Satoko Tahara-Hanaoka

CD226 (DNAM-1) Is Involved in Lymphocyte Function–associated Antigen 1 Costimulatory Signal for Naive T Cell Differentiation and Proliferation

Upon antigen recognition by the T cell receptor, lymphocyte function–associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12–independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1–mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation.

Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

Lentiviral vector-mediated transduction of murine CD34- hematopoietic stem cells

OBJECTIVE Efficient gene transfer into murine hematopoietic stem cells (HSCs) provides a powerful tool for exploring hematopoietic stem cell biology. In this study, we evaluated the efficiency of lentiviral vector-mediated gene transfer into murine CD34(-/low)c-Kit(+)Sca-1(+)Lin(-) (CD34(-) KSL) cells that are highly enriched for HSCs. MATERIALS AND METHODS FACS-sorted CD34(-) KSL cells were transduced with the vesicular stomatitis virus G glycoprotein-pseudotyped HIV-1-based lentiviral vector containing the green fluorescent protein (GFP) gene under the control of the cytomegalovirus promoter, and then 50 transduced cells were transplanted into lethally irradiated mice. Transduction efficiency was assessed by FACS analysis for GFP expression in peripheral blood (PB) cells. FACS-sorted GFP(+) KSL bone marrow (BM) cells from primary recipients were used for secondary transplantation, and GFP expression in PB cells of reconstituted mice was analyzed by FACS. RESULTS GFP expression was detected in PB cells of all primary recipients (n = 10) at an average of 40% (range 26-58%) when the lentiviral vector containing the woodchuck hepatitis virus posttranscriptional regulatory element was used. GFP(+) cells were found in multilineage cells in PB, BM, spleen, and thymus for at least 8 months posttransplantation. In secondary recipients, donor-derived GFP(+) KSL BM cells could reconstitute and GFP expression was detected in both myeloid and lymphoid cells in PB. CONCLUSION Our results indicate that lentiviral vectors can efficiently transduce highly enriched murine HSCs and sustain long-term expression of the transgene in the multilineage differentiated progeny in reconstituted mice.

Critical role of M. tuberculosis for dendritic cell maturation to induce collagen-induced arthritis in H-2b background of C57BL/6 mice

Collagen‐induced arthritis (CIA) can be induced even in CIA‐resistant H‐2b background of C57BL/6 mice when these mice are immunized with type II collagen (CII) emulsified in complete Freunds adjuvant (CFA) containing high, but not low, dose of Mycobacterium tuberculosis. Here, we investigated the pathogenesis of CIA in C57BL/6 mice induced by the immunizing protocol. We examined expressions of cytokines, costimulatory molecules and major histocompatibility complex (MHC) class II in draining lymph nodes (DLN) in CIA‐induced C57BL/6 mice by quantitative reverse transcription–polymerase chain reaction. We also examined an effect of M. tuberculosis on the expression of these molecules on dendritic cells (DC) in vitro by flow cytometry. We finally examined an effect of M. tuberculosis in CFA used for immunization with CII antigen on priming of CD4+ helper T cells specific to CII in DLN of CIA‐induced C57BL/6 mice. The expression of interferon‐γ (IFN‐γ), Interleukin‐12p40 (IL‐12p40), costimulatory molecules CD40, CD80 and CD86 and MHC class II were up‐regulated in DLN of CIA‐induced C57BL/6 mice. Expressions of these costimulatory molecules were also up‐regulated on DC after stimulation with high, but not low, dose of M. tuberculosis in vitro. Furthermore, priming of CD4+ helper T cells specific to CII antigen in DLN required immunization with CII using CFA containing high, but not low, dose of M. tuberculosis. These results suggested that high dose of M. tuberculosis were required for maturation of DC enough to prime CD4+ helper T cells specific to CII antigen in DLN of H‐2b background of C57BL/6 mice.

Dual Assemblies of an Activating Immune Receptor, MAIR-II, with ITAM-Bearing Adapters DAP12 and FcRγ Chain on Peritoneal Macrophages

Certain activating immune receptors expressed on myeloid cells noncovalently associate with either DAP12 or FcεRIγ (FcRγ 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-α 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 FcRγ chain homodimers in peritoneal macrophages. LPS enhanced the FcRγ chain expression and FcRγ 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 FcRγ chain as well as DAP12. Our findings present the first case of an activating receptor that uses either DAP12 or FcRγ chain as a signaling adapter. The FcRγ chain may provide cooperation with and/or compensation for DAP12 in MAIR-II-mediated inflammatory responses by peritoneal macrophages.

