Anaphylaxis is a life-threatening immediate hypersensitivity reaction triggered by antigen capture by immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcvarepsilonRI) on mast cells. However, the regulatory mechanism of mast cell activation is not completely understood. Here we identify an immunoglobulin-like receptor, Allergin-1, that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain, and show it was preferentially expressed on mast cells. Mouse Allergin-1 recruited the tyrosine phosphatases SHP-1 and SHP-2 and the inositol phosphatase SHIP. Coligation of Allergin-1 and FcvarepsilonRI suppressed IgE-mediated degranulation of bone marrow-derived cultured mast cells. Moreover, mice deficient in Allergin-1 developed enhanced passive systemic and cutaneous anaphylaxis. Thus, Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice.
The myeloid-associated Ig-like receptor family (CD300) consists of nine activating or inhibitory cell surface receptors preferentially expressed on myeloid cells and are encoded by the genes in a small cluster on mouse chromosome 11. One of the receptors, CD300LF (MAIR-V), has a long cytoplasmic tail containing two consensus ITIMs and an immunoreceptor tyrosine-based switching motif, suggesting that CD300LF regulates the activation of myeloid cells. However, the functional characteristics of this receptor are still incompletely understood. In this study, we demonstrate that cross-linking CD300LF with anti-CD300LF mAb induced cell death in peritoneal macrophages as well as in several transfectants expressing CD300LF. CD300LF-mediated cell death was dependent on the cytoplasmic region but did not require an ITIM or immunoreceptor tyrosine-based switching motif. Scanning electron microscopy revealed a loss of blebs from the surface of the dead cells mediated by CD300LF, a morphological feature similar to that observed in apoptotic cells. However, CD300LF-mediated cell death was not inhibited by a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, or autophagy inhibitors, 3-methyladenine or N-acetyl-l-cystein. Moreover, the splicing isoform of a transcription factor, X-box binding protein-1, which is produced in dead cells as a response to endoplasmic reticulum stress, was not detected. Together, these results indicate that CD300LF mediates caspase and endoplasmic reticulum stress-independent cell death by a novel mechanism.
Invariant natural killer T cells (iNKT cells) are activated by lipid antigens presented by CD1d, but the pathway leading to lipid antigen presentation remains incompletely characterized. Here we show a whole-genome siRNA screen to elucidate the CD1d presentation pathway. A majority of gene knockdowns that diminish antigen presentation reduced formation of glycolipid-CD1d complexes on the cell surface, including members of the HOPS and ESCRT complexes, genes affecting cytoskeletal rearrangement, and ABC family transporters. We validated the role in vivo for the multidrug resistance protein 1 (Mrp1) in CD1d antigen presentation. Mrp1 deficiency reduces surface clustering of CD1d, which decreased iNKT cell activation. Infected Mrp1 knockout mice show decreased iNKT cell responses to antigens from Streptococcus pneumoniae and were associated with increased mortality. Our results highlight the unique cellular events involved in lipid antigen presentation and show how modification of this pathway can lead to lethal infection.
Although airway hyperresponsiveness (AHR) is a prominent feature of asthma, how it is regulated remains incompletely understood. Allergin-1, an inhibitory immunoglobulin-like receptor containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), is expressed on human and mouse mast cells (MCs) and inhibits high-affinity receptor for IgE (FcεRI)-mediated signaling. Using MC-deficient KitW-sh/W-sh mice and Mas-TRECK mice, which carries a diphtheria toxin (DT)-induced MC deletion system based on il4 enhancer elements, we demonstrate here that MCs are involved in the induction of house dust mite (HDM)-induced AHR. Further, we show that MCs deficient in Allergin-1 exacerbated HDM-induced AHR, but had no effect on airway inflammation. In vitro analysis demonstrated that Allergin-1 inhibited anti-HDM allergen antibody-dependent HDM allergen-mediated degranulation by MCs. Thus, Allergin-1 on MCs plays an important role in the regulation of HDM-induced AHR.
