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Originally published online as doi:10.1189/jlb.0508329 on December 12, 2008

Published online before print December 12, 2008
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(Journal of Leukocyte Biology. 2009;85:508-517.)
© 2009 by Society for Leukocyte Biology

Expression and functional role of MDL-1 (CLEC5A) in mouse myeloid lineage cells

Naoko Aoki*,1, Yuka Kimura*, Shoji Kimura{dagger}, Toshihiro Nagato*, Makoto Azumi*, Hiroya Kobayashi*, Keisuke Sato* and Masatoshi Tateno*

* Department of Pathology and
{dagger} School of Nursing, Asahikawa Medical College, Asahikawa, Japan

1 Correspondence: Department of Pathology, Asahikawa Medical College, Midorigaoka-higashi 2-1-1-1, Asahikawa, Japan. E-mail: aoq{at}asahikawa-med.ac.jp


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ABSTRACT
 
Myeloid DNAX activation protein 12 (DAP12)-associating lectin-1 (MDL-1), also known as C-type lectin domain family 5, member A, is a type II transmembrane protein belonging to the C-type lectin family and associates with DAP12 (also called KARAP or TYROBP). It has been reported that two isoforms of MDL-1—long form (MDL-1L) and short form (MDL-1S)—exist in mice. Previously, we observed the marked induction of MDL-1 mRNA expression during the pulmonary mycobacterial infection in mice. The data suggested that the MDL-1-expressing cells were involved in immune responses against mycobacterial infection; however, little is known about the function of MDL-1 as yet. In this study, we demonstrated the significant protein expression of MDL-1L and MDL-1S in mouse neutrophils and macrophages. MDL-1L was highly glycosylated by N-linked glycan and sialic acid. Interestingly, the expression pattern of MDL-1 was different between neutrophils and macrophages. MDL-1 expression was notably induced during the differentiation of the mouse myeloid cell line 32Dcl3 into neutrophils. Additionally, we observed that MDL-1 stimulation induced a significant amount of RANTES and macrophage-derived chemokine production in 32Dcl3 cells in cooperation with signaling through TLR. MDL-1 stimulation also up-regulated CD11b expression and maintained cell survival. Our findings indicate that MDL-1, therefore, plays an important role in immune defense as a result of an innate immunity, which involves neutrophils and macrophages.

Key Words: DAP12 • DAP10 • innate immunity • Toll-like receptor


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INTRODUCTION
 
Myeloid DNAX activation protein 12 (DAP12)-associating lectin-1 (MDL-1), also known as C-type lectin domain family 5, member A (CLEC5A), is the first discovered DAP12 (also called KARAP and TYROBP)-associating molecule in myeloid cells [1 ]. MDL-1 has a short cytoplasmic region, which lacks particular signaling motifs and associates noncovalently with DAP12 to transmit downstream signaling via DAP12 immunoreceptor tyrosine-based activation motif (ITAM) (Fig. 1A ) [1 , 2 ]. MDL-1 is a type II transmembrane protein belonging to the C-type lectin family and is expressed on the monocytes and macrophages [1 ]. It has been reported that mouse and pig have two isoforms of MDL-1 as a result of possible alternative splicing, whereas human MDL-1 exists as a single form [1 , 3 ]. In comparison to MDL-1S, mouse MDL-1L has an additional 25 aa containing an extra-putative N-linked glycosylation site in the stalk region and 69% identity with human MDL-1 at the amino acid level (Fig. 1A) [1 ]. However, little is known about the functional role of MDL-1 at the present time.


Figure 1
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  		Figure 1. The structure of MDL-1 and characterization of mAb against mouse MDL-1, which (A) is associated noncovalently with DAP12. TM, Transmembrane; K, lysine (positively charged amino acid); D, aspartic acid (negatively charged amino acid). MDL-1 long form (MDL-1L) contains an additional 25 aa in the stalk (arrow). The potential N-linked glycosylation site is depicted with an asterisk. (B) 293T cells were transfected with FLAG-MDL-1 short form (MDL-1S; #4-1-1) or FLAG-MDL-1 long form (MDL-1L; #1-2-1), respectively. The cells were stained with mAb N354.1, N20.7, N16.10, or anti-FLAG mAb (M2) followed by FITC-conjugated goat anti-Armenian hamster IgG or FITC-conjugated rabbit anti-mouse IgG antibody and analyzed by flow cytometry (solid line). The first antibody was omitted for the negative control (dotted line). The numbers on the histograms show the mean fluorescence intensity (MFI) of the total viable cells. FL1-H, Fluorescence 1-height.

We demonstrated previously the important role of DAP12 in myeloid cell differentiation using a murine M1 leukemia cell line [4 , 5 ]. DAP12-transfected M1 cells revealed morphological and phenotypical change to macrophages by signaling through DAP12 [4 ], which affected the cell survival and apoptosis under certain conditions [5 ]. We also observed that Zymosan A-induced granuloma formation in mouse liver was enhanced markedly by adenoviral-mediated DAP12 gene transfer [6 ]. These data suggested that DAP12-associating receptors on mouse myeloid cells, such as the triggering receptor expressed on myeloid cells (TREM) family, signal regulatory protein β, or MDL-1, strongly contributed to macrophage differentiation and activation. In fact, we observed remarkably strong induction of MDL-1 and TREM-1 mRNA in mouse lung tissues during mycobacterial infection [7 ]. We obtained a similar result by in vitro mycobacterial infection of isolated lung macrophages from naïve C57/B6 mouse.

