Identification of Allergin-1
By using the signal sequence trap method , we identified a cDNA (MILR1) encoding an Ig-like receptor, designated Allergin-1, which consists of a 19–amino acid (aa) leader sequence, a 208-aa extracellular region composed of two Ig-like domains, a 21-aa transmembrane domain, and a 95-aa cytoplasmic domain (Fig. 1) . We also identified two cDNAs encoding Allergin-1 isoforms, which lacked the first or second Ig-like domain in the extracellular portion and were designated Allergin-1 short form 1 (Allergin-1S1) and Allergin-1 short form 2 (Allergin-1S2), respectively (Fig. 1). The extracellular portions of mouse Allergin-1 and human Allergin-1L, and those of human Allergin-1S1 and Allergin-1S2, contained six and three potential N-linked glycosylation sites, respectively. Genomic DNA database analyses demonstrated that MILR1 consists of ten exons. The genes encoding Allergin-1 (MILR1 and milr1) are located on chromosome 17q23.3 in humans and chromosome 11E1 in mice, near CD300 (or Cd300) family genes that encode Ig-like receptors mediating positive or negative signals in myeloid cells [19, 20]. MILR1 and milr1 were also close to the gene, which encodes PECAM-1 (CD31 or Cd31), an ITIM-bearing Ig-like receptor expressed on myeloid cells and platelets.
Expression of Allergin-1
Flow cytometry analyses of spleen cells demonstrated that Allergin-1 is expressed on dendritic cells, macrophages and neutrophils, but not on T, B, or natural killer (NK) cells in mice. However, Allergin-1 was most strongly expressed on mast cells in the peritoneal cavity. In contrast, Allergin-1 was not detected on basophils in the bone marrow. In agreement with mouse Allergin-1, human Allergin-1L and/or Allergin-1S1 were also expressed in myeloid cell lineages, including monocytes, neutrophils, and dendritic cells, in the peripheral blood. It was noted that, unlike mouse Allergin-1, human Allergin-1L and/or Allergin-1S1 were considerably expressed on peripheral blood basophiles .
Anti-human Allergin-1 monoclonal antibodies (mAbs) EX32 and EX33 recognize an epitope of the first and the second Ig-like domain of Allergin-1L, respectively. By using these mAbs simultaneously together with mAbs against the lineage markers (CD3, CD19, CD56, CD11b, and CD11c), c-Kit and FcεRIα, we developed a multi-color flow cytometric method to characterize the human primary mast cells in the bronchial alveolar lavage fluid (BALF). Mast cells were defined as PI−CD45+Lin−c-Kit+FcεRIα+cells, which comprised 0.153 % ± 0.041 % (n = 28) of the total cell population in BALF. We found diverse expression profiles of Allergin-1 isoform on BAL mast cells among donors; however, Allergin-1S1 was dominantly expressed on BAL-MC, compared with Allergin-1L1 or Allergin-1S2 (Fig. 2) .
Function of Allergin-1
In Vitro Analyses of Allergin-1
Allergin-1 contains immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences in the cytoplasmic portion, suggesting that Allergin-1 is tyrosine phosphorylated and recruits the SH2-containing phosphatases, such as SHP-1, SHP-2, or SHIP. Because Allergin-1 is strongly expressed on mast cells, we examined whether mouse Allergin-1 mediates an inhibitory signal against FcεRI-mediated degranulation, by using a rat basophil leukemia cell line, RBL-2H3. Co-ligation of Allergin-1 with FcεRI significantly decreased degranulation, as determined by β-hexosaminidase release from the transfectants, when compared with FcεRI stimulation alone, suggesting that mouse Allergin-1 inhibits IgE-mediated degranulation of mast cells (Fig. 3).
Because an insufficient number of human primary mast cells can be obtained to conduct the ELISA assay for chemical mediators such as histamine or β-hexosaminidase released from mast cells of BALF samples, we performed an activation assay by utilizing a multicolor flow cytometry to detect cell surface CD107a as a marker of mast cell degranulation. We detected mast cells derived from the culture of cord blood and peripheral blood stem cells that turned out to be positive for CD107a expression on the cell surface when the mast cells were stimulated with anti-TNP IgE followed by TNP-conjugated control mAb. However, CD107a+ cells were significantly decreased when FcεRIα was colligated with Allergin-1 with anti-TNP IgE and TNP-conjugated anti-Allergin-1 mAb . We then examined the inhibitory function of Allergin-1 on mast cells in BALF. BALF cells were also stimulated and then stained with anti-CD107a and mAbs for the identification of BAL mast cells, as described above, and analyzed by using multi-color flow cytometry. We detected a subpopulation of BAL mast cells that was positive for CD107a when the BALF cells were stimulated with anti-TNP IgE followed by TNP-conjugated control mAb. However, the population of CD107a+ mast cells was significantly decreased when BAL mast cells were stimulated via anti-TNP IgE and TNP-conjugated anti-Allergin-1 mAb . Since BAL mast cells also preferentially express Allergin-1S1 rather than Allergin-1L or Allergin-1S2, these results indicated that Allergin-1S1 inhibits the IgE-mediated activation of BAL mast cells.
In Vivo Analyses of Allergin-1
Since Allergin-1 inhibited FcεRI-mediated degranulation of mast cells in vitro, we next examined whether Allergin-1 was involved in passive systemic and cutaneous anaphylaxises (PSA), an IgE-mediated type 1 immediate hypersensitivity reaction. WT and Allergin-1-deficient mice were passively sensitized with anti-TNP IgE mAb and then intravenously injected with ovalbumin (OVA) or TNP-conjugated OVA (TNP-OVA). WT mice challenged with TNP-OVA showed a progressive decrease in rectal temperature to 4 °C below the basal temperature by 18 min after injection (Fig. 4). Allergin-1-deficient mice challenged with TNP-OVA showed significantly lower rectal temperatures than the corresponding WT mice (Fig. 4) .
WT and Allergin-1-deficient mice were also passively sensitized by intravenous injection of anti-dinitrophenol (DNP) IgE mAb and then challenged with epicutaneous application of dinitrofluorobenzene (DNFB) in acetone/olive oil in the left ear and acetone/olive oil alone in the right ear. The ear swelling in Allergin-1-deficient mice was significantly greater than the ear swelling in WT mice during the entire 50 h observation period after the antigen challenge . Taken together, these results suggest that Allergin-1 negatively regulates IgE-mediated mast cell activation in vivo and suppresses the type 1 immediate hypersensitivity reactions.