Hyeon-Woo Lee

Cross-linking of 4-1BB activates TCR-signaling pathways in CD8+ T lymphocytes

Cross-linking of 4-1BB, a member of the TNFR family, increased tyrosine phosphorylation of TCR-signaling molecules such as CD3ε, CD3ζ, Lck, the linker for activation of T cells, and SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76). In addition, incubation of activated CD8+ T cells with p815 cells expressing 4-1BBL led to redistribution of the lipid raft domains and Lck, protein kinase C-θ, SLP-76, and phospholipase C-γ1 (PLC-γ1) on the T cell membranes to the areas of contact with the p815 cells and recruitment of 4-1BB, TNFR-associated factor 2, and phospho-tyrosine proteins to the raft domains. 4-1BB ligation also caused translocation of TNFR-associated factor 2, protein kinase C-θ, PLC-γ1, and SLP-76 to detergent-insoluble compartments in the CD8+ T cells, and cross-linking of 4-1BB increased intracellular Ca2+ levels apparently by activating PLC-γ1. The redistribution of lipid rafts and Lck, as well as translocation of PLC-γ1, and degradation of IκB-α in response to 4-1BB were inhibited by disrupting the formation of lipid rafts with methyl-β-cyclodextrin. These findings demonstrate that 4-1BB is a T cell costimulatory receptor that activates TCR-signaling pathways in CD8+ T cells.

Bisphosphonates modulate the expression of OPG and M-CSF in hMSC-derived osteoblasts

Bisphosphonates have been known to suppress osteoclast activity, survival, and recruitment. In this study, we tested effects of BPs on expression of two critical genes for osteoclastogenesis, M-CSF, and OPG in the process of osteoblast differentiation from hMSC. (1) The cells were cultured in osteogenic induction medium together with 0 (control group) and 10–8xa0M alendronate, pamidronate for up 2 and 3xa0weeks (for real-time PCR) and 3 and 4xa0weeks (for ELISA). (2) The real-time PCR protocol for M-CSF, OPG, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) consist of 40 cycles. (3) Enzyme-linked immunosorbent assay (ELISA): the amounts of M-CSF and OPG in the culture medium were determined using commercially available ELISA kits for M-CSF and OPG. Treatment of differentiating cells with alendronate or pamidronate, nitrogen-containing BPs increase the expression of OPG, which suppresses osteoclastogenesis, whereas it decreases the expression of M-CSF, which enhances preosteoclast formation. These results suggest a new mechanism by which BPs inhibit osteoclastogenesis. Results support hypothesis that progressive accumulation of bisphosphonate in jaws causes imbalance in osteogenesis and bone absorption and collateral osteoclast–osteoblast interaction. Bisphosphonate-related osteonecrosis of jaw (BPONJ) is one of the most serious complications of bisphosphonate (BP) therapy. However, the mechanism behind the this process of BPONJ is still unclear and there are so many hypotheses. Among many hypotheses, we focused on osteoclast–osteoblast interaction in this study. The findings of this study show new light on the present BPONJ occurrence theory based on the osteoclastic activity of BPs. Also, a more advanced and developed theory for BRONJ occurrence may be obtained by combining the osteoclast inhibition mechanism and the effects on osteoblastic differentiation by BPs.

CD137 ligand-mediated reverse signals increase cell viability and cytokine expression in murine myeloid cells: involvement of mTOR/p70S6 kinase and Akt.