Recently, it has been demonstrated that DAP12 plays an inhibitory role in signaling in macrophage and dendritic cell (DC) activation in contrast to the activating effector function of DAP12 reported in the initial studies [8 9 10 ]. Additionally, we showed that the lack of DAP12 leads to pronounced proinflammatory and Th1 cytokine responses in pulmonary mycobacterial or influenza infection [11 ]. The mechanisms of DAP12 dual functionality are still unknown; however, there may be a possibility that positive or negative signaling by DAP12 is accounted for by the ligands avidity of DAP12-associating receptors. Thus, it is likely that many of the DAP12-associating receptors, including MDL-1, precisely tune these activatory or inhibitory immune responses in innate and adaptive immunity.

In this study, we demonstrated that MDL-1L and MDL-1S expressed vigorously in mouse bone marrow cells and thioglycollate-induced peritoneal neutrophils. We also observed a higher molecular weight of MDL-1L in neutrophils than that observed in peritoneal macrophages. We found that MDL-1 stimulation induced a significant amount of RANTES and macrophage-derived chemokine (MDC) production in cooperation with signaling through TLR in the mouse myeloid cell line 32Dcl3. MDL-1 stimulation also up-regulated CD11b expression and maintained cell survival in 32Dcl3 cells. These findings indicate that MDL-1-expressing neutrophils and macrophages have an important role in immune defense by innate and adaptive immunity in cooperation with other molecules such as TLR.


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MATERIALS AND METHODS
 
Mice, cells, and culture conditions
The C57BL/6 mice were bred in our facility and used at 8–14 weeks of age. The mouse myeloid precursor cell line 32Dcl3 was obtained from the Riken Cell Bank (Tsukuba, Japan). Dr. Kyuhei. Tomonari (Fukui Medical School, Fukui, Japan) provided the P388D1 and J774-1 macrophage cell lines derived from DBA/2 and BALB/c, respectively. The cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% FCS and 5 x 10–5 M 2-ME. Culture media containing 2 ng/ml IL-3 were used for the 32Dcl3 cell culture. Thioglycollate-induced peritoneal neutrophils and macrophages were obtained as described previously [12 ]. Briefly, 2 ml 3% thioglycollate medium (Sigma-Aldrich) was injected i.p. to C57BL/6 mice. Peritoneal neutrophils were obtained 4 h later, and peritoneal macrophages were harvested 4–5 days later. The cells were characterized by morphology or a flow cytometry analysis.

MDL-1 expression vectors and transfection
The expression constructs containing coding sequences of mouse MDL-1L and MDL-1S tagged with a c-myc peptide epitope (EQKLISEEDL) at the C terminus were amplified by PCR with the following primers: sense, 5' CGG ATA TCA TCA TGA ACT GGC ACA TG 3'; antisense, 5' TCA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC TTT GGC ATT CAT TTC GCA GAT 3'. C57/BL6 mouse bone marrow cells were used as templates. The PCR products were cloned into pIRESneo2 (Clontech, Palo Alto, CA, USA). The coding sequences of mouse MDL-1L and MDL-1S tagged with a 3x FLAG peptide epitope (DYKDHDGDYKDHDIDYKDDDDK) at the C terminus were amplified by PCR with the following primers: sense, 5' CG GAA TTC ATC ATG AAC TGG CAC ATG 3'; antisense, 5' TCT AGA TTT GGC ATT CAT TTC GCA GAT 3'. Myc-MDL-1L and Myc-MDL-1S were used as templates. The PCR products were cloned into p3x FLAG-CMV-14 expression vector (Sigma-Aldrich). 293T cells were transiently transfected with these expression constructs using Lipofectamin TM 2000 (Invitrogen, Carlsbad, CA, USA).

Generation of anti-MDL-1 mAb
Hamster anti-mouse MDL-1 mAb N20.7 was generated by immunizing Armenian hamsters (Oriental Yeast Co., Ltd., Tokyo, Japan) with the c-Myc-tagged MDL-1S-transfected Armenian hamster lung 1 (AHL-1) cells (American Type Culture Collection, Manassas, VA, USA). To generate N354.1, the Armenian hamsters were immunized with N20.7 immune precipitates from c-Myc-tagged, MDL-1L-transfected 293 T cells. N16.10 was generated by immunizing Armenian hamsters with the c-Myc-tagged, MDL-1L-transfected AHL-1 cells. After immunization, the spleen cells were fused with SP2/0 mouse myeloma cells. The hybridoma supernatants were screened by a FACS analysis or Western blotting using FLAG-tagged MDL-1L or MDL-1S.

Flow cytometry and antibodies
The cells (1x106) were incubated with saturating amounts of primary mAb for 30 min in staining buffer (PBS, 1% FCS, 0.1% sodium azide) at 4°C. Then, the cells were washed and incubated with the second antibody for 30 min at 4°C. After washing, the stained cells were analyzed by FACScan (BD Biosciences, Franklin Lakes, NJ, USA). As second antibodies, FITC-conjugated goat anti-Armenian hamster IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or FITC-rabbit anti-mouse IgG (Dako, Glostrup, Denmark) was used. For two-color staining, PE-conjugated anti-mouse Ly-6G (Gr-1) and PE-conjugated anti-mouse CD11b were purchased from eBioscience (San Diego, CA, USA). Armenian hamster IgG (eBioscience) was used as a control. To isolate the lineage-negative cells, the MACS lineage cell depletion kit was used (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Cells are labeled magnetically with a cocktail of biotynylated antibodies against lineage antigens (CD5, CD45, CD11b, anti-Ly6G/C, 7-4, and Ter 119). After staining with antibiotin microbeads, the lineage-negative cells were isolated using MACS magnetic separation system. A flow cytometric analysis was also used to detect apoptotic cells. The cells were incubated with FITC-conjugated Annexin V (Roche, Basel, Switzerland) and propidium iodide for 15 min at room temperature.

