptors (CLEC2, CLEC5a, CLEC9a, DCAR, DECTIN1, DECTIN2, DCSIGN-short, DCSIGNlong, MGL1, MINCLE, SIGNR1, and SIGNR3) and immunoglobulin family (TREM1, TREM2, TREM3, LMIR2, LMIR4, LMIR5, LMIR7, LMIR8, SIRP␣, and SIRP-1) were included. DCSIGN-short and DCSIGN-long are human receptors while the others are mouse receptors. Anti-inflammatory activity was not observed for all the receptor fusions examined. Interestingly, NF-B activation was only induced by LMIR5-Fcin a dose dependent manner. Addition of LMIR5-Fc did not obviously affect the NF-B activation induced by LPS or zymosan. The possibility that the fused human IgG Fc was
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Fig. 1. NF-B activation by LMIR5-Fc in human monocytes. (A) 5 × 104 TBRed cells were incubated overnight with 240 ng/well (1.2 g/ml) fusion proteins in the presence or absence of 10 ng/ml LPS. A number of NF-B-positive cells were analyzed by flow cytometry. (B) TBRed cells were incubated with various concentrations of LMIR5-Fc in the presence or absence of 10 ng/ml LPS or 20 g/ml zymosan. (C) 240 ng fusion protein and 10 ng/ml LPS samples were heated at 100 ◦ C for 40 min and subjected to TBRed assay as previously mentioned. Data are representative of two independent experiments and reported as mean ± SEM.
responsible for NF-B activation could be ruled out because the human IgG Fc and the other fusion proteins neither induced NFB activation nor affected LPS activity (Fig. 1A and B). Importantly, heated LMIR5-Fc was not able to trigger NF-B activation whereas LPS sample with or without heating showed a similar inflammatory activity (Fig. 1C). To demonstrate the inflammatory activity of LMIR5-Fc in vivo, mice were given with 20 g LMIR5-Fc or 4 g LPS per mouse by i.p. injection. All mice were sacrificed at 3 or 5 h after injection and blood samples were collected for cytokine assays. IL-6, IL12-p40, and MCP-1 were rapidly increased at the early time point and significantly decreased at 5 h. The similar expression pattern of these cytokines was observed for LPS administration but LPS induced a higher cytokine production. Although LPS-induced NF-B activation in TBRed cells was unchanged in the presence of LMIR5-Fc in vitro experiment, LMIR5-Fc statistically increased LPS-induced production of IL-12p40, MCP-1, and IFN-␥ at 3 h. In contrast to LPS, LMIR5-Fc was unable to induce IFN-␥ production (Fig. 2A–D). IL-10 production was not induced by LPS or LMIR5-Fc at both time points (data not shown). Collectively, these results showed the unique inflammatory property of LMIR5 extracellular domain. T cell Ig domain and mucin domain protein 1 (TIM1) is recently identified as an endogenous ligand for LMIR5 (Yamanishi et al., 2010). TIM1 is predominantly expressed on B rather than T cells. It is the costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation (Ding et al., 2011). TIM-1 is expressed by a large majority of IL-10-expressing regulatory B cells that can be induced through TIM1 ligation to promote tolerance (Ding et al., 2011). A TIM1-LMIR5 interaction results in cytokine production and is pivotal in neutrophil accumulation in the mouse model of kidney injury (Yamanishi et al., 2010). However, the same group reports the controversial finding
