Eun-Ju Kim

CD300F Blocks Both MyD88 and TRIF-Mediated TLR Signaling through Activation of Src Homology Region 2 Domain-Containing Phosphatase 1

CD300F is known to exhibit inhibitory activity in myeloid cells through its intracellular ITIM. To investigate the effect of CD300F stimulation on TLR signaling, the human acute monocytic leukemia cell line THP-1 was treated with CD300F-specific mAbs or two synthetic peptides that represented the ITIM-like domains of CD300F. Treatment with these agents blocked TLR2-, 3-, 4-, and 9-mediated expression of proinflammatory mediators such as IL-8 and matrix metalloproteinase-9. The luciferase reporter assay in 293T cells and Western blot analysis of THP-1 cells revealed that these inhibitory actions were effective in pathways involving MyD88 and/or TRIF of TLR signaling and associated with marked suppression of IκB kinase activation, phosphorylation/degradation of IκB, and subsequent activation of NF-κB. Use of specific inhibitors and immunoprecipitation analysis further indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatase-1, a protein tyrosine phosphatase with inhibitory activity in hematopoietic cells. These data indicate that CD300F is an active regulator of TLR-mediated macrophage activation through its association with Src homology 2 domain-containing phosphatase-1 and that the synthetic peptides can be applied for the regulation of immune responses that are induced by TLRs.

CD300a and CD300f differentially regulate the MyD88 and TRIF-mediated TLR signalling pathways through activation of SHP-1 and/or SHP-2 in human monocytic cell lines

CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4+ T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll‐like receptor (TLR) ‐mediated macrophage activation has not been investigated. The human monocytic cell lines THP‐1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4‐mediated and TLR9‐mediated expression of pro‐inflammatory mediators without affecting TLR3‐mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor‐κB binding sites indicated that co‐transfection of CD300f blocked reporter expression induced by over‐expression of both myeloid differentiation factor 88 (MyD88) and toll‐interleukin 1 receptor‐domain‐containing adapter‐inducing interferon‐β (TRIF), whereas CD300a blocked only MyD88‐induced events. Synthetic peptides representing immunoreceptor tyrosine‐based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88‐mediated signalling was regulated mainly by SH2‐containing tyrosine phosphatase 1 (SHP‐1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF‐mediated pathway required the combined action of SHP‐1 and SHP‐2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF‐mediated TLR signalling pathways through differential activation of SHP‐1 and SHP‐2.

Highly sensitive and novel point-of-care system, aQcare Chlamydia TRF kit for detecting Chlamydia trachomatis by using europium (Eu) (III) chelated nanoparticles.

Background The bacterium Chlamydia trachomatis is one of the leading causes of sexually transmitted diseases worldwide. Since no simple and effective tool exists to diagnose C. trachomatis infections, we evaluated a novel point-of-care (POC) test, aQcare Chlamydia TRF kit, which uses europium-chelated nanoparticles and a time-resolved fluorescence reader. Methods The test performance was evaluated by comparing the results obtained using the novel POC testing kit with those obtained using a nucleic acid amplification test (NAAT), using 114 NAAT-positive and 327 NAAT-negative samples. Results The cut-off value of the novel test was 20.8 with a detection limit of 0.27 ng/mL. No interference or cross-reactivity was observed. Diagnostic accuracy showed an overall sensitivity of 93.0% (106/114), specificity of 96.3% (315/327), positive predictive value (PPV) of 89.8% (106/118), and negative predictive value (NPV) of 97.5% (315/323). The sensitivity of the novel test was much higher than that of currently available POC tests. Furthermore, the relative ease and short turnaround time (30 min) of this assay enables C. trachomatis-infected individuals to be treated without a diagnostic delay. Conclusions This simple and novel test is a potential tool to screen a larger population, especially those in areas with limited resources.

Stimulation of Fas (CD95) induces production of pro-inflammatory mediators through ERK/JNK-dependent activation of NF-κB in THP-1 cells.

Although Fas is known to be an apoptosis triggering molecule, accumulating studies indicate that Fas has non-apoptotic functions in certain cases. In an effort to identify the role of Fas in macrophage function, the human macrophage-like cell line THP-1 was analyzed after treatment with agonistic anti-Fas monoclonal antibody or co-incubation with FasL-expressing cells. Stimulation of Fas induced the expression of pro-inflammatory mediators such as matrix metalloproteinase (MMP)-9 and IL-8. The specificity of the reaction was confirmed by the transfection of Fas-specific siRNAs which resulted in a suppression of Fas expression as well as the responsiveness to the agonistic antibody. Utilization of various signaling inhibitors and ELISA-based NF-κB DNA binding assay demonstrated that the signaling initiated from Fas is mediated by mitogen activated protein kinases (MAPKs) including extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) which induce subsequent activation of NF-κB. Furthermore, mixed cell culture experiment demonstrated that Fas can be activated through interaction with membrane-bound form of FasL during cell-to-cell interaction. These data indicate that Fas plays a role as an activation inducing molecule through interaction with its counterpart and Fas-mediate events are mediated by ERK/JNK MAPKs which subsequently activate NF-κB for the transcriptional activation of pro-inflammatory mediators.

