Yuzuru Sakamoto

Inhibitory Immunoglobulin-Like Receptors LILRB and PIR-B Negatively Regulate Osteoclast Development

Osteoclasts, multinucleated cells of myeloid-monocytic origin, are responsible for bone resorption, which is crucial for maintenance of bone homeostasis in concert with bone-forming osteoblasts of nonhematopoietic, mesenchymal origin. Receptor activator of NF-κB ligand (RANKL) and M-CSF, expressed on the surface of and secreted by osteoblasts, respectively, are essential factors that facilitate osteoclast formation. In contrast to the activation processes for osteoclast formation, inhibitory mechanisms for it are poorly understood. Herein we demonstrate that inhibitory Ig-like receptors recruiting Src homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) are expressed on osteoclast precursor cells like other myeloid cells, and that they play a regulatory role in the development of osteoclasts. We detected cell-surface expression of paired Ig-like receptor (PIR)-B and four isoforms of leukocyte Ig-like receptor (LILR)B on cultured osteoclast precursor cells of mouse and human origin, respectively, and showed that all of these ITIM-harboring inhibitory receptors constitutively recruit SHP-1 in the presence of RANKL and M-CSF, and that some of them can suppress osteoclast development in vitro. Fluorescence energy transfer analyses have suggested that the constitutive binding of either murine PIR-B or its human ortholog LILRB1 to MHC class I molecules on the same cell surface comprises one of the mechanisms for developmental regulation. These results constitute the first evidence of the regulation of osteoclast formation by cell-surface, ITIM-harboring Ig-like receptors. Modulation of these regulatory receptors may be a novel way to control various skeletal system disorders and inflammatory arthritis.

Cis binding between inhibitory receptors and MHC class I can regulate mast cell activation

Allergy is caused by immune effector cells, including mast cells and basophils. Cellular signaling that activates these effector cells is regulated by different inhibitory receptors on their surface. We show that human leukocyte immunoglobulin (Ig)-like receptor (LILR) B2 and its mouse orthologue, paired Ig-like receptor (PIR)–B, constitutively associate to major histocompatibility complex (MHC) class I on the same cell surface (in cis). The IgE-mediated effector responses were augmented in β2-microglobulin (β2m) and PIR-B–deficient mast cells. In addition, the increased cytokine production of β2m-deficient mast cells was not affected by the co-culture with MHC class I–positive mast cells, showing that less cis interaction between PIR-B and MHC class I on mast cells led to the increased cytokine release. Thus, the constitutive cis binding between LILRB2 or PIR-B and MHC class I has an essential role in regulating allergic responses.

Regulation of cytotoxic T lymphocyte triggering by PIR-B on dendritic cells

Priming of cytotoxic T lymphocytes (CTLs) by dendritic cells (DCs) is crucial for elimination of pathogens and malignant cells. To activate CTLs, DCs present antigenic peptide-complexed MHC class I molecules (MHC-I) that will be recognized by the CTLs with T cell receptors and CD8 molecules. Here we show that paired Ig-like receptor (PIR)-B, an MHC-I receptor expressed on antigen-presenting cells, can regulate CTL triggering by blocking the access of CD8 molecules to MHC-I. PIR-B-deficient DCs evoked CTLs more efficiently, leading to accelerated graft and tumor rejection. PIR-B+ non-DC transfectant cells served as less efficient stimulators and targets for CTLs than PIR-B− cells at the effector phase in vitro. On surface plasmon resonance analysis, PIR-B and CD8αα were revealed to compete in binding to MHC-I. Our results may provide a novel strategy for regulating CTL-mediated immunity and diseases in a sterical manner.

Knockdown of hypoxia-inducible factor-1α by siRNA inhibits C2C12 myoblast differentiation

We analyzed the role of Hypoxia‐inducible factor (HIF)‐1α in myoblast differentiation by examining the expression and regulation of HIF‐1α in proliferating and differentiating C2C12 myoblast, and by knocking down HIF‐1α of C2C12 myoblasts with small interfering RNA (siRNA), given that HIF‐1α has been shown to be involved in differentiative process in non‐muscle tissues. Although HIF‐1α mRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF‐1α protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF‐1α accumulated in the nuclei of myogenin‐positive myoblasts. The inhibition of proteasome in the growth phase led to HIF‐1α protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF‐1α, suppressed HIF‐1α accumulation. Therefore, we suggest that the level of HIF‐1α protein expression is regulated by a proteasome‐and chaperon‐dependent pathway in C2C12 myoblast. Knockdown of HIF‐1α effectively blocked myotube formation and myosin heavy chain (MHC) expression. Finally, HIF‐1α expression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF‐1α is required for C2C12 myogenesis in vitro, and suggest that HIF‐1α may have an essential role in regenerative muscle tissue in vivo. J. Cell. Biochem. 98: 642–649, 2006.

Differential but Competitive Binding of Nogo Protein and Class I Major Histocompatibility Complex (MHCI) to the PIR-B Ectodomain Provides an Inhibition of Cells

Binding of class I MHC molecules (MHCI) to an inhibitory receptor, PIR-B, expressed on B cells and myeloid cells provides constitutive cellular inhibition, thus ensuring peripheral tolerance. Recent unexpected findings pointed to a novel inhibitory role of PIR-B in neurite regeneration through binding to three axonal outgrowth inhibitors of myelin, including Nogo. Thus, it becomes interesting to determine whether the actions of the inhibitory myelin proteins and MHCI could coexist independently or be mutually exclusive as to the PIR-B-mediated immune and neural cell inhibition. Here, we present data supporting the competition of Nogo- and MHCI-mediated inhibition where they coexist. Kinetic analyses of Nogo and MHCI binding to the whole or a part of the recombinant PIR-B ectodomain revealed that PIR-B binds with higher affinity to Nogo than MHCI and that the MHCI binding only occurred with the N-terminal domains of PIR-B, whereas Nogo binding occurred with either the N- or C-terminal ectodomains. Importantly, kinetic tests indicated that the binding to PIR-B of Nogo and MHCI was competitive. Both endogenous and exogenous Nogo intensified the PIR-B-mediated suppression of interleukin-6 release from lipopolysaccharide-stimulated wild-type, but not PIR-B-deficient, cultured mast cells, indicating that PIR-B mediates Nogo-induced inhibition. Thus, we propose a novel mechanism by which PIR-B-mediated regulation is achieved differentially but competitively via MHCI and Nogo in cells of the immune system.

Effect of exercise, aging and functional capacity on acute secretory immunoglobulin A response in elderly people over 75 years of age.

Background:  Age‐associated decline in immune function and regulation, referred to as immunosenescence, brings about an increased incidence of infectious diseases in the aged; however, there are few data on the effect of aging and exercise on mucosal immune function in elderly people. Moreover, there is no evidence on whether the change in functional capacity affects mucosal immunity in elderly people. Therefore, the aim of the present study was to examine the effects of exercise, aging and functional capacity on mucosal immune function in elderly people over 75 years of age.