that LMIR5-Fc could induce cytokine production in the peritoneal cells in which TIM1 expression is undetectable (Yamanishi et al., 2012). One explanation is that other receptors are responsible for the inflammatory activity of LMIR5-Fc. 3.2. Requirement of TLR4 in the inflammatory activity of LMIR5-Fc Immunoreceptor tyrosine-based activation motifs (ITAMs) and toll-like receptors play a crucial role in antigen receptor signalings. MyD88 is the adaptor protein essential for TLR signaling (Akira and Takeda, 2004). ITAM receptors are associated with several adaptor proteins, including FcR␥, whose downstream signaling event is regulated by the adaptor protein CARD9 (Hara et al., 2007). To identify signaling pathways involved in the inflammatory activity of LMIR5, BMDMs from mutant mice lacking these adaptors were stimulated with LMIR5-Fc. MyD88−/− , CARD9−/− , and FcR␥−/− mice were included in this study. Production of IL-6 and IL-12p40 was examined. LMIR5-Fc-induced cytokine production was observed in WT BMDMs but it was substantially lost in the absence of MyD88 and slightly reduced or unchanged in the absence of CARD9 and FcR␥. Notably, IL-12p40 production was reduced in FcR␥−/− BMDMs, suggesting the involvement of FcR␥-associated receptor(s). However, this observation was specific for IL-12p40 as LMIR5-Fc-induced IL6 production was not different between WT and FcR␥−/− BMDMs (Fig. 3A and B). These results suggested that MyD88-associated receptors might be the key players responsible for the inflammatory activity of LMIR5-Fc. To address if TLR2 was the action target of LMIR5-Fc, WT and TLR2−/− BMDMs were stimulated with LMIR5-Fc. Cytokines IL-6 and IL-12p40 were measured from the cell-culture supernatant. Zymosan stimulation was used as a positive control for TLR2
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Fig. 2. Production of inflammatory mediators upon intra-peritoneal injection of LMIR5-Fc. ((A)–(D)) cytokines were measured from the blood samples at 3 or 5 h after i.p. injection of LMIR5-Fc (20 g/mouse) and/or LPS (4 g/mouse). Three mice were included for each group. Data are representative of six experimental replicates of one experiment (* , P < 0.05). Data are represented as mean ± SEM.
signaling. Zymosan-induced IL-6 production was observed in WT BMDMs and significantly reduced in TLR2−/− BMDMs. In contrast, LMIR5-Fc stimulation resulted in IL-6 production in both WT and TLR2−/− at the similar level. IL-12p40 production was also unchanged regardless of the expression of TLR2. Unexpectedly, IL12p40 production was not reduced in TLR2−/− BMDMs in response to zymosan stimulation when compared to WT BMDMs (Fig. 3C and D). Possibly, the expression of IL-12p40 is regulated by several immune receptors associated with Myd88 and FcR␥ as previously shown in Fig. 3A. These results suggested that LMIR5-Fc might act on other TLRs. To examine whether LMIR5-Fc acted on TLR4, WT and TLR4−/− BMDMs were stimulated with LMIR5-Fc or LPS. Pam3 as a ligand for TLR1 and TLR2 was also included. Production of IL-6 and IL-12p40 was examined. As expected, cytokine production was completely lost in TLR4−/− BMDMs in response to LPS, but not Pam3. LMIR5Fc stimulation resulted in a lower cytokine production in TLR4−/− BMDMs when compared to WT BMDMs. Heated LMIR5-Fc failed to induce cytokine production while heating did not affect the inflammatory activity of LPS (Fig. 3E and F). To further ensure TLR4-mediated inflammatory activity of LMIR5-Fc, TLR4 reporter cells (HB-4) were stimulated with LMIR5Fc or LPS. The control reporter cells without TLR4 (HB-N) was included for a comparative analysis. Both HB-4 and HB-N are originally derived from HEK 293 cells. LPS stimulation in HB-4 resulted in the activation of NF-B leading to the production of reporter gene product SEAP. LMIR5-Fc stimulation induced NF-B activation in HB-4. As expected, HB-N showed unresponsiveness to LPS or LMIR5-Fc stimulation (Fig. 4A). It was noteworthy that the lowest amount of Escherichia coli endotoxin standard required for