Deglycosylation
To examine the state of glycosylation of MDL-1, the total cell lysates from bone marrow cells were deglycosylated using an enzymatic protein deglycosylation kit (Sigma-Aldrich), according to the manufacturer’s protocol. Briefly, the samples were incubated with PNGase F, O-Glycosidase, or {alpha}-2(3,6,8,9) neuraminidase for 3 h under reducing conditions and then analyzed by Western blot using N354.1

Immunoprecipitation, electrophoresis, and blotting
The cells were lysed in lysis buffer [0.5% Triton X-100, 50 mM Tris (pH 8.0), 140 mM NaCl, and 10 mM EDTA] containing the protease inhibitor cocktail Complete Mini, EDTA-free (Roche). We used 1% n-dodecyl-β-D-maltoside instead of 0.5% Triton X-100 to examine the association between MDL-1 and DAP12. Rabbit anti-DAP12 polyclonal antibodies (pAb) were generated as described previously. For hamster anti-DAP10 mAb (SK118-4), Armenian hamsters were immunized with the immunoprecipitate of FLAG-tagged mouse DAP10-transfected 293 T cells using anti-FLAG agarose beads (Sigma-Aldrich), and hybridoma was established as described previously. The lysates were clarified by centrifugation and immunoprecipitated with antibodies bound to Protein G-Sepharose 4 Fast Flow (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) for 1 h at 4°C. The resulting immunocomplexes were washed and run on 4–12% NuPage Bis-Tris SDS-PAGE gels (Invitrogen) under reducing or nonreducing conditions. The proteins were then blotted onto Immobilon-P (Millipore, Bedford, MA, USA), blocked in 5% skim milk, and probed with goat anti-MDL-1 pAb (R&D Systems, Inc., Minneapolis, MN, USA) or hamster anti-MDL-1 mAb N354.1. The ECL system (GE Healthcare Bio-Science Corp.) was used for the detection.

RT-PCR analysis
Total RNA was prepared using Sepasol-RNA I (Nacaraitesque, Inc., Kyoto, Japan), according to the manufacturer’s protocol. cDNA was prepared from 1.5 µg total RNA by using a first-strand cDNA synthesis kit for RT-PCR (Avian Myeloblastosis virus,Roche). The primers used were as follows: β-actin (forward, 5'-TGGAATCCTGTGGCATCCATGAAAC-3', reverse, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'); RANTES (forward, 5'-GCCTCACCATATGGCTCGGACACCACT-3', reverse, 5'-CTTCTCTGGGTTGGCACACACTTGGCGG-3'); IFN-inducible protein 10 (IP-10; forward, 5'-CCATGAACCCAAGTGCTGCCGTCATTT-3', reverse, 5'-CTTAGATTCCGGATTCAGACATCTCTGCTCAT-3'); MDC (forward, 5'-GTGGCTCTCGTCCTTCTTGCTGTGGC-3', reverse, 5'-GGACAGTTTATGGAGTAGCTTCTTC-3'); TNF-{alpha} (forward, 5'-GGCAGGTCTACTTTGGAGTCATTGC-3', reverse, 5'-ACATTCGAGGCTCCAGTGAATTCGG-3'); myeloperoxidase (MPO; forward, 5'-ATGCAGTGGGGACAGTTTCTGGATCATGAC-3', reverse, 5'-GTCGTTGTAGGATCGGTACTGCGGTAGGTA-3'); and MIP-2 (forward, 5'-AAGGCTAACTGACCTGGAAAGGAGGAGCCT-3', reverse, 5'-CTGTGTGGGTGGGATGTAGCTAGTTCCCAA-3'). PCR was performed by using the following program: 1 min at 94°C, 30x (5 s at 94°C, 30 s at 60°C, 90 s at 72°C), 7 min at 72°C.

Assays for 32Dcl3 cell stimulation with anti-MDL-1 antibodies and ELISA
Culture dishes were coated with 20 µg/ml N354.1, N20.7, N16.10, Armenian hamster IgG (eBioscience), or Syrian hamster IgG (Jackson ImmunoResearch Laboratories, Inc.) overnight at 4°C and washed with culture medium twice. In the experiment to exclude the possible contamination of LPS in the antibody preparations, antibodies were boiled for 10 min for the heat treatment. The 32Dcl3 cells were stimulated with G-CSF (10 ng/ml) in the absence of IL-3 for 4 days and then washed twice with RPMI medium supplemented with 10% FCS and 2-ME. Cells (1x107) were cultured on antibody-coated culture dishes for 24 h. The mRNA expression was examined by RT-PCR. To analyze the protein levels of cytokine production, the cells were cultured on antibody-coated culture dishes for 72 h. Then, the culture supernatants were measured by specific ELISA, according to the manufacturer’s protocol. All ELISA kits were purchased from PeproTech (Rocky Hill, NJ, USA).


