Hyuk Soon Kim

Tumor necrosis factor α–induced interleukin-32 is positively regulated via the Syk/protein kinase Cδ/JNK pathway in rheumatoid synovial fibroblasts

OBJECTIVE Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS. METHODS FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase-polymerase chain reaction, and small interfering RNA (siRNA) transfection. RESULTS IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor alpha (TNFalpha). TNFalpha-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase Cdelta (PKCdelta), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKCdelta mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKCdelta. CONCLUSION The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKCdelta-mediated signaling events.

Interleukin-33 stimulates formation of functional osteoclasts from human CD14(+) monocytes.

Interleukin (IL)-33 is a recently described pro-inflammatory cytokine. Here we demonstrate IL-33 as a regulator of functional osteoclasts (OCs) from human CD14+ monocytes. IL-33 stimulates formation of tartrate-resistant acid phosphatase (TRAP)+ multinuclear OCs from monocytes. This action was suppressed by anti-ST2 antibody, suggesting that IL-33 acts through its receptor ST2, but not by the receptor activator of NF-κB ligand (RANKL) decoy, osteoprotegerin, or anti-RANKL antibody. IL-33 stimulated activating phosphorylations of signaling molecules in monocytes that are critical for OC development. These included Syk, phospholipase Cγ2, Gab2, MAP kinases, TAK-1, and NF-κB. IL-33 also enhanced expression of OC differentiation factors including TNF-α receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor. IL-33 eventually induced bone resorption. This study suggests that the osteoclastogenic property of IL-33 is mediated through TRAF6 as well as the immunoreceptor tyrosine-based activation motif-dependent Syk/PLCγ pathway in human CD14+ monocytes.

Characterisation of allergen-specific responses of IL-10-producing regulatory B cells (Br1) in Cow Milk Allergy

CD19+CD5+ regulatory B cells regulate immune responses by producing IL-10. IL-10-producing regulatory B cell (Br1) responses by allergen stimulation were investigated in human food allergy. Six milk allergy patients and eight milk-tolerant subjects were selected according to DBPCFC. PBMCs were stimulated by casein in vitro and stained for intracellular IL-10 and apoptosis. In response to allergen stimulation, Br1 decreased from 26.2+/-18.3 to 15.5+/-8.9% (p=0.031, n=6) in the milk allergy group and increased from 15.4+/-9.0 to 23.7+/-11.2% (p=0.023, n=8) in the milk-tolerant group. Apoptotic non-IL-10-producing regulatory B cells increased from 21.8+/-9.3 to 38.0+/-16.1% (p=0.031, n=6) in the milk allergy group and unchanged from 28.8+/-13.8 to 28.0+/-15.0% (p=0.844, n=8) in the milk-tolerant group. Br1 may be involved in the immune tolerance of food allergies by producing IL-10 and simultaneously undergoing apoptosis in humans. The exact roles for Br1 in immune tolerance needs to be further investigated.

Morus bombycis Koidzumi extract suppresses collagen-induced arthritis by inhibiting the activation of nuclear factor-κB and activator protein-1 in mice.

ETHNOPHARMACOLOGICAL RELEVANCE Morus bombycis Koidzumi is widely distributed in Asia. In Korea, it has been used in traditional medicine because of its apparent anti-inflammatory, antioxidant, and hepatoprotective properties. AIM OF THE STUDY Although the extract of Morus bombycis Koidzumi (MB) has long since been used as a traditional anti-inflammatory medicine in Korea, its effect on arthritis remains unknown. We aimed to investigate the anti-arthritis activity of MB and the mechanism underlying it. MATERIALS AND METHODS The anti-arthritis activity of MB was assessed by using mouse models of type II collagen-induced arthritis (CIA). The clinical arthritis index and histopathological changes were evaluated in mice. Reverse transcriptase-polymerase chain reaction (RT-PCR), electrophoretic mobility shift assay (EMSA), and other biologic approaches were used for measuring the effect of MB on arthritis and understanding the underlying mechanism. RESULTS MB significantly decreased the clinical arthritis index in CIA mice; this was confirmed by examining histological changes in joints. Infiltration of immune cells, synovial hyperplasia, cartilage destruction, and bone erosion in the hind paw were largely suppressed by MB. The mRNA levels of matrix metalloproteinase (MMP)-1/MMP-3, inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6), and chemokines (macrophage inflammatory protein (MIP)-1, monocyte chemoattractant protein (MCP)-1, RANTES) were significantly suppressed by MB in a dose-dependent manner. The number of osteoclasts in the hind tibia was also significantly decreased. With regard to the mechanism, MB suppressed the activation of nuclear factor (NF)-κB and activator protein (AP)-1 in CIA mice. CONCLUSIONS MB produced an anti-arthritis effect in CIA mice by inhibiting the production of critical inflammatory mediators and osteoclasts through the downregulation of NF-κB and AP-1.

