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The Journal of Immunology, 2007, 178: 765-770.
Copyright © 2007 by The American Association of Immunologists, Inc.

Dual Assemblies of an Activating Immune Receptor, MAIR-II, with ITAM-Bearing Adapters DAP12 and FcR{gamma} Chain on Peritoneal Macrophages1

Chigusa Nakahashi*,{dagger}, Satoko Tahara-Hanaoka*, Naoya Totsuka*, Yasushi Okoshi*, Toshiyuki Takai{ddagger},§, Nobuhiro Ohkohchi{dagger}, Shin-ichiro Honda*, Kazuko Shibuya* and Akira Shibuya2,*

* Department of Immunology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences and {dagger} Department of Surgery, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan; {ddagger} Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan; and § Core Research for Evolutional, Science and Technology, Kawaguchi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Certain activating immune receptors expressed on myeloid cells noncovalently associate with either DAP12 or Fc{epsilon}RI{gamma} (FcR{gamma} chain), the ITAM-bearing transmembrane adapter proteins. An activating receptor, myeloid-associated Ig-like receptor (MAIR) II, is expressed on a subset of B cells and macrophages in the spleen and peritoneal cavity of mice and associates with DAP12 in these cells. However, we demonstrate here that cross-linking MAIR-II with mAb induced secretion of a significant amount of the inflammatory cytokines TNF-{alpha} and IL-6 from DAP12–/– as well as wild-type (WT) peritoneal macrophages. We show that MAIR-II associates with not only DAP12 but also FcR{gamma} chain homodimers in peritoneal macrophages. LPS enhanced the FcR{gamma} chain expression and FcR{gamma} chain-dependent cell surface expression of MAIR-II and had additive effects on MAIR-II-mediated inflammatory cytokine secretion from peritoneal macrophages. The lysine residue in the transmembrane region of MAIR-II was involved in the association with FcR{gamma} chain as well as DAP12. Our findings present the first case of an activating receptor that uses either DAP12 or FcR{gamma} chain as a signaling adapter. The FcR{gamma} chain may provide cooperation with and/or compensation for DAP12 in MAIR-II-mediated inflammatory responses by peritoneal macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antigen recognition by the TCR and the BCR initiates signals for activation, cell differentiations, and effector function via the noncovalently associating transmembrane (TM)3 adapter molecules CD3{gamma}, CD3{delta}, CD3{epsilon}, and {zeta} subunits of the TCR and {alpha} and beta subunits of BCR, respectively. These adapter proteins bear ITAMs in their cytoplasmic regions, which are tyrosine phosphorylated by src family protein tyrosine kinases (PTKs) after Ag recognition, resulting in activation of downstream signaling molecules.

Fc receptors, including the high-affinity IgE receptor (Fc{epsilon}RI), the IgG receptors (Fc{gamma}RI, III, and IV), and the receptor for IgA (Fc{alpha}RI; CD89), also associate with the ITAM-bearing adapter Fc{epsilon}RI{gamma} (FcR{gamma} chain) that is responsible for FcR-mediated cell activation (1). The FcR{gamma} chain is also an adapter that associates with several myeloid cell-specific receptors, including mouse PIR-A (2, 3) and human ILT-1 (4) and ILT-7 (5).

DAP12 (6), also named killer cell-activating receptor-associated protein (7), is another family member of ITAM-containing TM adapter proteins. DAP12 is expressed in NK cells and associates with several activating NK receptors (reviewed in Ref. 8), and is also expressed in myeloid cells. Several myeloid cell-specific receptors that associate with DAP12 have been reported, including human and mouse triggering receptor expressed on myeloid cell (TREM) 1, TREM-2, myeloid DAP12-associating lectin 1 (MDL), and paired Ig-like receptor (PILR) beta, human signal regulatory protein beta, and mouse TREM-3 and CD200RLa (reviewed in Ref. 9).

We have recently identified paired activating and inhibitory Ig-like receptors, designated myeloid-associated Ig-like receptor (MAIR) I and MAIR IIa (MAIR-II), whose extracellular domains are highly conserved with each other (10). MAIR-I, also named LMIR-1 (11) or CMRF-35-like molecule 8 (12), contains ITIMs in its cytoplasmic domain and inhibits IgE-mediated degranulation of mast cells (10, 13). In contrast, MAIR-II, also named LMIR-II (11), CMRF-35-like molecule 4 (12), or DIgR1 (14), is expressed on subsets of peritoneal macrophages and B cells and associates with DAP12, which mediates activating signals, resulting in inflammatory cytokine secretion from macrophages (10). By screening a macrophage cDNA library and the mouse genome, we and another group found that MAIR-I and MAIR-II are members of a multigene family consisting of at least nine genes on a small segment of mouse chromosome 11 (our unpublished observation and Ref. 12). MAIR family genes are most similar to the human CMRF (CD300) family, which is located on human chromosome 17 (15), syntenic region of mouse chromosome 11. MAIR-I and MAIR-II were assigned to be CD300a and CD300d, respectively.