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RESULTS
 
Protein expression pattern of MDL-1 in mouse-immune organs
To gain more precise knowledge about MDL-1L and MDL-1S, we generated mAb against MDL-1 and selected three antibodies: N354.1, N20.7, and N16.10. We performed a flow cytometry analysis to assess the reactivity of these mAb using 293T cells transiently transfected with expression vectors of FLAG-tagged MDL-1L and MDL-1S. Anti-MDL-1 mAb N20.7 and N16.10 reacted with MDL-1L and MDL-1S. On the other hand, N354.1 reacted solely with MDL-1L (Fig. 1B) . As the molecular weight of these antibodies was approximately 160 kD under nonreducing conditions, it is highly possible that these antibodies belong to the IgG subclass (data not shown).

As we previously found the MDL-1 mRNA expression to be induced markedly during pulmonary mycobacterial infection, we investigated the expression pattern of the MDL-1L and MDL-1S in mouse immune organs by Western blotting (Fig. 2A ) [7 ]. MDL-1L and MDL-1S were constitutively expressed at high levels in mouse bone marrow cells. N354.1 demonstrated a higher affinity to MDL-1L than N20.7, which was also observed in a flow cytometry analysis (Fig. 1B) . In comparison with bone marrow cells, the expression of MDL-1 in spleen cells and residential peritoneal macrophages was considerably weaker. We could not detect MDL-1 protein expression in thymocytes and mesenteric lymph node cells by Western blotting (data not shown). In comparison with bone marrow cells and spleen cells, the molecular weight of MDL-1L protein in peritoneal macrophages is slightly smaller. This finding suggests that there is a difference of modification such as glycosylation between MDL-1L in bone marrow cells and the one in macrophages. MDL-1L and MDL-1S are constitutively associated with DAP12 in bone marrow cells and macrophages (Fig. 2A) . The mouse-activating lectin-like NK receptor NKG2D exists in two splice isoforms, and only a short form of NKG2D can pair with DAP10 and DAP12 [13 ]. Similarly, MDL-1 splice forms may associate with DAP10 in addition to DAP12. To evaluate the association of DAP10 and MDL-1, Western blotting using anti-DAP12 pAb and anti-DAP10 mAb was performed (Fig. 2B) . MDL-1L and MDL-1S made a pair with DAP10 as well as DAP12 in bone marrow cells.


Figure 2
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  		Figure 2. Analysis of the MDL-1 expression in mouse myeloid cells. (A) Cell lysates prepared from 5 x 106 spleen cells, 5 x 106 bone marrow cells, or 3 x 106 peritoneal macrophages were immunoprecipitated (IP) with N354.1, N20.7, or rabbit anti-mouse DAP12 pAb. The samples were analyzed by Western blotting [immunoblotting (IB)] using anti-MDL-1 pAb under the nonreducing condition. (B) Cell lysates prepared from 4 x 106 bone marrow cells were immunoprecipitated with rabbit anti-DAP12 pAb or hamster anti-DAP10 mAb SK118-4. The samples were analyzed by Western blotting using anti-MDL-1 pAb under the nonreducing condition. (C) Cell lysates prepared from 3 x 106 bone marrow cells (BM), thioglycollate-induced peritoneal neutrophils (PN), or thioglycollate-induced macrophages (MP) were immunoprecipitated with N354.1, N20.7. N16.10, or anti-MDL-1 pAb (poly). Armenian hamster IgG (Ham Ig) was used as a control. The samples were analyzed by Western blotting using anti-MDL-1 pAb under nonreducing conditions.

To confirm the MDL-1 expression in myeloid lineage cells and to specify the reactivity of respective anti-MDL-1 mAb, we examined the MDL-1 expression in bone marrow cells, thioglycollate-induced neutrophils, or thioglycollate-induced macrophages by immune precipitation using N354.1, N20.7, N16.10, or goat anti-MDL-1 pAb. As shown in Figure 2C , MDL-1 was detected in thioglycollate-induced neutrophils, thioglycollate-induced macrophages, and bone marrow cells. Although N20.7, N16.10, and anti-MDL-1 pAb reacted with MDL-1L and MDL-1S, N354.1 detected only MDL-1L in these types of cells. Interestingly, bone marrow cells and thioglycollate-induced neutrophils revealed vigorous expression of MDL-1 in comparison with the expression level of MDL-1 in thioglycollate-induced macrophages. Of note, the molecular weight of MDL-1L in the thioglycollate-induced macrophages is the same as the one in the residential macrophages and is slightly lower than the one in neutrophils. We observed a DAP12 and DAP10 association with MDL-1L and MDL-1S in thioglycollate-induced mouse neutrophils and macrophages (data not shown.).

To determine what subsets of bone marrow cells express MDL-1, a flow cytometry analysis was performed. Figure 3A shows the surface expression of MDL-1 on mouse bone marrow cells using N354.1 and N16.10. After deduction of the nonspecific binding by control hamster Ig, approximately 35% total bone marrow cells were double-positive for Gr-1 and MDL-1L or MDL-1 L/S by Gr-1 and anti-MDL-1 mAb double-staining (Fig. 3A) . As these Gr-1-positive cells were positive for CD11b (data not shown), it is presumed that neutrophils were a major fraction of the MDL-1-expressing cells in bone marrow. We confirmed MDL-1 cell-surface expression in thioglycollate-induced neutrophils (Fig. 3A) . After deduction of the nonspecific binding by control hamster Ig, 45.70% total cells were positive for N354.1 (MDL-1L) and Gr-1, and 33.74% total cells were positive for N16.10 (MDL-1L/S) and Gr-1. We also examined the cell-surface expression of MDL-1 using thioglycollate-induced macrophages. However, we could not observe a significant increase of MDL-1 in macrophage populations because of high background by control hamster Ig (data not shown). To investigate the function of MDL-1 in bone marrow neutrophils, it is important to clarify the expression of MDL-1 in bone marrow stem cells. We performed the MDL-1 staining against a lineage marker-negative population in bone marrow cells. As shown in Figure 3B , lineage marker-negative cells that include bone marrow stem cells were negative for N354.1 or N16.10.