House dust mite (HDM) allergens are leading causes of allergic asthma characterized by Th2 responses. The lung-resident CD11b + dendritic cells (DCs) play a key role in Th2 cell development in HDM-induced allergic asthma. However, the regulatory mechanism of HDM-induced CD11b + DC activation remains incompletely understood. In this study, we demonstrate that mice deficient in an inhibitory immunoreceptor, Allergin-1, showed exacerbated HDM-induced airway eosinophilia and serum IgE elevation. By using bone marrow-chimeric mice that were sensitized with adoptively transferred HDM-stimulated wild-type or Allergin-1-deficient CD11b + bone marrow-derived cultured DCs (BMDCs), followed by challenge with HDM, we show that Allergin-1 on the BMDCs suppressed HDM-induced allergic airway inflammation. We also show that Allergin-1 suppressed HDM-induced PGE 2 production from CD11b + BMDCs by inhibiting Syk tyrosine kinase activation through recruitment of SHP-1, subsequently leading to negative regulation of Th2 responses. These results suggest that Allergin-1 plays an important role in regulation of HDM-induced allergic airway inflammation.
Immunohistochemical studies were made on the regeneration of T cells of host- and donor-type in the thymus and spleen of radiation bone marrow chimeras by using B10- and B10.BR-Thy-1 congenic mice. Both the thymic cortex and the medulla were first repopulated with thymocytes of irradiated host origin, restoring the normal histologic appearance by days 11 to 14, regardless of the H-2 compatibility between the donor and the host. In Thy-1 congenic chimeras, thymocytes of donor bone marrow origin, less than 100 cells in one thymic lobe, were first recognized at day 7, when the thymus involuted to the smallest size after the irradiation. The thymocytes of donor-type then proliferated exponentially, showing a slightly faster rate when higher doses of bone marrow cells were used for reconstitution, reaching a level of 100 million by day 17 and completely replacing the cortical thymocytes of host origin by day 21. The replacement of cortical thymocytes started from the subcapsular layer in a sporadic manner. The replacement of medullary thymocytes from host- to donor-type occurred gradually between days 21 and 35, after the replacement in the cortex was completed. In the spleen, about 1 million survived cells were recovered at day 3 after the irradiation, and approximately 60% of them were shown to be host-type T cells that were observed in the white pulp areas. The host-type T cells in the spleen increased gradually after day 10, due to the influx of host-type T cells from the regenerating thymus. Thus a pronounced increase of T cells of host-type was immunohistochemically observed in the splenic white pulp between days 21 and 28, when thymocytes of host-type were present mainly in the thymic medulla. These host-type T cells were shown to persist in the spleen for a long time, as long as 420 days after the treatment. Phenotypically, they were predominantly Lyt-1+2+ when examined at day 28, but 5 mo later, they were about 50% Lyt-1+2+ and 50% Lyt-1+2-. Donor-type T cells in the spleen began to appear at about day 14 in chimeras that were transplanted with a larger dose of bone marrow cells, whereas this was slightly delayed in those grafted with a smaller dose of bone marrow cells, starting at about day 28.(ABSTRACT TRUNCATED AT 400 WORDS)
Although most T lymphocytes recognize peptides presented by major histocompatibility complex (MHC)-encoded class I and class II molecules, there also are significant populations of T cells that recognize nonpeptide antigens. Prominent among these T lymphocytes are the type I or invariant natural killer T cells (iNKT cells). These T lymphocytes recognize lipids presented by CD1d, a nonpolymorphic, class I-like, antigen-presenting molecule. We have carried out a whole genome siRNA screen in a macrophage cell line for genes that affect the presentation of a potent glycosphingolipid antigen, GalGal Cer, to iNKT cells. In order to stimulate iNKT cells, this antigen requires internalization and lysosomal carbohydrate antigen processing to remove the terminal galactose. After several rounds of validation, functional classification and gene expression analysis, we have identified genes that lead to altered antigen presentation in macrophages. A majority of the identified genes do not perturb surface CD1d expression, but we can demonstrate they effect the formation of surface CD1d complexes with the stimulating glycolipid. Members of the HOPS and ESCRT complexes have been identified to play an important role in Cd1d dependent antigen presentation. Interestingly, our data show that ABCC1 and several other ABC family transporters are involved in lipid antigen presentation by CD1d. CD1d and MHC-II antigen presentation pathways both depends on antigen loading in the lysosome, therefore we also analyzed the role of these genes in MHC-II antigen presentation. Although the majority of genes identified doesn’t affect MHC-II antigen presentation, there are a few molecules that affect both antigen presentation pathways.
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