Identification of the Fcα/μR isoform specifically expressed in the kidney tubules

Fcalpha/muR is expressed not only in lymphoid, but also in non-lymphoid organs, including kidney. However, molecular and functional characteristics of Fcalpha/muR, particularly expressed in non-lymphoid organs, have remained unclear. Here we identified an isoform of Fcalpha/muR in the murine kidney on the C57BL/6J background. The kidney expressed only the isoform, which was not expressed in other lymphoid and non-lymphoid organs, and this isoform binds both IgA and IgM. Immunohistochemical analyses suggested that the Fcalpha/muR isoform was expressed in the tubular epithelial cells, but not in the glomeruli. This was confirmed by flow cytometry analysis of isolated tubular epithelial cells and by RT-PCR analyses using the separately excised glomerular and tubular regions by the laser microdissection system. These results suggest that Fcalpha/muR may not be involved in IgA deposition in glomerular mesangium cells in IgA nephropathy. Rather, it may play an important role in immunity in the renal tubular regions.

Differential Level in Co‐Down‐Modulation of CD4 and CXCR4 Primed by HIV‐1 gp120 in Response to Phorbol Ester, PMA, among HIV‐1 Isolates

HIV‐1 enters cells through interacting with cell surface molecules such as CD4 and chemokine receptors. We generated recombinant soluble gp120s derived from T‐cell line‐tropic (T‐tropic) and macrophage‐tropic (M‐tropic) HIV‐1 strains using a baculovirus expression system and investigated the association of CD4‐gp120 complex with the chemokine receptor and/or other surface molecule(s). For monitoring the co‐down‐modulations of the CD4‐gp120 complex, a cytoplasmic domain deletion mutant (tailless CD4), which is not capable of undergoing down‐modulation by itself in response to phorbol ester PMA, was used. Our studies revealed both cell‐type and HIV‐1 strain‐specific differences. We found that T‐tropic gp120s were capable of priming co‐down‐modulation with tailless CD4 by interacting with CXCR4, whereas M‐tropic SF162 gp120 could not after PMA treatment even in the presence of CCR5. Among the T‐tropic HIV‐1 envelopes, IIIB gp120 was the most potent. Furthermore, the ability of gp120 to prime the PMA induced co‐down‐modulation of tailless CD4 appeared to be dependent on the concentration of the principal coreceptor CXCR4. Nevertheless, the observation that IIIB gp120 strongly primed tailless CD4 co‐down‐modulation on human osteosarcoma HOS cells that express undetectable levels of surface CXCR4 raised the possibility that membrane component(s) other than those recently identified can be involved in down‐modulation of the CD4/gp120 complexes.

Comment on “CD226 Is Specifically Expressed on the Surface of Th1 Cells and Regulates Their Expansion and Effector Functions”

The Th1/Th2 cell plays a distinct role in immune responses. However, cell surface markers specific for each cell type have not yet been identified. In a recent article, Dardalhon et al. ([1][1]) reported that CD226 (DNAM-1) is a cell surface molecule expressed selectively on differentiated Th1, but

Allergin-1 on mast cells suppresses house dust mite-induced airway hyperresponsiveness in mice

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.

A long-chain fatty acid elongase Elovl6 regulates mechanical damage-induced keratinocyte death and skin inflammation

Mechanical damage on the skin not only affect the barrier function but also induce various immune responses, which trigger or exacerbate the inflammation in healthy individuals and patients with inflammatory skin diseases. However, how mechanical damage-induced skin inflammation is regulated remains largely unknown. Here, we show that mechanical damage due to tape stripping triggered keratinocyte death and release of danger-associated molecular patterns (DAMPs) such as high-mobility group box 1 protein (HMGB-1) and IL-1α, which induced production of proinflammatory cytokines and chemokines IL-1β and CXCL-1 by keratinocytes in mice. We also show that a long-chain fatty acid elongase Elovl6 is expressed in keratinocytes. Mice deficient in Elovl6 had increased epidermal levels of cis-vaccenic acid (CVA); this accelerated keratinocyte death triggered by tape stripping and release of DAMPs and exacerbated skin inflammation. Our results demonstrate that Elovl6 regulates mechanical damage–triggered keratinocyte death and skin inflammation.