Figure 3
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  		Figure 3. Analysis of the MDL-1 expression in mouse myeloid cell surface. (A) After RBC lysis using NH4Cl, mouse bone marrow cells or thioglycollate-induced peritoneal neutrophils were stained with 10 µg/ml N354.1 or N16.10. FITC-conjugated goat anti-Armenian hamster IgG was used as a second antibody. The cells were double-stained with Gr-1. Armenian hamster IgG was used as a control. The numbers on the dot-blot represent the percentage of positive cells in each quadrant relative to the total number of cells scored. (B) Lineage-negative cells were negatively isolated from mouse bone marrow cells using MACS lineage cell depletion kit. Lineage markers are a cocktail of biotin-conjugated mAb against CD5, CD45R, CD11b, Ly-6G/C, 7-4, and Ter-119. Cells were stained with 10 µg/ml N354.1 or N16.10. FITC-conjugated goat anti-Armenian hamster IgG was used as a second antibody. Armenian hamster IgG was used as a control. R-PE-conjugated streptavidin was used to detect lineage marker-positive cells. The numbers on the dot-blot represent the percentage of positive cells in each quadrant relative to the total number of cells scored.

The glycosylation analysis of MDL-1L in mouse bone marrow cells
Although the predicted MW of MDL-1L is ~22 kD, we consistently observed the MDL-1L at ~45 kD. To assess the glycosylation state of MDL-1L, cell lysates prepared from bone marrow cells were digested with PNGase F, O-Glycosidase, and/or {alpha}-2(3,6,8,9) neuraminidase. We used anti-MDL-1L mAb N354.1 for the immune blotting of total cell lysate prepared from bone marrow. As shown in Figure 4A , PNGase F deglycosylated MDL-1L effectively, and the MW of MDL-1L was reduced from 45 kD to 30 kD. The amino acid sequence of MDL-1L contains three possible, potential N-linked glycosylation sites, and one of them is located in an additional 25 aa in the stalk (Fig. 1A) . Probably approximately 15 kD N-linked glycan binds to these sites. We observed the further migration of MDL-1L by neuraminidase digestion in cooperation with PNGase F, and this finding indicates that glycans in MDL-1L contain sialic acid moieties. Furthermore, O-glycosidase treatment in conjunction with PNGase F and neuraminidase decreased the molecular weight of MDL-1L slightly in comparison with the treatment with PNGase F and neuraminidase. Although we could not find any predicted O-linked glycosylation sites using an O-glycosylation prediction program (http://www.cbs.dtu.dk/services/NetOGlyc/) in MDL-1L and MDL-1S, it might be possible that O-linked glycosylation sites exist in MDL-1L. After treatment with these three enzymes, we found the MW of MDL-1L at ~20 kD, which is consistent with the predicted MW.


Figure 4
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  		Figure 4. The glycosylation analysis of MDL-1 L in mouse bone marrow cells. (A) Total cell lysates prepared from bone marrow cells were deglycosylated using PNGase F, O-Glycosidase, and/or {alpha}-2(3,6,8,9) neuraminidase for 3 h and analyzed by Western blotting using N354.1 under the reducing condition. no, No treatment; B, digestion buffer; P, PNGase F; O, O-Glycosidase; and N, neuraminidase. (B) Lysates prepared from 1 x 107 mouse bone marrow cells were precleared twice with N20.7- or N354.1-conjugated protein G sepharose beads (P: preclear) or not (C: control). The supernatants were immunoprecipitated with N20.7 or N354.1 and analyzed by Western blotting using anti-MDL-1 pAb under the nonreducing condition.

In the present study, we performed SDS-PAGE in some experiments under nonreducing conditions to eliminate bands of Ig heavy chain and light chain, which may be overlaid on MDL-1L and MDL-1S in the gel. However, the size of MDL-1L in the nonreduced conditions and the one in the reduced conditions was almost the same (Fig. 2A , nonreduced conditions; Fig. 4A , reduced conditions). Although there remains a possibility of a noncovalently bound multimer of MDL-1, this finding may indicate that a certain amount of MDL-1 exists as monomers and still can associate DAP12 or DAP10 (Fig. 2) .

The MW of completely deglycosylated MDL-1L is close to MDL-1S. To confirm the specificity of N354.1 against MDL-1L, we performed a sequential immunoprecipitation experiment using a Western blotting analysis. Lysates prepared from bone marrow cells were precleared twice with N354.1- or N20.7-conjugated protein G sepharose beads, and then, the supernatants were immunoprecipitated with N354.1 or N20.7. N354.1 did not cross-react with MDL-1S and detected only MDL-1L (Fig. 4B) .