NF-B activation in HB-4 cells was 0.055 EU/ml. LMIR5-Fc used in this experiment contained 0.031 EU/ml endotoxin per 100 ng LMIR5-Fc. LMIR5-Fc at 15 and 60 ng still triggered a potent NFB activation. Indeed, LMIR5-Fc used in this study derived from the same lot of preparation in which endotoxin was detected at 0.075 ± 0.106 EU/ml per 240 ng LMIR5-Fc as previously mentioned. The detected endotoxin in 240 ng LMIR5-Fc, which was the highest amount used in this study, did not possess any inflammatory activity. NF-B activation was not induced by heated LMIR5-Fc. Heating did not affect the inflammatory activity of LPS (Fig. 4B). These results suggested that TLR4-mediated NF-B activation in HB-4 was due to the activity of LMIR5-Fc, not contaminating endotoxin. 3.3. A direct interaction between TLR4 and LMIR5 To demonstrate a direct interaction between LMIR5 and TLR4, we initially examined the binding capacity of LMIR5-Fc to HB-4 by cell surface staining. LMIR5-Fc-binding cells were detected using anti-human IgG Fc-PE conjugate. HB-N cells without TLR4 were used as a negative control. Unexpectedly, LMIR5-Fc interacted with all the cells examined at the same level regardless of the expression of TLR4 (data not shown). Alternative approaches are needed to demonstrate the LMIR5-TLR4 binding. TLR4 is composed of N-terminal, central, and C-terminal domains. MD-2 binds to the concave surface of the N-terminal and central TLR4 domains (Roger et al., 2009). To alternatively demonstrate the LMIR5-TLR4 interaction, we first generated a soluble recombinant chimeric protein composed of the N-terminal half of the mouse TLR4 ectodomain (amino acids 1-334) fused to the Fc
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Fig. 3. Requirement of MyD88 and TLR4 in the inflammatory activity of LMIR5-Fc. ((A)–(F)) BMDMs from wild type (WT) and gene-deficient mice (−/− ) were stimulated with 1.2 g/ml fusion protein, 100 ng/ml Pam3, 50 g/ml zymosan, or 10 ng/ml LPS for 24 h. Cytokines were measured using ELISA kits. In some experiments, heating was performed at 100 ◦ C for 40 min. Data are representative of two independent experiments and represented as mean ± SEM (* , P < 0.05).
domain of human IgG1 (TLR4-Fc). The recombinant TLR4-Fc protein was produced in HEK 293 cells to ensure posttranscriptional modifications as previously described (Roger et al., 2009). In the presence of serum as a source of soluble MD-2, LPS was shown to bind to TLR4-Fc as also demonstrated previously (data not shown) (Roger et al., 2009). Next, we examined the binding capacity of TLR4-Fc to the lymphocyte 2B4 cells, which express the surface LMIR5 in association with its adaptor protein DAP12. As expected, TLR4-Fc interacted with LMIR5-positive cells, but not the control cells without LMIR5 (Fig. 4C). We next examined if TLR4-Fc was able to induce signal transduction through LMIR5 using LMIR5 reporter cells 2B4. This reporter expresses a surface LMIR5-HA fusion in association with DAP12 and a fluorescent maker GFP under the control of transcriptional factor NFAT. Cross-linking of anti-HA antibody resulted in NFAT activation, which could be determined by a number of GFP-positive cells. However, cross-linking of TLR4-Fc did not trigger NFAT activation (Fig. 4D). Because TLR4-Fc interacted with both LMIR5 and LPS, we asked whether LPS could inhibit TLR4-LMIR5 interaction. Addition of LPS did not affect the binding capacity of TLR4-Fc to LMIR5-expressing 2B4 cells (Fig. 4E) or coated LMIR5 on ELISA plate (Fig. 4F). However, TLR4 could inhibit the inflammatory activity of LPS and LMIR5-Fc in a dose dependent manner (Fig. 4G and H). These results suggested that the binding sites on TLR4 might be different for LMIR5-Fc and LPS. Binding capacity of LMIR5-Fc was examined for TBRed, NKT cells, CD8+ T cells B3Z, RAW-Blue cells, microglia cells BV2, lymphocyte 2B4, epithelial cells HEK-293, BMDMs, and BMDCs. LMIR5 interacted with most cells examined, except for RAWBlue and 2B4 (Fig. 5A). Attempts have been made to explore the