The family of TM adapter proteins described above are all characterized by a small extracellular region, an aspartic acid (D), a negatively charged amino acid, in the TM domain, and a cytoplasmic domain that contains one or more ITAM (16). Receptors that associate with these TM adapters have a positively charged amino acid, such as lysine (K) or arginine (R), in their TM region and possess short cytoplasmic regions without any signaling motif. Certain activating immune receptors expressed on lymphoid and myeloid cells selectively associate either with DAP12 or FcR{gamma}; however, there is not prior evidence that a receptor can pair with both of these adapter proteins. We demonstrate here that MAIR-II physically and functionally associates with not only DAP12 but also FcR{gamma} chain in peritoneal macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6J mice were purchased from Clea. DAP12-deficient and FcR{gamma} chain-deficient mice were previously described (17, 18). These mutant mice were backcrossed to C57BL/6J mice for nine generations. All experiments were performed according to the guidelines of the animal ethics committee of the University of Tsukuba Animal Research Center.

Cells and transfectant

Mouse peritoneal macrophages were collected by peritoneal lavage with 5 ml of PBS from 8- to 12-wk-old mice before or 3 days after i.p. injection of 2 ml of 4% sterile thioglycolate. B cells and macrophages from the spleen and peritoneal cavity were purified by using a MACS selection system with biotin-conjugated anti-B220 mAb and streptavidin-coated MicroBeads (Miltenyi Biotec) and anti-CD11b MicroBeads, respectively. The purities of B220+ B cells and CD11b+ macrophages were >95 and 88%, respectively, as determined by flow cytometry. Bone marrow cells were cultured in the presence of M-CSF (10 ng/ml) for 4 days and analyzed by flow cytometry, showing that >80% of cultured cells were CD11b+ macrophages. RBL-2H3 transfectant stably expressing MAIR-II-IRES GFP and DAP12 tagged with Flag or the N terminus and the BW5147 transfectant stably expressing the FcR{gamma} chain tagged with Flag at the N terminus were as described previously (10). The 2B4 transfectant stably expressing N terminus Flag-tagged DAP12 was provided by H. Arase (Osaka University, Osaka, Japan). These BW5147 and 2B4 transfectants were further transfected with WT or mutated MAIR-II, which contained a codon for A195 (GCG) instead of K195 (AAG), as described elsewhere (10).

Antibodies

Control rat IgGs, anti-CD11b, and anti-CD16/CD32 2.4G2 mAbs were purchased from BD Biosciences; anti-phosphotyrosine (4G10) and anti Fc{epsilon} RI{gamma} (FcR{gamma} chain) mAbs were purchased from Upstate Biotechnology; anti-p42/44 ERK and anti-phospho-p42/44 ERK were purchased from Cell Signaling; and rabbit anti-Flag polyclonal Ab was purchased from Sigma-Aldrich. Anti-mouse DAP12 polyclonal Ab was a gift from M. Ono (Tohoku University School of Medicine, Sendai, Japan). TX52 (anti-MAIR-II) mAb was generated in our laboratory as described previously (10). F(ab')2 of TX52 mAb were prepared, as described previously (10). Purity of the F(ab')2 were determined by SDS-PAGE. To exclude LPS contamination, Abs were treated with polymixin B (10 µg/ml; Sigma-Aldrich) before use.

Biochemistry

Cells were lysed in 1% digitonin (Calbiochem), 0.12% Triton X-100 (Sigma-Aldrich), 150 mM NaCl, 20 mM triethanolamine, protease inhibitors (1 mM PMSF and 10 U/ml aprotinin), and phosphatase inhibitors (1 mM EGTA, 10 mM NaF, 1 mM Na4P2O7, 0.1 mM beta-glycerophosphate, and 1 mM Na3VO4). After overnight preclearing with 2.4G2mAb-conjugated agarose beads (ImmunoPure Immobilized Protein L; Pierce), immunoprecipitations and/or immunoblotting experiments were performed as described previously (10). For tyrosine phosphorylation studies, cells were stimulated with F(ab')2 of control rat IgG or anti-MAIR-II (TX52), followed by cross-linking with F(ab')2 of rabbit anti-rat IgG (Southern Biotechnology Associates). For stripping and reblotting, Restore Western Blot Stripping Buffer (Pierce) was used according to the manufacturer’s instruction.