Protein expression of MDL-1 in mouse myeloid cell lines and induction of MDL-1 during differentiation to neutrophil
J774-1 and P388D1 are mouse macrophage cell lines. Two variants of MDL-1 were constitutively expressed in macrophage cell lines and associated with DAP12 (Fig. 5A ). The MW of MDL-1L in J774-1 appears to be the same as MDL-1L in bone marrow. In contrast, the MW of MDL-1L in P388D1 is close to the one in macrophages (Fig. 5A) . In comparison with macrophage cell lines, MDL-1 was hardly detectable in the myeloid precursor cell line 32Dcl3 at the protein level (Fig. 5B) . However, neutrophil differentiation of 32Dcl3 by stimulation with G-CSF for 7 days induced a significant amount of MDL-1L and MDL-1S (Fig. 5B) . MDL-1L and MDL-1S made a pair with DAP10 and/or DAP12 in G-CSF-treated 32Dcl3 cells (data not shown). The differentiation of 32Dcl3 was confirmed morphologically by Diff-Quick staining (Fig. 5C) . Initially, the 32Dcl3 cells had a myeloblast-like morphology, having a high nucleus-to-cytoplasm ratio. On Day 4, many vacuoles appeared in their cytoplasm, and the cells looked like monocytes. The cell size became bigger, and the nuclei were kidney-shaped. By Day 7, the cells shrank and demonstrated nuclear segmentation. Morphologically, they looked closely similar to murine neutrophilic-segmented granulocytes.


Figure 5
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  		Figure 5. MDL-1 expression in different mouse myeloid lineage cell lines. (A) Cell lysates (300 µg) were prepared from J774-1 or P388D1 and coimmunoprecipitated with anti-DAP12 pAb. The samples were analyzed by Western blotting using anti-MDL-1 pAb under the nonreducing condition. The cell lysates prepared from 2 x 107 bone marrow cells and 7 x 106 peritoneal macrophages were used as controls. (B) 32Dcl3 cells were stimulated with G-CSF (10 ng/ml) in the absence of IL-3 for 7 days (d). Cell lysates (300 µg) were immunoprecipitated with N354.1 or N20.7, respectively, at the indicated time-points. Samples were analyzed by Western blotting using anti-MDL-1 pAb under the nonreduced condition. (C) The cells were stained using Diff-Quick at the indicated time-points and observed under the microscope.

Signal through MDL-1 up-regulated RANTES, IP-10, and MDC mRNA expression in G-CSF-pretreated 32Dcl3 cells, and TLR-4 signal synergistically augmented the MDL-1 signal in RANTES and MDC protein expression
To examine the functional role of MDL-1 in mouse myeloid cells, we performed RT-PCR to evaluate the cytokine and chemokine production in G-CSF-pretreated 32Dcl3 cells during MDL-1 stimulation. As shown in Figure 6A , Th1-related chemokine RANTES and IP-10 mRNA were induced by N354.1 and N20.7 stimulation. Interestingly, Th2-related chemokine MDC mRNA was also induced markedly during stimulation. In comparison, TNF-{alpha}, MPO, and MIP-2 were expressed constitutively at the mRNA level, and there was no significant change of expression during MDL-1 stimulation. In agreement with our findings by RT-PCR, we observed a small amount of RANTES and MDC expression at the protein level in G-CSF-pretreated 32Dcl3 cells by MDL-1 stimulation, whereas we could not detect them in the PBS or hamster Ig-treated groups (Fig. 6B) . As previous studies showed that one of the DAP12-associating molecules, TREM-1, enhanced the production of cytokines in conjunction with signaling through TLRs, we examined whether TLR signaling affected MDL-1 signaling or not [14 , 15 ]. Surprisingly, signaling through TLR by LPS synergistically augmented the production of RANTES and MDC during MDL-1 stimulations (Fig. 6B) . These findings indicate a strong cross-talk between TLR and MDL-1 signaling. To exclude the possibility of endotoxin contamination in anti-MDL-1 mAb, we performed heat-treated antibody experiments (Fig. 6C) . Addition of heat-treated anti-MDL-1 antibodies did not reveal any increase in the MDC production, indicating that there is no heat-stable stimulant such as LPS or nucleic acid in the antibody preparation.


Figure 6
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  		Figure 6. Signal through MDL-1 up-regulated RANTES, IP-10, and MDC mRNA expression in G-CSF-pretreated 32Dcl3 cells, and the TLR-4 signal synergistically augmented the MDL-1 signal in RANTES and MDC protein expression. (A) 32Dcl3 cells were stimulated with G-CSF (10 ng/ml) in the absence of IL-3 for 4 days and then washed twice with RPMI medium supplemented with 10% FCS and 2-ME. Cells (1x107) were cultured on the indicated antibody-coated 6-cm culture dish for 24 h. mRNA expression was examined by RT-PCR. Syrian Hamster IgG was used as a control. (B) 32Dcl3 cells were stimulated with G-CSF (10 ng/ml) in the absence of IL-3 for 4 days and then washed twice with RPMI medium supplemented with 10% FCS and 2-ME. Cells (2x105) were cultured on the indicated antibody-coated 96-well culture dishes for 72 h, with or without LPS (1 µg/ml). RANTES or MDC production in culture supernatants was measured by ELISA. Armenian hamster IgG was used as a control. (C) 32Dcl3 cells were stimulated with G-CSF (10 ng/ml) in the absence of IL-3 for 4 days and then washed twice with RPMI medium supplemented with 10% FCS and 2-ME. Cells (2x105) were cultured on the indicated antibody-coated 96-well culture dishes for 72 h with LPS (1 µg/ml) or soluble heat-treated antibodies (10 µg/ml). MDC production in the culture supernatants was measured by ELISA. Armenian hamster IgG was used as a control.