biological significance of LMIR5-Fc bindings to these cells, including BMDCs. Although maturation marker CD80 was not up-regulated by LMIR5-Fc stimulation, LMIR5-Fc was able to induce IL-6 and IL12p40 secretion from BMDCs (Fig. 5B–D). This capacity was not due to endotoxin contamination as heated LMIR5-Fc failed to induce cytokine production (Fig. 5C and D). Not only microbial products but also self-components are able to activate TLR4 signaling. LPS from Gram-negative bacteria has long been known as an exogenous activator of TLR4 (Akira and Takeda, 2004). TLR4 is recently reported to recognize Shiga toxins and mediates the lethal cardiac toxicity of anthrax (Brigotti et al., 2013; Kandadi et al., 2012). Pertussis toxin-induced activation of several signaling molecules is nearly abolished in the absence of TLR4 (Kerfoot et al., 2004). Endogenous activators of TLR4 include S100 proteins, HMGB1, histones, cold-inducible RNA-binding protein, and heat shock proteins (Vogl et al., 2007; Xu et al., 2009, 2011; Yang et al., 2010; Qiang et al., 2013; Quintana and Cohen, 2005). The release of these endogenous activators contributes to septic death (Vogl et al., 2007; Xu et al., 2009; Qiang et al., 2013). In this study, the LMIR5 extracellular domain is identified as another endogenous activator of TLR4. It will be interesting to examine the involvement of other toll-like receptors in addition to TLR2 and TLR4. A single receptor LMIR5 is capable of inducing different activation signals through its extracellular and intracellular domains. For extracellular domain, this study finds that LMIR5 delivers the activation signal through a TLR4-MyD88-NF-B pathway. In addition, FcR␥-associated receptors may be involved in LMIR5-induced IL-12p40 production (see, Fig. 3A). For intracellular domain, the membranous LMIR5 is able to deliver the activation signal through DAP10 and DAP12 in LMIR5-transducing mast cells upon LMIR5specific antibody cross-linking (Yamanishi et al., 2008). However,
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Fig. 4. Interaction between TLR4 and LMIR5. (A) 5 × 104 HB-4 and HB-N were incubated overnight with 10 ng/ml LPS (+), reagent (−), and various concentrations of LMIR5Fc. Reporter cell assay was measured at A630. A630 > 0.3 is considered as positive. Data are representative of at least five independent experiments and represented as mean ± SEM. (B) 240 ng fusion protein and 10 ng/ml LPS samples were heated at 100 ◦ C for 40 min and subjected to HB-4 assay. (C) TLR4-Fc was examined for its binding capacity to LMIR5-expressing cells 2B4. Black histogram is for the control cells without LMIR5. Empty histogram is for LMIR5-expressing 2B4. Data are representative of two independent experiments. The most obvious results are shown. (D) TLR4-Fc, hIgG Fc, medium (negative) or anti-HA antibody (positive) was cross-linked to LMIR5 reporter cells DAP12 + LMIR5+ or control cells DAP12+ for assay of NFAT-GFP activation. (E) TLR4-Fc was examined for its binding capacity to LMIR5 or LMIR4-expressing 2B4 in the presence or absence of 10 g LPS low and 30 g LPS high. (F) TLR4-Fc-biotin conjugate was premixed with or without 10 g LPS for 30 min. The mixture or reagent (negative) was added to ELISA wells containing of coated LMIR5-Fc or LPS. ((G)–(H)) HB-4 was incubated overnight with LPS or LMIR5-Fc in the presence or absence of TLR4-Fc. Data are represented as mean ± SEM (* , P < 0.05).