Stimulation of macrophages and cytokine measurement

Peritoneal macrophages (5 x 105/well) were stimulated for 24 h in 96-well flat-bottom plates coated with F(ab')2 of control rat IgG or anti-MAIR-II (TX52). The amounts of TNF-{alpha} and IL-6 were measured by ELISA (BD Pharmingen).

Statistics

Statistical analyses were performed using the unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MAIR-II mediates proinflammatory cytokine production from DAP12-deficient peritoneal macrophages

We previously demonstrated that MAIR-II was coimmunoprecipitated with DAP12, but not FcR{gamma} chain, in primary spleen cells (10). Because MAIR-II was expressed on subsets of B cells and macrophages in spleen, we first examined whether MAIR-II associated with DAP12 either in B cells, macrophages or both. We demonstrate that MAIR-II was coimmunoprecipitated with DAP12 in both B220+ B cells and CD11b+ macrophages purified from spleen (Fig. 1A). Additional experiments showed similar results in B220+ B cells and CD11b+ macrophages purified from peritoneal exudate cells (PEC) (Fig. 1A), suggesting that DAP12 is a physiological partner of MAIR-II in B cells and macrophages in the spleen and peritoneal cavity. In fact, whereas splenic macrophages from WT mice produced TNF-{alpha} when stimulated with F(ab')2 of anti-MAIR-II mAb, DAP12–/– macrophages did not respond to the Ab stimulation for TNF-{alpha} secretion (Fig. 2), indicating that DAP12 is required for MAIR-II-mediated activating signal in splenic macrophages. Unexpectedly, however, cross-linking MAIR-II produced significant amount of both TNF-{alpha} and IL-6 from DAP12–/– as well as WT peritoneal macrophages (Fig. 2). These results suggest that another MAIR-II-associating adapter molecule, in addition to DAP12, may mediate activating signals specifically in peritoneal macrophages.


Figure 1
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  		FIGURE 1. MAIR-II associates not only with DAP12 but also with the FcR{gamma} chain in peritoneal and bone marrow-derived cultured macrophages. A, B cells (B) and macrophages (M{phi}) purified from the spleen (Sp) and PEC or bone marrow (BM)-derived cultured macrophages were cultured overnight in the presence or absence of LPS (100 ng/ml). Cells were lysed in 1% digitonin buffer, immunoprecipitated (IP) with control Ig (cIg) or anti-MAIR-II, and immunoblotted with anti-DAP12, anti-FcR{gamma} chain, or anti-MAIR-II. Relative density of each signal was measured by densitometry. B, Three x 106 B cells (B) and macrophages (M{phi}) purified from the spleen and PEC from WT mice were stimulated or not with LPS (100 ng/ml) overnight, lysed with 1% Nonidet P-40 buffer, and immunoblotted with anti-DAP12 or the anti-FcR{gamma} chain. Data are representative of three independent experiments.

 

Figure 2
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  		FIGURE 2. MAIR-II mediates cytokine production from DAP12–/– as well as WT peritoneal macrophages. Macrophages (M{phi}) from the spleen and PEC derived from WT and DAP12–/– mice were stimulated with plate-coated F(ab')2 of anti-MAIR-II or isotype-matched control Ig for 68 and 24 h, respectively. Culture supernatants were harvested, and TNF-{alpha} and IL-6 concentrations were measured by ELISA. Data are the mean ± SD and are representative of three independent experiments.

 
MAIR-II associates with FcR{gamma} chain as well as DAP12 in peritoneal macrophages

We then performed immunoblotting analyses of B cells and macrophages from the spleen and PEC, demonstrating that both cell types express FcR{gamma} chain as well as DAP12 (Fig. 1B), although macrophages expressed the adaptors much more than B cells, consistent with our previous report (10). These results led us to investigate whether MAIR-II also associates with FcR{gamma} chain in these cells from the spleen and PEC. We observed coimmunoprecipitation of MAIR-II with FcR{gamma} chain in lysates of peritoneal macrophages and bone marrow-derived cultured macrophages (Fig. 1A). Moreover, it was enhanced after culture of peritoneal macrophages in the presence of LPS (Fig. 1A), suggesting that not only DAP12 but also FcR{gamma} chain associates with MAIR-II in peritoneal macrophages. In contrast, association of MAIR-II with FcR{gamma} chain was not detected in splenic B cells and macrophages and peritoneal B cells, even after culture in the presence of LPS, although these cells expressed the FcR{gamma} chain as well as DAP12 (Fig. 1B).