Cross‐linking of CD137 ligand (CD137L), a member of the TNF family, with recombinant CD137‐Fc (rCD137‐Fc) protein enhanced adherence of bone marrow‐derived macrophages, and increased the expression of ICAM‐1, IL‐1β, IL‐6, M‐CSF and phosphotyrosine proteins. In RAW264.7 cells, a murine myeloid cell line, rCD137‐Fc not only increased adherence but also cell multiplication, in a manner comparable to LPS or M‐CSF. In addition, it up‐regulated expression of IL‐1β, IL‐1 receptor antagonist, IL‐6, COX2, tenascin C, neuropeptide Y and M‐CSF mRNA. Neutralization of M‐CSF by incubating the RAW264.7 cells with anti‐M‐CSF mAb did not prevent the CD137L signal‐induced viability. Viability was blocked by PP2, an Src tyrosine kinase inhibitor, rapamycin, an mTOR inhibitor and LY294002, a PI3K inhibitor, but not by Wortmannin, another PI3K inhibitor. Cross‐linking of CD137L increased phosphorylation of Akt and p70S6 kinase. The latter was blocked by PP2, rapamycin or LY294002, but not by Wortmannin, whereas phosphorylation of Akt was blocked by LY294002 or Wortmannin. These findings demonstrate that reverse signals evoked by CD137L regulate immune functions in macrophages.

The Therapeutic Potential of 4-1BB (CD137) in Cancer

Techniques for modulating immune cells for cancer therapy have been widely studied. One key approach that is being clinically tested is developing tumor-destructive cell-mediated immune responses by regulating co-stimulatory molecules. 4-1BB (CD137), a member of the TNF receptor family, is expressed following activation of T and NK cells. Recently, it has been reported that DCs also express 4-1BB. Cross-linking of 4-1BB provides a potent co-stimulatory signal for lymphocytes via signal transduction pathways that modulate a number of cellular responses. One remarkable response is stimulation of anti-tumor activity in vivo and in vitro. We here review the potential role of 4-1BB in cancer immunotherapy focusing on the cellular and molecular mechanisms involved.

Effects of moderate intensity static magnetic fields on human bone marrow-derived mesenchymal stem cells.

This study aimed to explore effects of static magnetic fields (SMFs) of moderate intensity (3-50u2009mT) as biophysical stimulators of proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs). MSCs were exposed to SMFs of three intensities: 3, 15, and 50u2009mT. Proliferation was assessed by cell counting and bromodeoxyuridine incorporation, and differentiation by measuring alkaline phosphatase (ALP) activity, calcium content, mineralized nodule formation, and transcripts of osteogenic markers. Moderate intensity SMFs increased cell proliferation, ALP activity, calcium release, and mineralized nodule formation in a dose- and time-dependent manner, which peaked at 15u2009mT. In the same manner, they upregulated expression of osteogenic marker genes such as ALP, bone sialoprotein 2 (BSP2), collagen1a1 (COL1a1), osteocalcin (OCN), osteonectin (ON), osteopontin (OPN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) with peak at 15u2009mT after 14 or 21 days of exposure. Results demonstrate that moderate intensity SMFs promote proliferation and osteoblastic differentiation of MSCs. This effect could help to improve MSC responses during osseointegration between a dental implant and surrounding bone.

Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells

Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit(+) Lin(-) HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit(+) Lin(-) HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.

CD137L-and RANKL-mediated reverse signals inhibit osteoclastogenesis and T lymphocyte proliferation

Members of the tumor necrosis factor-related family of ligands and receptors appear to be critical regulators of osteoclastogenesis and various cellular responses in T cells. In the present study, we have investigated CD137L and RANKL (receptor activator of nuclear factor (NF)-kappaB ligand)-induced biological responses in osteoclasts and T cells, respectively. Osteoclast-like cells were generated from murine bone marrow in the presence of RANKL and monocyte-macrophage colony-stimulating factor (M-CSF). RAW264.7 cells (murine monocytic cell line) constitutively express CD137L. Ligation of CD137L with anti-CD137L mAb (TKS-1) inhibits RANKL-induced osteoclast formation in a dose-dependent manner. Bone marrow cells expressed CD137L only when induced by treatment with M-CSF. In bone marrow cells, cross-linking of CD137L with anti-CD137L mAb (TKS-1) inhibits M-CSF/RANKL-evoked formation of multi-nucleated osteoclasts. Further we examined RANKL-mediated regulation of T cell proliferation. Both mouse CD4(+) and CD8(+) T cells expressed RANKL following their activation by anti-CD3 Ab and anti-CD137 Ab. Ligation of RANKL with OPG-Fc, the decoy receptor for RANKL, inhibited both mouse CD4(+) and CD8(+) T cell proliferation. From the above results, we suggest that the cellular responses in cell-to-cell interactions between T cells and osteoclasts are regulated through reciprocal regulations of CD137/CD137L and RANK/RANKL interactions.