BAFF and APRIL induce inflammatory activation of THP-1 cells through interaction with their conventional receptors and activation of MAPK and NF-κB

BackgroundBAFF and APRIL, as closely related members of the TNF superfamily, are important regulators of B-cell survival. They share two receptors, TACI and BCMA, and BAFF can stimulate an additional receptor, BAFF-R. Although these molecules have been under intense investigation in order to identify their role in immune reactions, the effect of BAFF and APRIL on macrophage function has not been tested.MethodsThe human macrophage-like cell line THP-1, which expresses BAFF/APRIL and all three of their receptors, was stimulated with recombinant human BAFF or APRIL or monoclonal antibodies against the receptors and the resulting cellular responses were investigated. Treatment of the cells with these agents induced the expression of pro-inflammatory mediators such as matrix metalloproteinase (MMP)-9 and IL-8. Suppression of the expression of these receptors using specific siRNAs resulted in the blocking of the response, confirming that these responses require specific interaction between BAFF/APRIL and their receptors. Inhibitors of MAPK and NF-κB blocked the expression of IL-8. Furthermore, inhibitors of MAPK blocked the BAFF-induced specific DNA binding activity of NF-κB.ConclusionThese data indicate that BAFF and APRIL can induce inflammatory activation of THP-1 cells through the activation of MAPK, which leads to the subsequent activation of NF-κB.

Stimulation of FasL Induces Production of Proinflammatory Mediators Through Activation of Mitogen-Activated Protein Kinases and Nuclear Factor-κB in THP-1 Cells

FasL is a member of the tumor necrosis factor (TNF) superfamily involved in the various immune reactions such as activation-induced cell death, cytotoxic effector function, and establishment of immune privileged sites through its interaction with Fas. On the other hand, FasL is known to transmit a reverse signal that serves as a T cell co-stimulatory signal. However, the role of FasL-mediated reverse signaling in macrophage function has not been investigated. In order to investigate the presence of FasL-mediated signaling in macrophages, the human macrophage-like cell line THP-1 was analyzed after treatment with FasL ligating agents such as recombinant Fas:Fc fusion protein or anti-FasL monoclonal antibody. Stimulation of FasL induced the expression of proinflammatory mediators such as matrix metalloproteinase-9, TNF-α, and IL-8. The specificity of the reaction was confirmed by the transfection of the FasL-specific siRNAs, which suppressed FasL expression as well as the production of proinflammatory mediators. Utilization of various inhibitors of signaling adaptors and ELISA-base nuclear factor (NF)-κB binding assay demonstrated that the signaling initiated from FasL is mediated by mitogen-activated protein kinases including extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase which induce subsequent activation of NF-κB. These data indicate that membrane expression of FasL and its interaction with its counterpart may contribute to the inflammatory activation of macrophages during immune reactions or pathogenesis of chronic inflammatory diseases.

Synthetic peptides containing ITIM-like sequences of IREM-1 inhibit BAFF-mediated regulation of interleukin-8 expression and phagocytosis through SHP-1 and/or PI3K.

B‐cell activation factor of the tumour necrosis factor family (BAFF), an important regulator of B‐cell survival, has recently been found to be expressed on the surface of murine and human macrophages and engagement with its receptor was shown to trigger induction of pro‐inflammatory mediators and block phagocytic activity. In an effort to generate immunomodulatory agents that can regulate BAFF‐mediated signal, decapeptides representing the intracellular immunoreceptor tyrosine‐based inhibitory motifs (ITIMs) of immune receptor expressed on myeloid cells (IREM)‐1, an inhibitory transmembrane protein expressed on myeloid cells, were synthesized in conjugation with HIV‐transactivator of transcription (TAT)48–57, which facilitates the internalization of peptides into cells. Interestingly, all five of these synthetic peptides, representing the five ITIM‐like sequences present in the cytoplasmic tail of IREM‐1, exhibited inhibitory action against BAFF‐mediated induction of matrix metalloproteinase‐9 and interleukin‐8 expression. Inhibitor assay and immunoprecipitation assay followed by Western blotting demonstrated that the inhibitory action was mediated by Src homology 2 (SH2)‐containing tyrosine phosphatase (SHP)‐1 and/or phosphoinositide 3‐kinase (PI3K). ELISA‐based nuclear factor‐κB DNA binding assay observed that the synthetic peptides blocked the activation of nuclear factor‐κB in an SHP‐1 and phosphoinositide 3‐kinase‐dependent manner. Three of these synthetic peptides exhibited varying degrees of inhibitory action against BAFF‐mediated blockage of phagocytosis in a SHP‐1 and PI3K‐dependent manner. These data indicate that the synthetic peptides are capable of blocking BAFF‐mediated regulation of macrophage activities through the activation of SHP‐1 and PI3K as well as inhibition of nuclear factor‐κB activation.