MAIR-II mediates activating signals through both DAP12 and the FcR{gamma} chain in peritoneal macrophages

The signaling pathways downstream of DAP12 and the FcR{gamma} chain have well been characterized; both adapter proteins activate a common pathway, including stimulation of the Src family PTKs, which phosphorylate ITAMs on tandem tyrosine residues, thereby leading to the recruitment and activation of PTKs of the Syk family and subsequent downstream signal transduction (9, 19). ERK is a tyrosine-phosphorylated signaling molecule downstream of both DAP12 and the FcR{gamma} chain. To determine whether either DAP12, the FcR{gamma} chain, or both mediate signals after cross-linking MAIR-II, we stimulated peritoneal macrophages derived from WT, DAP12–/–, FcR{gamma}–/–, and double-deficient mice (DKO) with F(ab')2 of the anti-MAIR-II mAb. Immunoblotting studies demonstrated that ERK 1/2 were tyrosine phosphorylated after stimulation with anti-MAIR-II in peritoneal macrophages from WT, DAP12–/–, and FcR{gamma}–/–, but not DKO, mice (Fig. 3A). Consistent with these signaling events, cross-linking MAIR-II also induced TNF-{alpha} and IL-6 productions from WT, DAP12–/–, and FcR{gamma}–/– peritoneal macrophages, but not from DKO mice (Fig. 3B). Taken together, these results suggest that both DAP12 and the FcR{gamma} chain are responsible for MAIR-II-mediated activating signals in peritoneal macrophages.


Figure 3
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  		FIGURE 3. Both DAP12 and the FcR{gamma} chain are involved in MAIR-II-mediated signaling in peritoneal macrophages Peritoneal macrophages from the mice indicated were stimulated or not with plate-coated F(ab')2 of anti-MAIR-II or isotype-matched control Ig for 5 min (A) or for 24 h (B). The lysates were immunoblotted with anti-phospho-p42/44 ERK or anti-p42/44 ERK (A). Culture supernatants were harvested and TNF-{alpha} and IL-6 concentrations were measured by ELISA (B). Data are the mean ± SD and are representative of five independent experiments.

 
LPS enhances FcR{gamma} chain-dependent cell surface expression of MAIR-II in peritoneal macrophages

We demonstrated that LPS enhanced the association of MAIR-II with the FcR{gamma} chain, but not DAP12 (Fig. 1), suggesting that LPS up-regulates FcR{gamma} chain expression, accelerating the complex formation and cell surface expression of MAIR-II and the FcR{gamma} chain. In fact, we found that overnight culture with LPS induced up-regulation of FcR{gamma} chain expression in WT peritoneal macrophages (Fig. 4A). In contrast, DAP12 expression was not affected by LPS in peritoneal macrophages (Fig. 4A), although it was enhanced after culture in the presence of LPS in spleen B cells (10). Moreover, whereas peritoneal macrophages derived from WT, DAP12–/–, FcR{gamma} –/–, and DKO mice expressed comparable amounts of MAIR-II on the cell surface, to culture in the presence of LPS up-regulated cell surface expression of MAIR-II in WT and DAP12–/–, but not FcR{gamma}–/– and DKO, peritoneal macrophages (Fig. 4B). Together, these results suggested that LPS enhanced FcR{gamma} chain-dependent cell surface expression of MAIR-II. In contrast with LPS, both a Th1 cytokine IFN-{gamma} and a Th2 cytokine IL-4, which are thought to induce classical and alternative activation of macrophages, respectively (20), did not have any effects on cell surface expression of MAIR-II on peritoneal macrophages (data not shown).


Figure 4
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  		FIGURE 4. LPS has additive effects on MAIR-II-mediated inflammatory cytokine secretion from peritoneal macrophages. A, Three x 106 peritoneal macrophages from WT mice were stimulated or not with LPS (100 ng/ml) for 48 h, lysed, and immunoblotted with anti-DAP12 or the anti-FcR{gamma} chain (left). Relative density of each signal was measured by densitometry (right). B, PEC from the mice indicated were cultured in the presence (LPS (+)) or absence of LPS (100 ng/ml; LPS (–)) for 48 h and then stained with Alexa 647-conjugated F(ab')2 of anti-MAIR-II (open histogram) or isotype-matched control Ig (shaded histogram), in combination with anti-CD11b and anti-F4/80. CD11b+ and F4/80+ cells were analyzed by flow cytometry. C, Peritoneal macrophages from the mice indicated were cultured overnight in the presence of LPS (100 ng/ml) with plate-coated F(ab')2 of anti-MAIR-II or isotype-matched control Ig for 24 h. Culture supernatants were harvested and the TNF-{alpha} concentration was measured by ELISA. Data are representative of four to five experiments.

