Cutting Edge: Signal-Regulatory Protein β1 Is a DAP12-Associated Activating Receptor Expressed in Myeloid Cells1

Signal-regulatory proteins (SIRPs) are cell-surface glycoproteins expressed on myeloid and neural cells that have been shown to recruit SH2 domain-containing protein phosphatase 1 (SHP-1) and SHP-2 and to regulate receptor tyrosine kinase-coupled signaling. One SIRP of unknown function, designated SIRPβ1, contains a short cytoplasmic domain that lacks sequence motifs capable of recruiting SHP-1 and SHP-2. Using a SIRP-specific mAb, we show that SIRPβ1 is expressed in monocytes and dendritic cells and associates with the signal transduction molecule DAP12. SIRPβ1/DAP12 complex formation was required for efficient cell-surface expression of SIRPβ1. Stimulation of this complex induced tyrosine phosphorylation, mitogen-activated protein kinase activation, and cellular activation. Thus, SIRPβ1 is a new DAP12-associated receptor involved in the activation of myeloid cells.

Another subset of SIRP receptors, called SIRP␤, contains short cytoplasmic domains that lack cytoplasmic sequence motifs capable of recruiting SHP-2 and SHP-1. In addition, they contain a single basic lysine residue within the hydrophobic transmembrane domain (1). These characteristics are reminiscent of those of a group of NK cell receptors which includes the killer cell Ig-like receptors with short tails (KIR2DS), the CD94-NKG2C/E heterodimers, and Ly49D/H homodimers. These NK cell receptors activate cell-mediated cytotoxicity and cytokine release by associating with a separate 12-kDa protein, called DAP12, which contains a single cytoplasmic immunoreceptor tyrosine-based activating motif (14 -16). To examine the possible association of SIRP␤1 with DAP12, we generated a specific mAb and examined cellular distribution, biochemical composition, and signaling properties of SIRP␤1.

Materials and Methods
Generation of mAb BALB/c mice were immunized with human DCs. Myeloid cell-specific mAbs were selected by staining peripheral blood leukocytes and DCs. Anti-SIRP␤1 mAb 148 was further selected by screening against SIRP␤1-COS transfectants. Superfect (Quiagen, Hilden, Germany). SIRP␤1 and DAP12 cDNAs were cotransfected in Jurkat cells by electroporation, and SIRP␤1-DAP12 stable transfectants were selected in G418-containing medium. SIRP␤1 expression on transfected cells was assessed by FACS analysis and immunoblot using mAb 148. Coexpression of DAP12 in double transfectants was determined by anti-DAP12 immunoblot on either whole cell lysates or SIRP␤1 immunoprecipitates (see below).

Immunoprecipitation and immunoblotting
Pervanadate-treated (200 M sodium orthovanadate and 200 M H 2 O 2 at 37°C for 10 min) monocytes, monomac-6 cells, and SIRP␤1-DAP12-Jurkat cells were lysed in 1% Triton X-100, precleared with protein G beads (Amersham Pharmacia, Uppsala, Sweden) and normal mouse serum, and subjected to immunoprecipitation with mAb 148 as previously described (18). For whole cell lysate analysis, cells were lysed in Laemmli sample buffer. Immunoprecipitates and whole cell lysates were separated by standard SDS-PAGE, transferred to polyvinylidene difluoride membranes (Amersham Pharmacia), and immunoblotted with mAb 148 and/or rabbit anti-DAP12 antiserum (kindly provided by Dr. Kerry S. Campbell, Philadelphia, PA). In some experiments the precipitates were treated overnight with N-glycanase F (Boehringer Mannheim, Mannheim, Germany) according to the manufacture's protocols.

Cell stimulations
Two million cells/ml were incubated at 37°C with mAb 148 or control IgG (Immunotech, Marseille, France) and F(abЈ) 2 goat anti-mouse Ab (GAM) (Jackson Laboratories, West Grove, PA) as cross-linker. After stimulation, one cell aliquot was lysed and subjected to antiphosphotyrosine and antiextracellular signal-regulated kinase (ERK) blotting using PY-20 (Transduction Laboratories, Lexington, KY) and either anti-phospho-ERK or ERK Abs (New England Biolabs, Beverly, MA). Alternatively, after 12 h of stimulation, another cell aliquot was analyzed for expression of CD69 by FACS analysis using a PE-conjugated anti-CD69 mAb (Immunotech).
Following immunoprecipitation of SIRP␤1 from SIRP␤1-COS cells with mAb 148 and N-glycanase F treatment of the immunoprecipitate, SIRP␤1 was seen as a ϳ40-kDa protein (Fig. 1D). A similar band was observed in monomac-6 cells, together with an additional band of ϳ65 kDa, which corresponds to the predicted m.w. of nonglycosylated SIRP␣1 (Fig. 1E). Thus, mAb 148 recognizes SIRP␤1 as well as SIRP␣1. Using mAb 148 we next studied the cellular distribution of SIRP␤1. SIRP␤1 was detected in lysates from U937, monomac-6, and DCs, but were not found in peripheral NK/CD8 T cells, NK, or Jurkat cells (Fig. 1F). The presence in some cell lysates of more than one band in the 50-kDa region suggests that SIRP␤1 may be present in more that one glycosylated form. SIRP␤1 was always coexpressed with a more abundant ϳ90-kDa protein, which corresponds to SIRP␣. Taken together, these results indicate that SIRP␤1 is a 50-kDa cell-surface glycoprotein preferentially expressed in myeloid cells.