natural ligands for the membranous LMIR5 have not been identified. Furthermore, the 29-aa cytoplasmic tail of LMIR5 contains the tyrosine-based motif that becomes a docking site for Grb2 upon c-Fyn phosphorylation (Martinez-Barriocanal and Sayos, 2006). 3.4. Examination of NF-B-activating contaminants in LMIR5-Fc samples To exclude the possibility that contaminants were responsible for the inflammatory activity of LMIR5-Fc, the following experiments were performed. Firstly, LMIR5-Fc samples were fractionated using a gel filtration and 14 fractions were collected. The presence of LMIR5-Fc was examined by incubating each fraction with HB-4 cells. Binding of LMIR5-Fc to HB-4 was detected using anti-hIgG Fc-PE conjugate and analyzed by flow cytometry. Seven fractions (4–10) were positive for LMIR5-Fc and
consistently responsible for NF-B activation. As expected, other factions were negative for both LMIR5-Fc and NF-B activation (Fig. 6A and B). Secondly, LMIR5-Fc samples were purified using a protein G. Protein G-binding components were eluted and loaded on the gel filtration. Eighteen fractions were collected for HB-4 and TBRed assays. Ten fractions (3–12) induced NF-B activation in both reporter cells. The other factions were negative. Notably, factions 2 and 13 were weakly positive for HB-4 but negative for TBRed. This inconsistent result might be due to the fact that HB-4 is more sensitive than TBRed (Fig. 6C and D). It was noted that endotoxin was detected at 0.100 ± 0.005 EU/ml for 5 l fraction 7 (Fig. 6A and B) and 0.111 ± 0.017 EU/ml for 5 l fraction 7 (Fig. 6C and D). The same amount of LMIR5-Fc fraction (5 l) was used for TBRed and HB-4 assays. The contribution of contaminated endotoxin could be excluded because this level of endotoxin did not possess inflammatory
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Fig. 5. Binding capacity of LMIR5-Fc to various cells. (A) Binding of LMIR5-Fc was examined for various cell lines, BMDMs, and BMDCs. Black histogram is for hIgG binding. Empty histogram is for LMIR5-Fc binding. (B) BMDCs were stimulated with 240 ng fusion protein or 10 ng/ml LPS for 48 h. Black and empty histograms are unstimulated and stimulated cells, respectively. ((C) and (D)) BMDCs were stimulated with 240 ng fusion protein or 10 ng/ml LPS for 24 h for assay of cytokine production. Heat denaturation was performed at 100 ◦ C for 40 min. Data are represented as mean ± SEM (* , P < 0.05).
activity as previously shown. Thirdly, coated anti-human IgG Fc antibody was used to cross-link LMIR5-Fc to HB-4 cells. Nonspecific or unbound components were washed away before adding HB-4. LMIR4-Fc was used as a negative control. Cross-linking of LMIR5-Fc, but not LMIR4-Fc, could induce NF-B activation
and this was not observed in the wells without antibodies (Fig. 6E). Finally, LMIR5-Fc or LPS samples were incubated with polymyxin B-agarose conjugate (PMB) for 2 h in order to remove LPS. After centrifugation, the supernatant was collected and concentrated before incubation with HB-4. As expected, 5 ng/ml LPS
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Fig. 6. Examination of NF-B-inducing contaminants in LMIR5-Fc samples. ((A) and (B)) LMIR5-Fc samples were loaded on PD MiniTrap G-25 columns and 14 fractions (1–14) were collected. Each fraction was examined for the presence of LMIR5-Fc by HB-4 binding assay and the NF-B activation by HB-4 reporter cell assay. ((C) and (D)) LMIR5-Fc samples were purified using a protein G. Protein G-binding components were eluted and fractioned using a gel filtration as previously mentioned. Eighteen factions were collected and subjected to HB-4 and TBRed assays. (E) Anti-human IgG Fc antibody was coated in PBS on 96-well plate at 4 ◦ C overnight, washed twice with PBS, incubated with fusion protein for 2 h, and washed with PBS, consecutively. HB-4 reporter cell assay was performed. (F) LMIR5-Fc samples were mixed with polymyxin B (PMB) for 2 h at room temperature and the supernatant was concentrated using Ultracel-10 K before addition of HB4. Data are representative of two independent experiments and reported as mean ± SEM.