 
We next examined whether LPS had any effect on MAIR-II-mediated signaling in peritoneal macrophages as a result of enhancement of cell surface expression of MAIR-II. To address this question, peritoneal macrophages were cultured in the presence of LPS and then stimulated with F(ab')2 of the anti-MAIR-II mAb. Stimulation with LPS alone significantly induced TNF-{alpha} secretion from WT, DAP12–/–, FcR{gamma}–/–, and DKO peritoneal macrophages. Cross-linking MAIR-II further increased TNF-{alpha} secretion from WT and DAP12–/–, but not from FcR{gamma}–/– or DKO, peritoneal macrophages that had been stimulated with LPS (Fig. 4C), indicating that LPS-induced, FcR{gamma} chain-dependent MAIR-II expression could amplify inflammatory responses by peritoneal macrophages.

MAIR-II associates with DAP12 or the FcR{gamma} chain homodimers

To determine whether DAP12 and FcR{gamma} can form heterodimers, we transfected a rat basophilic leukemia, RBL-2H3, which does not express MAIR-II and DAP12, but does the FcR{gamma} chain (21), with a MAIR-II-IRES-GFP construct along with Flag-tagged DAP12. Experiments were performed to examine whether both DAP12 and the FcR{gamma} chain were coimmunoprecipitated with MAIR-II by using anti-Flag or anti-FcR{gamma} chain mAbs. As demonstrated in Fig. 5, MAIR-II was coimmunoprecipitated with either homodimers of DAP12 or the FcR{gamma} chain. Heterodimers between DAP12 and FcR{gamma} were not observed.


Figure 5
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  		FIGURE 5. MAIR-II associates with the FcR{gamma} chain or DAP12 homodimers RBL-2H3 that constitutively expresses the FcR{gamma} chain was transfected with a MAIR-II-IRES-GFP construct along with Flag-tagged DAP12 in a retrovirus vector. Two days after infection, MAIR-II-expressing cells were sorted, based on GFP expression, lysed, and immunoprecipitated (IP) with control Ig (cIg), anti-Flag, or anti-FcR{gamma} chain, and immunoblotted (IB) with anti-MAIR-II, anti-Flag, or anti-FcR{gamma} chain under reducing and nonreducing conditions.

 
The lysine residue in the TM region of MAIR-II is involved in association with both DAP12 and the FcR{gamma} chain

Because MAIR-II contains a lysine TM region, but not an arginine in the TM region, a question was raised whether the lysine residue is involved in the association with the FcR{gamma} chain as well as DAP12. We generated WT and mutant MAIR-II cDNA, which encoded MAIR-II containing an alanine instead of the lysine in the TM region. These cDNA were introduced into the 2B4 and BW5147 mouse T cell transfectants stably expressing Flag-tagged DAP12 and Flag-tagged FcR{gamma} chain, respectively. As demonstrated in Fig. 6A, whereas introducing WT MAIR-II into Flag-tagged DAP12-expressing 2B4 induced cell surface expression of DAP12, introduction of the mutant MAIR-II did not. Similarly, in contrast to WT MAIR-II, mutant MAIR-II also did not induce cell surface expression of the FcR{gamma} chain. Moreover, the FcR{gamma} chain as well as DAP12 were coimmunoprecipitated with WT, but not mutant MAIR-II (Fig. 6B). Of note, mutant MAIR-II was expressed higher than WT MAIR-II on the cell surface of both 2B4 and BW5147 transfectants (Fig. 6A). This difference might be dependent on the retrovirus transduction efficiency because the total amount of mutant MAIR-II was higher than that of WT MAIR-II in each transfectant (Fig. 6B). Together, these results indicated that the lysine residue in the TM region of MAIR-II was involved in the association with the FcR{gamma} chain as well as DAP12.