MDL-1 transmitted the survival signal in a certain circumstance
We were interested in whether a signal through MDL-1 affected 32Dcl3 cell viability. As shown in Figure 7A , the cell size of 32Dcl3 cells after MDL-1 stimulation became bigger than the ones stimulated with PBS or hamster Ig. The activation of 32Dcl3 cells and the avoidance of apoptosis may contribute to this phenomenon. The percentage of Annexin V-positive apoptotic cells in the MDL-1-stimulated group was smaller than the one in the control PBS or the hamster Ig group (Fig. 7B ; P<0.05). The cell morphology of a 48-h culture showed increased cytoplasm and vacuoles in the MDL-1-stimulated group in comparison with the PBS or hamster Ig group. Interestingly, 48 h of MDL-1 stimulation did not induce the differentiation of monocyte-like 32Dcl3 cells to neutrophils (Fig. 7C) . It is suspected that the signal through MDL-1 is not positively involved in neutrophil differentiation. To evaluate 32Dcl3 cell activation, a cell-surface phenotypic analysis was performed. The signal through MDL-1 revealed an up-regulation of CD11b in 32Dcl3 cells with MDL-1 stimulation (Fig. 7D) . No differences in CD11c, Gr-1, F4/80, and I-A staining were seen (data not shown). There is a possibility that the stimulation through MDL-1 produces not only MDC or RANTES but also other chemokines and cytokines in 32Dcl3 cells, and those factors may therefore help to maintain 32Dcl3 cell activation/differentiation and survival.


Figure 7
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  		Figure 7. MDL-1 transmitted the survival signal in a certain circumstance. 32Dcl3 cells were stimulated with G-CSF (10 ng/ml) in the absence of IL-3 for 4 days and then washed with RPMI medium supplemented with 10% FCS and 2-ME twice. Cells (7.5x105) were cultured on the indicated antibody-coated 48-well culture dishes for 72 h. Syrian hamster IgG was used as a control. (A) The cell size was determined by flow cytometry using forward light-scatter (FSC) after 24 h. The numbers on the histograms show the MFI of the total viable cells. (B) The percentage of Annexin V-positive cells was analyzed by FACScan at the indicated time-point. *, P < 0.05, for N20.7 versus hamster Ig or N354.1 versus hamster Ig. The data shown indicate the mean values for three independent experiments ± SEM. (C) The cells were stained using Diff-Quick after 48 h and observed under the microscope. (D) The expression of CD11b was analyzed by flow cytometry after 30 h (solid line). PE-conjugated anti-mouse CD11b was omitted for the negative control (dotted line). The numbers on the histograms show the percentage of CD11b high population. Any dead cells were gated out using FSC and side light-scattering.


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DISCUSSION
 
The C-type lectin-like super domain (CTLD) family has diverse functions and in particular, is important in innate immunity including NK function or pathogen recognition [16 ]. CTLD-containing proteins (CTLDcps) are classified into 17 groups based on their domain architectures in vertebrates [17 ]. MDL-1 (CLEC5A) belongs to the Group V "NK cell receptors" family that is characterized by non-Ca+ binding type II transmembrane CTLDcps. In this group, not only killer cell lectin-like receptors such as NKG2 and Ly49 but also many molecules expressed on other cell types were included, for example, CD72 on B cells, CD69 on various hematopoietic cells, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) on vascular endothelial cells, Dectin-1 on macrophages and DCs, myeloid inhibitory C-type lectin-like receptor (MICL; CLEC12A) on myeloid cells, CLEC1 on DCs, and CLEC2 in liver [17 ]. The genes of these molecules are located mostly within the NK complex, which exists in human chromosome 12p13 or mouse chromosome 6F3 [18 ]. Uniquely, human and mouse MDL-1 genes locate in chromosome 7q33 or 6B2, respectively [17 ] (National Center for Biotechnology Information Map Viewer: http://www.ncbi.nlm.nih.gov/mapview/).

A previous report showed that human MDL-1 mRNA is constitutively expressed in monocytes and macrophages but not in granulocytes [1 ]. A significantly higher expression of MDL-1 was observed in CD14+ monocytes in comparison with CD34 stem cells [19 ]. On the contrary, a markedly reduced MDL-1 mRNA expression was observed when human monocytes were differentiated to DCs [1 ]. In mice, MDL-1 transcripts were detected only in a macrophage cell line but not in T cell lines, B cell lines, or mast cell lines [1 ]. However, all of these studies assessed only mRNA expression, and there has been no study that tried to examine the protein expression of MDL-1. We herein demonstrated the massive expression of MDL-1L and MDL-1S in bone marrow cells or thioglycollate-induced neutrophils. We also detected a certain amount of MDL-1 expression in peritoneal residential macrophages and thioglycollate-induced macrophages. In our previous report, we observed a significant induction of MDL-1 mRNA in mouse lung tissue during pulmonary mycobacterial infection [7 ]. As a significant number of neutrophils infiltrated in the mouse mycobacterial granuloma, it may be suspected that not only macrophages but also neutrophils are a predominant source of MDL-1 in mouse mycobacterial infection [20 ].