TL1A induces the expression of TGF-β-inducible gene h3 (βig-h3) through PKC, PI3K, and ERK in THP-1 cells

βig-h3, an extracellular matrix protein involved in various biological processes including cellular growth, differentiation, adhesion, migration, and angiogenesis, has been shown to be elevated in various inflammatory processes. Death receptor 3 (DR3), a member of the TNF-receptor superfamily that is expressed on T cells and macrophages, is involved in the regulation of inflammatory processes through interaction with its cognate ligand, TNF-like ligand 1A (TL1A). In order to find out whether the TL1A-induced inflammatory activation of macrophages is associated with the up-regulation of βig-h3 expression, the human acute monocytic leukemia cell line (THP-1) was stimulated with either recombinant human TL1A- or DR3-specific monoclonal antibodies. Stimulation of DR3 up-regulated the intracellular levels as well as the secretion of βig-h3. Utilization of various inhibitors and Western blot analysis revealed that activation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), phosphoinositide kinase-3 (PI3K), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is required for TL1A-induced βig-h3 expression. PKC appears to be the upstream regulator of PI3K since the presence of PKC inhibitor blocked the phosphorylation of AKT without affecting ERK phosphorylation. On the other hand, suppression of either PI3K or ERK activity resulted in the suppression of IκB phosphorylation. These findings indicate that TL1A can regulate the inflammatory processes through modulation of the βig-h3 expression through two separate pathways, one through PKC and PI3K and the other through ERK, which culminates at NF-κB activation.

SHPS-1 and a synthetic peptide representing its ITIM inhibit the MyD88, but not TRIF, pathway of TLR signaling through activation of SHP and PI3K in THP-1 cells.

BackgroundSrc homology 2 domain-containing protein tyrosine phosphatase substrate (SHPS)-1 is known to have regulatory effects on myeloid cells. However, its role in macrophage activation is not clearly understood.Methods and resultsIn order to investigate the role of SHPS-1 in Toll-like receptor (TLR)-mediated activation, human monocytic cell lines were treated with anti-SHPS-1 monoclonal antibody. The triggering of SHPS-1 blocked the expression of IL-8 and TNF-α in cells treated with a TLR4 ligand that induces a signaling pathway involving myeloid differentiation factor 88 (MyD88) and Toll-interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF). Interestingly, SHPS-1 inhibited TLR9/MyD88-mediated, but not TLR3/TRIF-mediated, expression of IL-8. Accordingly, a synthetic peptide representing the immunoreceptor tyrosine-based inhibition motif (ITIM) of SHPS-1 suppressed only the MyD88 pathway. Utilization of specific inhibitors and Western blot analysis indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatases (SHPs) and phosphoinositide 3-kinase (PI3K).ConclusionSHPS-1 negatively regulates the MyD88-dependent TLR signaling pathway through the inhibition of NF-κB activation.

A novel derivative of decursin, CSL-32, blocks migration and production of inflammatory mediators and modulates PI3K and NF-κB activities in HT1080 cells

Decursin and related coumarin compounds in herbal extracts have a number of biological activities against inflammation, angiogenesis and cancer. We have analysed a derivative of decursin (CSL‐32) for activity against inflammatory activation of cancer cells, such as migration, invasion and expression of pro‐inflammatory mediators. The human fibrosarcoma cell line, HT1080, was treated with TNFα (tumour necrosis factor α) in the presence or absence of CSL‐32. The cellular responses and modification of signalling adapters were analysed with respect to the production of pro‐inflammatory mediators, as also migration, adhesion and invasion. Treatment of HT1080 cells with CSL‐32 inhibited their proliferation, without affecting cell viability, and TNFα‐induced expression of pro‐inflammatory mediators, such as MMP‐9 (matrix metalloproteinase‐9) and IL‐8 (interleukin‐8). CSL‐32 also suppressed phosphorylation and degradation of IκB (inhibitory κB), phosphorylation of p65 subunit of NF‐κB (nuclear factor‐κB) and nuclear translocation of NF‐κB, which are required for the expression of pro‐inflammatory mediators. In addition, CSL‐32 inhibited invasion and migration of HT1080 cells, as also cellular adhesion to fibronectin, an ECM (extracellular matrix) protein. CSL‐32 treatment resulted in a dose‐dependent inhibition of PI3K (phosphoinositide 3‐kinase) activity, required for the cellular migration. The analyses show that CSL‐32 inhibits processes associated with inflammation, such as the production of pro‐inflammatory mediators, as well as adhesion, migration and invasion in HT1080 cells.