Figure 6
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  		FIGURE 6. The lysine residue of MAIR-II is involved in association of the FcR{gamma} chain as well as DAP12 homodimers. 2B4 and BW5147 transfectants stably expressing Flag-tagged DAP12 (Flag-DAP12/2B4) and Flag-tagged FcR{gamma} chain (Flag-FcR{gamma}/BW5147), respectively, were transfected with WT or mutated (K195A) MAIR-II in a retrovirus vector. Two days after infection, cells were stained with Alexa 647-conjugated F(ab')2 of anti-MAIR-II and biotin-conjugated anti-Flag mAbs, followed by FITC-conjugated streptavidin, and analyzed by flow cytometry (A). These cells were also lysed, immunoprecipitated with anti-MAIR-II, and immunoblotted (IB) with anti-Flag or anti-MAIR-II (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have presented here the first case of an activating receptor that uses either DAP12 or the FcR{gamma} chain as a signaling adapter. At present, it is unclear whether any other activating receptors on myeloid or lymphoid cells have a similar property. Although NKG2D noncovalently associates with DAP10 in CD8+ T cells, a mouse NKG2D isoform pairs with either DAP10 or DAP12 in activated mouse NK cells (22, 23). Unlike DAP12, DAP10 has a YxxM motif in the cytoplasmic domain, which recruits the p85 subunit of the PI3K, thereby providing a different function from DAP12. On the contrary, DAP12 and the FcR{gamma} chain seem to initiate the same signal transduction pathways by recruiting Syk and/or ZAP70 (19). Because LPS enhanced FcR{gamma} chain expression and FcR{gamma} chain-dependent cell surface expression of MAIR-II on peritoneal macrophages, the FcR{gamma} chain may provide cooperation with and/or compensation for DAP12 in MAIR-II-mediated inflammatory responses by peritoneal macrophages. This may be reasonable, because peritoneal macrophages are required to be activated more quickly and sufficiently for innate immunity than spleen macrophages or B cells that are involved in adaptive immunity or link between innate and adaptive immunity.

Lanier and colleagues (24) demonstrated that, although cross-linking of DAP12-associated receptors on macrophages can initiate cytokine production in some circumstances, these same cytokine secretions were increased from DAP12–/– macrophages when they are induced by TLR under different conditions. Very recently, triggering receptor expressed on myeloid cells (TREM-2) has been found to mediate inhibition of cytokine secretion induced by TLR signaling from macrophages through DAP12 (25, 26). In our study, it was interesting that TNF-{alpha} secretion induced by LPS seemed to be decreased, rather than increased, from FcR{gamma}–/– peritoneal macrophages upon cross-linking with MAIR-II (Fig. 4C), suggesting a possibility that MAIR-II also inhibited LPS-mediated TNF-{alpha} secretion through DAP12. Further analysis is required to determine this possibility.

Several activating receptors, including CD16, NKp46, and NKp30, associate with heterodimers of FcR{gamma} and {zeta}, as well as the homodimer of these adapter proteins (27), suggesting that FcR{gamma} and {zeta} are redundant. We have demonstrated that MAIR-II associated with either the homodimers of DAP12 or FcR{gamma}, but not the heterodimers of DAP12 and FcR{gamma}. These results were reasonable, because, unlike FcR{gamma} and {zeta}, DAP12 and the FcR{gamma} chain have the extracellular cysteines and the TM aspartic acid residues at quite different positions from each other, which may prevent the heterodimer formation by these two adapter proteins. Specifically, DAP12 has two cysteine residues per chain in the membrane-proximal extracellular region, which may be involved in dimer formation, whereas the FcR{gamma} chain has one cysteine per chain in the TM domain (16). Moreover, the negatively charged residue aspartic acid, which is involved in physical association with activating receptors containing a positively charged amino acid residue in the TM domain, is located close to the center of the TM region of DAP12, but it is in the N-terminal segment of the TM domain of the FcR{gamma} chain (16). DAP12, which has an aspartic acid close to the center of the TM region, preferentially associates with immune receptors, including NK receptors and activating myeloid receptors, which bear a charged amino acid residue (usually lysine) at the center, rather than at the N-terminal segment, of the TM domain (16). In contrast, FcR{gamma} chain has an aspartic acid in the N-terminal segment of the TM region and noncovalently associates with immune receptors, including several Fc receptors, and others, which bear a charged amino acid residue (often arginine) also at the N-terminal segment of the TM domain. Although the lysine residue in the TM region of MAIR-II is involved in the association of MAIR-II with both DAP12 and the FcR{gamma} chain, it cannot be concluded that the molecular and structural characteristics of the assemblies of MAIR-II with the FcR{gamma} chain are similar to those with DAP12, because not only a charged amino acid but also the orientation of receptors toward TM adaptors play an important role in association of receptors with adaptors (28).