was totally captured by PMB. The supernatant from PMB-treated LPS samples was unable to induce NF-B activation in HB-4. In contrast to LPS, LMIR5-Fc samples with or without PMB treatment showed a similar inflammatory activity (Fig. 6F). Notably, a new LMIR5-Fcsample was reproduced in HEK 293 and examined for its inflammatory property, which was reproducible in HB-4 (data not shown). Collectively, the contaminants were not the cause of NF-B activation. To our knowledge, the immune receptors that are responsible for the inflammatory activity of the LMIR5 extracellular domain
have not been identified. In this study, we provide the evidence that supports the existence of LMIR5-TLR4 signaling pathway as follows: (i) LMIR5-Fc-induced inflammation is substantially lost in the absence of TLR4, (ii) LMIR5-Fc could induce NF-B activation in TLR4 reporter cells, (iii) a direct interaction between TLR4-Fc and LMIR5-expressing cells could be demonstrated, and (iv) NF-Bactivating contaminants are undetectable from LMIR5-Fc samples. Activation of TLR4 by the LMIR5 extracellular domain may play an important role in the amplification of inflammatory responses. Indeed, the contribution of LMIR5 in sepsis is recently
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demonstrated. LMIR5 deficiency profoundly reduces a systemic cytokine production and a septic mortality in LPS-administered mice (Yamanishi et al., 2012). Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death is decreased by LMIR5-Fc administration (Yamanishi et al., 2012). In this study, LMIR5 extracellular domain amplifies LPS-induced inflammation in vivo and it constitutively activates myeloid cells through TLR4-MyD88-NF-B signaling pathway. Thus, identification of endogenous activator of TLR4 not only advances our understanding of additional inflammatory mediators but will also help in the development of new therapeutic strategies; for example, anti-LMIR5 neutralizing antibodies for treatment of sepsis. A recent study has demonstrated that fusion protein containing antigen of interest fused to the extra domain A from fibronectin, which is an endogenous TLR4 ligand, is highly immunogenic and induces a potent immune responses against the fused antigen (Arribillaga et al., 2013). Indeed, TLR4 activation is critical for the development of potent humoral and cellular responses and it mediates vaccine-induced protective immunity (Lartigue et al., 2009; Higgins et al., 2006; Hutchens et al., 2008; Hwang et al., 2009; Loser et al., 2010). We report here that LMIR5-Fc interacts with several cell types, including antigen-presenting cells, and induces cytokine production from dendritic cells. Therefore, the finding from here may open a new way to use the LMIR5 extracellular domain-based vaccine delivery for vaccination against infectious diseases and cancer. Acknowledgments This work was supported by grant-in-aid for scientific research from Japan Society for the Promotion of Science (JSPS, 11F01107 to S.Y.) and the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government (24390256 to H.H.). V.P. is the recipient of a JSPS fellowship under the FY2011-2013 program of JSPS Postdoctoral Fellowship for Foreign Researcher. References Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511. Arribillaga, L., Durantez, M., Lozano, T., Rudilla, F., Rehberger, F., Casares, N., Villanueva, L., Martinez, M., Gorraiz, M., Borras-Cuesta, F., Sarobe, P., Prieto, J., Lasarte, J.J., 2013. A fusion protein between streptavidin and the endogenous TLR4 ligand EDA targets biotinylated antigens to dendritic cells and induces T cell responses in vivo. BioMed Res. Int. 2013, 864720. Brigotti, M., Carnicelli, D., Arfilli, V., Tamassia, N., Borsetti, F., Fabbri, E., Tazzari, P.L., Ricci, F., Pagliaro, P., Spisni, E., Cassatella, M.A., 2013. Identification of TLR4 as the receptor that recognizes Shiga toxins in human neutrophils. J. Immunol. 191, 4748–4758. Ding, Q., Yeung, M., Camirand, G., Zeng, Q., Akiba, H., Yagita, H., Chalasani, G., Sayegh, M.H., Najafian, N., Rothstein, D.M., 2011. Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice. J. Clin. Invest. 121, 3645–3656. Gasiorowski, R.E., Ju, X., Hart, D.N., Clark, G.J., 2013. CD300 molecule regulation of human dendritic cell functions. Immunol. Lett. 149, 93–100. Hara, H., Ishihara, C., Takeuchi, A., Imanishi, T., Xue, L., Morris, S.W., Inui, M., Takai, T., Shibuya, A., Saijo, S., Iwakura, Y., Ohno, N., Koseki, H., Yoshida, H., Penninger, J.M., Saito, T., 2007. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat. Immunol. 8, 619–629. Higgins, S.C., Jarnicki, A.G., Lavelle, E.C., Mills, K.H., 2006. TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells. J. Immunol. 177, 7980–7989. Hutchens, M.A., Luker, K.E., Sonstein, J., Nunez, G., Curtis, J.L., Luker, G.D., 2008. Protective effect of Toll-like receptor 4 in pulmonary vaccinia infection. PLoS Pathog. 4, e1000153.
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