SIRP␤1 associates with DAP12
DAP12 and other signaling proteins like TCR, FcR␥, and the recently identified DAP10/KAP10 (19,20) have been shown to be tyrosine phosphorylated upon stimulation or after pervanadate treatment. Therefore, we tested whether SIRP␤1 associated with a phosphorylated protein in myeloid cells. SIRP␤1 precipitates from pervanadate-stimulated monocytes were analyzed by antiphosphotyrosine immunoblotting. SIRP␤1 did associate with a phosphorylated protein with a molecular mass of ϳ26 kDa under nonreducing conditions, which decreased to ϳ14 kDa under reducing conditions ( Fig. 2A). Subsequent analysis of SIRP␤1 immunoprecipitates from unstimulated cells with an anti-DAP12 antiserum revealed that this molecule corresponds to DAP12 (Fig. 2B). Control immunoblotting experiments revealed no association of SIRP␤1 with other myeloid adapter proteins, such as DAP10/ KAP10 or FcR␥ (data not shown). Thus, in myeloid cells, SIRP␤1 is constitutively associated with a DAP12 homodimer.

SIRP␤1/DAP12 complex triggers cell activation and ERK1/ERK2 phosphorylation
Because monocytes and DCs coexpress inhibitory and activating SIRP isotypes and mAb 148 recognizes both isotypes, it is difficult to investigate the function of SIRP␤1 independently from that of SIRP␣1 in primary cells. Therefore, to explore the possible stimulatory function of SIRP␤1, we cotransfected SIRP␤1 and DAP12 in Jurkat T cells, which do not express SIRP␣1, SIRP␤1, or DAP12 (Figs. 1F and 3). Expression of SIRP␤1 in transfected cells was confirmed by immunoblot and surface staining (Fig. 3, A and  B). Coexpression and association of DAP12 with SIRP␤1 were monitored by anti-DAP12 immunoblotting on whole cell lysates and SIRP␤1 immunoprecipitates (Fig. 3, C and D). Interestingly, cell surface expression of SIRP␤1 was significantly lower in Jurkat cells transfected with SIRP␤1 alone, indicating that DAP12 is required for efficient cell-surface expression of SIRP␤1 (Fig. 3E).
We first examined whether SIRP␤1 can trigger tyrosine phosphorylation. Aliquots of SIRP␤1-DAP12-transfected cells were either left untreated, or stimulated for 2 or 5 min with mAb 148 and then with a cross-linking Ab and then lysed. Whole cell lysates were subsequently analyzed by antiphosphotyrosine immunoblotting. As shown in Fig. 4A, SIRP␤1 stimulation led to a substantial increase in tyrosine phosphorylation of several proteins with apparent molecular masses of ϳ40, ϳ70, and ϳ100 kDa compared with the untreated cells. To examine whether the observed 40-kDa tyrosine-phosphorylated proteins corresponded to MAPK, immunoblotting analysis was performed using mAbs specific for acti-vated forms of ERK1 and ERK2. As shown in Fig. 4B, Ab crosslinking of SIRP␤1 did induce substantial phosphorylation of the ERK1 and ERK2. Finally, we tested whether stimulation of SIRP␤1 also triggered expression of early cell surface activation markers in SIRP␤1-DAP12-Jurkat cells. As shown in Fig. 4C, after overnight culture in the presence of anti-SIRP␤1 and crosslinking Ab, CD69 expression was up-regulated ϳ2.5-fold compared with expression in untransfected cells. The signaling observed following SIRP␤1 cross-linking was strictly dependent upon DAP12 expression because SIRP␤1 single transfectants induced little or no tyrosine phosphorylation, ERK activation, or CD69 up-regulation. Because SIRP␤1 expression is higher on the cell surface of SIRP␤1-DAP12 cells than on SIRP␤1 cells, it could be argued that cross-linking of SIRP␤1 in double-transfected cells may lead to unspecific cross-linking of TCR, which is also known to up-regulate CD69. However, in control experiments, cross-linking of a highly expressed cell-surface molecule, such as MHC class I, did not lead to CD69 up-regulation (Fig. 4C).
Taken together, our results show that SIRP␤1 is an activating myeloid receptor that forms a complex with a DAP12 homodimer. Association with DAP12 is required for efficient cell-surface expression of SIRP␤1. Upon stimulation, SIRP␤1/DAP12 complex  induces tyrosine phosphorylation of a number of proteins as well as cellular activation. The observed signaling properties of SIRP␤1/DAP12 are in agreement with previous results which demonstrate that NK cell receptor/DAP12 complexes induce tyrosine phosphorylation and MAPK activation in NK cells and CD69 up-regulation in KIR2DS2-DAP12-Jurkat cells (14 -16, 18, 21-23). Interestingly, SIRP␤1 was expressed in myeloid cells, but not in T cells or NK cells. This result, together with the recent discovery of a DAP12-associated C-type lectin called MDL-1 (24), which is also expressed in myeloid cells, indicates that myeloid cells, like NK cells, express a growing number of activating receptors that need to associate with DAP12 to transduce stimulatory signals. What could be the function of these receptors? Myeloid cell activation may lead to secretion of proinflammatory cytokines and chemokines. Particularly in DCs, activation may promote maturation, up-regulation of MHC, and costimulatory molecules, allowing DCs to prime T cells. We have also observed that myeloid cells express both inhibitory and activating SIRP isotypes. It will be important to determine whether these receptors mediate opposing functions or control separate signal transduction pathways in a coordinated manner.