We have demonstrated that, although MAIR-II associated with either DAP12 or the FcR{gamma} chain on peritoneal and bone marrow-derived cultured macrophages, it did with DAP12 alone on macrophages from the spleen. Macrophages are widely distributed in tissues and have marked phenotypic heterogeneity, which are likely to result from developmental signals encountered within individual tissue sites. In addition, each macrophage also acquires new functional capacities in response to stimuli encountered in the tissue microenvironment. These heterogeneity of tissue macrophages might lead to the functional difference also in the adapter usage of MAIR-II. In contrast, B cells from the peritoneal cavity as well as the spleen expressed MAIR-II that associated with DAP12 alone, probably because B cells may have little phenotypic heterogeneity between tissues. Future studies are required to clarify the molecular basis for the assembly of both adapters by MAIR-II.


   Acknowledgments
 
We thank Lewis Lanier for critical reading of this manuscript and Yurika Soeda for secretarial assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This research was supported in part by the grants provided by the Ministry of Education, Science and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Akira Shibuya. E-mail address: ashibuya{at}md.tsukuba.ac.jp Back

3 Abbreviations used in this paper: TM, transmembrane; PTK, protein tyrosine kinase; MAIR, myeloid-associated Ig-like receptor; WT, wild type; PEC, peritoneal exudate cell; TREM, triggering receptor expressed on myeloid cell. Back