We also found that the MW of MDL-1L in neutrophils is higher than the one in macrophages. This difference could be a result of the different glycosylation state between neutrophils and macrophages. In fact, Marshall et al. [21 ] demonstrated that one of the Group V CTLDcps, MICL (CLEC12A), revealed variable glycosylation in the different cell populations. Interestingly, MICL is more glycosylated in polymorphonuclear neutrophils in comparison with monocytes [21 ]. Several studies showed that the glycosylation state of Group V CTLDcps, including Ly49 and LOX-1, affects their ligand binding [22 , 23 ]. The varied glycosylation of MDL-1L may also affect the ligand binding and may contribute to the different function of neutrophils and macrophages in the immune response.

Interestingly, MDL-1L and MDL-1S were able to associate with adaptor molecules DAP12 and DAP10. Currently, it has been recognized that Group V CTLDcps, NKG2D, was the principal receptor that associates with DAP10 to transmit its signals [24 ]. Mouse NKG2D has long and short isoforms as a result of alternative splicing. It was considered that the long form of NKG2D associates exclusively with DAP10, whereas the short variant can interact with DAP10 and DAP12 [13 ]. A more recent study [25 ] showed that mouse NKG2D long form also could associate with DAP12 and DAP10, and the data suggested that signaling via these two adaptor molecules gives more complexity in the NK-activating function. Although we could not observe a big difference in the association between the MDL-1 isoforms and two adaptor molecules in the myeloid lineage cells, there still remains a possibility of a functional difference between MDL-1L and MDL-1S. For example, MDL-1L and MDL-1S may have a different ligand-binding affinity, and this affects the state of activation in these adaptor molecules. Accordingly, physiological ligand binding may result in distinct effector function depending on which types of adaptor molecules are stimulated effectively. Of note, judging from the result of the cytokine production or cell survival using N354.1 or N20.7, stimulation of MDL-1L alone appeared to be effective. In this case, MDL-1L might be functionally dominant.

The significant up-regulation of MDL-1 during mycobacterial infection in our previous study suggested the importance of MDL-1 for the host defense mechanisms in innate or adaptive immunity. As reported previously, one of the DAP12-associating molecules, TREM-1, amplifies cytokine production such as TNF, IL-6, IL-1 β, and GM-CSF in conjunction with certain kinds of TLR ligands in human PBMCs or monocytes [14 , 15 ]. Moreover, Dectin-1, Group V CTLDcps bearing an ITAM-like motif, also augmented IL-12 production in cooperation with TLR stimulation [26 ]. In our present study, it was observed that a significant synergistic effect existed between MDL-1 and TLR signaling by LPS in RANTES and MDC production. Although the ligand of MDL-1 is still unknown, the TLR pathways could potentiate MDL-1 signaling in innate immunity such as bacterial or viral infection. Of particular note, RANTES and IP-10 and MDC are considered to be contradictory chemokines, as RANTES and IP-10 are related to the Th1 type of immunity, and MDC is related to the Th2 type of immunity [27 , 28 ]. There is a possibility that the MDL-1 signal contributes not only to innate immunity but also to adaptive immunity and steers the course of adaptive immunity by tuning the type 1/type 2 immune balance as a result of a variety of chemokine productions.

Finally, we observed decent cell survival and resistance to apoptosis in anti-MDL-1 mAb-treated 32Dcl3 cells. 32Dcl3 is an IL-3-dependent cell line, and the replacement of IL-3 with G-CSF supported an initial proliferation, followed by the terminal differentiation into neutrophils [29 ]. Our result showed that the MDL-1 signal gives alternative survival signals to 32Dcl3 cells to some extent, although unlike the stimulation by G-CSF, differentiation into neutrophils was not observed morphologically (Fig. 7C) . As the MDL-1 expression was extremely poor at the protein level in bone marrow stem cells and some myeloid precursor cell lines (Figs. 3B and 5B , and data not shown), the contribution of the MDL-1 signal alone to neutrophil differentiation may be minimal at the earliest stage. Rubel et al. [30 ] have reported that the interaction between fibrinogen and CD11b delayed apoptosis in human neutrophils. In this regard, it is possible that the up-regulation of surface CD11b on 32Dcl3 cells during MDL-1 stimulation may involve cell survival in addition to the production of cytokines and chemokines.

In conclusion, we demonstrated the expression pattern and functional role of MDL-1 in mouse myeloid lineage cells. Our data in the present work, such as chemokine production or synergic effect with TLR, strongly suggest the important role of MDL-1 in innate immunity. During the preparation of our manuscript, Chen et al. [31 ] reported that anti-MDL-1 mAb treatment reduced Dengue virus infections. They observed that the blockade of MDL-1 suppresses the proinflammtory cytokines by Dengue virus-infected macrophages. Their observations are thus considered to strengthen our belief of the importance of MDL-1 as in infectious disease, not only in Dengue virus but also in other agents such as tuberculosis. Further work is under way to examine more precisely the function of MDL-1 during innate immunity and also adaptive immunity.


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ACKNOWLEDGEMENTS
 
This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture (Grant 17790252). We thank Drs. Toshiyuki Takai and Yohko Katagiri for helpful discussions, Dr. Zhou Xing for reading the manuscript, Dr. Hiroshi Aizawa for technical advice, and Ms. Rie Matsumoto for technical assistance.

Received May 29, 2008; revised November 15, 2008; accepted November 17, 2008.


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