4-1BB triggers IL-13 production from T cells to limit the polarized, Th1-mediated inflammation

4‐1BB (CD137) triggering typically induces Th1 response by increasing IFN‐γ from T cells upon TCR ligation. We found recently that 4‐1BB costimulation increased the expression of IL‐13 from CD4+ T cells, as well as CD8+ T cells. The enhanced IL‐13 expression by agonistic anti‐4‐1BB treatment was mediated via MAPK1/2, PI‐3K, JNK, mammalian target of rapamycin, NF‐AT, and NF‐κB signaling pathways. The signaling for IL‐13 induction was similar to that of IFN‐γ production by anti‐4‐1BB treatment in T cells. When the anti‐4‐1BB‐mediated IL‐13 expression was tested in an in vivo viral infection model such as HSV‐1 and vesicular stomatitis virus, 4‐1BB stimulation enhanced IL‐13 expression of CD4+ T, rather than CD8+ T cells. Although IL‐13 was enhanced by anti‐4‐1BB treatment, the increased IL‐13 did not significantly alter the anti‐4‐1BB‐induced Th1 polarization of T cells—increase of T‐bet and decrease of GATA‐3. Nevertheless, anti‐4‐1BB treatment polarized T cells excessively in the absence of IL‐13 and even became detrimental to the mice by causing liver inflammation. Therefore, we concluded that IL‐13 was coinduced following 4‐1BB triggering to maintain the Th1/2 balance of immune response.

Enhanced cytotoxicity of 5-FU by bFGF through up-regulation of uridine phosphorylase 1

Anti cancer agent 5-FU (Fluoro Uracil) is a prodrug that can be metabolized and then activated to interfere with RNA and DNA homeostasis. However, the majority of administered 5-FU is known to be catabolized in vivo in the liver where Dihydropyrimidine dehydrogenase (DPD) is abundantly expressed to degrade 5-FU. The biological factors that correlate with the response to 5-FU-based chemotherapy have been proposed to include uridine phosphorylase (UPP), thymidine phosphorylase (TPP), p53 and microsatellite instability. Among these, the expression of UPP is known to be controlled by cytokines such as TNF-α, IL1 and IFN-γ. Our preliminary study using a DNA microarray technique showed that basic fibroblast growth factor (bFGF) markedly induced the expression of UPP1 at the transcription level. In the present study, we investigated whether bFGF could modulate the expression of UPP1 in osteo-lineage cells and examined the sensitivity of these cells to 5-FU mediated apoptosis.

An Overview of Pathogen Recognition Receptors for Innate Immunity in Dental Pulp

Pathogen recognition receptors (PRRs) are a class of germ line-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of PRRs is crucial for the initiation of innate immunity, which plays a key role in first-line defense until more specific adaptive immunity is developed. PRRs differ in the signaling cascades and host responses activated by their engagement and in their tissue distribution. Currently identified PRR families are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain-like receptors (NLRs), the retinoic acid-inducible gene-I-like receptors (RLRs), and the AIM2-like receptor (ALR). The environment of the dental pulp is substantially different from that of other tissues of the body. Dental pulp resides in a low compliance root canal system that limits the expansion of pulpal tissues during inflammatory processes. An understanding of the PRRs in dental pulp is important for immunomodulation and hence for developing therapeutic targets in the field of endodontics. Here we comprehensively review recent finding on the PRRs and the mechanisms by which innate immunity is activated. We focus on the PRRs expressed on dental pulp and periapical tissues and their role in dental pulp inflammation.