Received for publication July 31, 2006. Accepted for publication November 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Nimmerjahn, F., J. V. Ravetch. 2006. Fc{gamma} receptors: old friends and new family members. Immunity 24: 19-28. [Medline]
  2. Kubagawa, H., P. D. Burrows, M. D. Cooper. 1997. A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells. Proc. Natl. Acad. Sci. USA 94: 5261-5266. [Abstract/Free Full Text]
  3. Hayami, K., D. Fukuta, Y. Nishikawa, Y. Yamashita, M. Inui, Y. Ohyama, M. Hikida, H. Ohmori, T. Takai. 1997. Molecular cloning of a novel murine cell-surface glycoprotein homologous to killer cell inhibitory receptors. J. Biol. Chem. 272: 7320-7327. [Abstract/Free Full Text]
  4. Nakajima, H., J. Samaridis, L. Angman, M. Colonna. 1999. Human myeloid cells express an activating ILT receptor (ILT1) that associates with Fc receptor {gamma}-chain. J. Immunol. 162: 5-8. [Abstract/Free Full Text]
  5. Cao, W., D. B. Rosen, T. Ito, L. Bover, M. Bao, G. Watanabe, Z. Yao, L. Zhang, L. L. Lanier, Y. J. Liu. 2006. Plasmacytoid dendritic cell-specific receptor ILT7-Fcvar{epsilon}RI{gamma} inhibits Toll-like receptor-induced interferon production. J. Exp. Med. 203: 1399-1405. [Abstract/Free Full Text]
  6. Lanier, L. L., B. C. Corliss, J. Wu, C. Leong, J. H. Phillips. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391: 703-707. [Medline]
  7. Tomasello, E., L. Olcese, F. Vely, C. Geourgeon, M. Blery, A. Moqrich, D. Gautheret, M. Djabali, M. G. Mattei, E. Vivier. 1998. Gene structure, expression pattern, and biological activity of mouse killer cell activating receptor-associated protein (KARAP)/DAP-12. J. Biol. Chem. 273: 34115-34119. [Abstract/Free Full Text]
  8. Lanier, L. L.. 2005. NK cell recognition. Annu. Rev. Immunol. 23: 225-274. [Medline]
  9. Lanier, L. L., A. B. Bakker. 2000. The ITAM-bearing transmembrane adaptor DAP12 in lymphoid and myeloid cell function. Immunol. Today 21: 611-614. [Medline]
  10. Yotsumoto, K., Y. Okoshi, K. Shibuya, S. Yamazaki, S. Tahara-Hanaoka, S. Honda, M. Osawa, A. Kuroiwa, Y. Matsuda, D. G. Tenen, et al 2003. Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation. J. Exp. Med. 198: 223-233. [Abstract/Free Full Text]
  11. Kumagai, H., T. Oki, K. Tamitsu, S. Z. Feng, M. Ono, H. Nakajima, Y. C. Bao, Y. Kawakami, K. Nagayoshi, N. G. Copeland, et al 2003. Identification and characterization of a new pair of immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrow-derived mast cells. Biochem. Biophys. Res. Commun. 307: 719-729. [Medline]
  12. Chung, D. H., M. B. Humphrey, M. C. Nakamura, D. G. Ginzinger, W. E. Seaman, M. R. Daws. 2003. CMRF-35-like molecule-1, a novel mouse myeloid receptor, can inhibit osteoclast formation. J. Immunol. 171: 6541-6548. [Abstract/Free Full Text]
  13. Okoshi, Y., S. Tahara-Hanaoka, C. Nakahashi, S. Honda, A. Miyamoto, H. Kojima, T. Nagasawa, K. Shibuya, A. Shibuya. 2005. Requirement of the tyrosines at residues 258 and 270 of MAIR-I in inhibitory effect on degranulation from basophilic leukemia RBL-2H3. Int. Immunol. 17: 65-72. [Abstract/Free Full Text]
  14. Luo, K., W. Zhang, L. Sui, N. Li, M. Zhang, X. Ma, L. Zhang, X. Cao. 2001. DIgR1, a novel membrane receptor of the immunoglobulin gene superfamily, is preferentially expressed by antigen-presenting cells. Biochem. Biophys. Res. Commun. 287: 35-41. [Medline]
  15. Clark, G. J., B. Cooper, S. Fitzpatrick, B. J. Green, D. N. Hart. 2001. The gene encoding the immunoregulatory signaling molecule CMRF-35A localized to human chromosome 17 in close proximity to other members of the CMRF-35 family. Tissue Antigens 57: 415-423. [Medline]
  16. Feng, J., D. Garrity, M. E. Call, H. Moffett, K. W. Wucherpfennig. 2005. Convergence on a distinctive assembly mechanism by unrelated families of activating immune receptors. Immunity 22: 427-438. [Medline]
  17. Kaifu, T., J. Nakahara, M. Inui, K. Mishima, T. Momiyama, M. Kaji, A. Sugahara, H. Koito, A. Ujike-Asai, A. Nakamura, et al 2003. Osteopetrosis and thalamic hypomyelinosis with synaptic degeneration in DAP12-deficient mice. J. Clin. Invest. 111: 323-332. [Medline]
  18. Takai, T., M. Li, D. Sylvestre, R. Clynes, J. V. Ravetch. 1994. FcR {gamma} chain deletion results in pleiotrophic effector cell defects. Cell 76: 519-529. [Medline]
  19. Vivier, E., J. A. Nunes, F. Vely. 2004. Natural killer cell signaling pathways. Science 306: 1517-1519. [Abstract/Free Full Text]
  20. Gordon, S.. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3: 23-35. [Medline]
  21. Tomasello, E., C. Cant, H. J. Buhring, F. Vely, P. Andre, M. Seiffert, A. Ullrich, E. Vivier. 2000. Association of signal-regulatory proteins beta with KARAP/DAP-12. Eur. J. Immunol. 30: 2147-2156. [Medline]
  22. Diefenbach, A., E. Tomasello, M. Lucas, A. M. Jamieson, J. K. Hsia, E. Vivier, D. H. Raulet. 2002. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D. Nat. Immunol. 3: 1142-1149. [Medline]
  23. Gilfillan, S., E. L. Ho, M. Cella, W. M. Yokoyama, M. Colonna. 2002. NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation. Nat. Immunol. 3: 1150-1155. [Medline]
  24. Hamerman, J. A., N. K. Tchao, C. A. Lowell, L. L. Lanier. 2005. Enhanced Toll-like receptor responses in the absence of signaling adaptor DAP12. Nat. Immunol. 6: 579-586. [Medline]
  25. Hamerman, J. A., J. R. Jarjoura, M. B. Humphrey, M. C. Nakamura, W. E. Seaman, L. L. Lanier. 2006. Cutting edge: inhibition of TLR and FcR responses in macrophages by triggering receptor expressed on myeloid cells (TREM)-2 and DAP12. J. Immunol. 177: 2051-2055. [Abstract/Free Full Text]
  26. Turnbull, I. R., S. Gilfillan, M. Cella, T. Aoshi, M. Miller, L. Piccio, M. Hernandez, M. Colonna. 2006. Cutting edge: TREM-2 attenuates macrophage activation. J. Immunol. 177: 3520-3524. [Abstract/Free Full Text]
  27. Lanier, L. L.. 2001. On guard–activating NK cell receptors. Nat. Immunol. 2: 23-27. [Medline]
  28. Bakema, J. E., S. de Haij, C. F. den Hartog-Jager, J. Bakker, G. Vidarsson, M. van Egmond, J. G. van de Winkel, J. H. Leusen. 2006. Signaling through mutants of the IgA Receptor CD89 and consequences for Fc receptor {gamma}-chain interaction. J. Immunol. 176: 3603-3610. [Abstract/Free Full Text]



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