The Scaffold Protein Prohibitin Is Required for Antigen-Stimulated Signaling in Mast Cells

Targeting a scaffold protein delivered by intracellular granules in mast cells may inhibit allergic responses. How to Prohibit Mast Cell Activation Mast cells are the major effector cells of the allergic response. Binding of antigen to immunoglobulin E (IgE) molecules bound to the cell-surface, high-affinity IgE receptor FcεRI results in receptor clustering, mast cell activation, and degranulation, resulting in the release of factors that mediate the allergic response. Phosphorylation and activation of the Src family kinases Lyn and Syk are critical for mast cell activation. Kim et al. found that the scaffold protein prohibitin (PHB), which mediates functions such as mitochondrial biogenesis and transcriptional regulation (see commentary by Yurugi and Rajalingam), was abundant in intracellular granules in mouse mast cells. Stimulation of mast cells with antigen resulted in the translocation of PHB to plasma membrane lipid rafts, which was required for the association of FcεRI with Syk, the activation of Syk, and degranulation. Knockdown of PHB in mice inhibited mast cell activation and anaphylaxis, suggesting that targeting PHB therapeutically may reduce allergic responses. The protein prohibitin (PHB) is implicated in diverse cellular processes, including cell signaling, transcriptional control, and mitochondrial function. We found that PHB was abundant in the intracellular granules of mast cells, which are critical for allergic responses to antigens. Thus, we investigated whether PHB played a role in signaling mediated by the high-affinity receptor for antigen-bound immunoglobulin E (IgE), FcεRI. PHB-specific small interfering RNAs (siRNAs) inhibited antigen-mediated signaling, degranulation, and cytokine secretion by mast cells in vitro. Knockdown of PHB inhibited the antigen-dependent association of the tyrosine kinase Syk with FcεRI and inhibited the activation of Syk. Fractionation studies revealed that PHB translocated from intracellular granules to plasma membrane lipid rafts in response to antigen, and knockdown of PHB suppressed the movement of FcεRIγ and Syk into lipid rafts. Tyrosine phosphorylation of PHB by Lyn was observed early after exposure to antigen, and point mutations in PHB indicated that Tyr114 and Tyr259 were required for the recruitment of Syk to FcεRIγ and mast cell activation. In mice, PHB-specific siRNAs inhibited antigen-initiated mast cell degranulation, passive cutaneous anaphylaxis, and passive systemic anaphylaxis. Together, these results suggest that PHB is essential for FcεRI-mediated mast cell activation and allergic responses in vivo, raising the possibility that PHB might serve as a therapeutic target for the treatment of allergic diseases.

DJ-1 regulates mast cell activation and IgE-mediated allergic responses

BACKGROUND DJ-1 is an antioxidant protein known to reduce levels of reactive oxygen species (ROS), but its presence or function in mast cells and allergic diseases is unknown. OBJECTIVES We sought to determine the role and mechanism of DJ-1 in allergic responses in vitro and in vivo. METHODS ROS and DJ-1 levels in serum or culture medium were measured with ELISA kits. The role of DJ-1 was evaluated in mast cell cultures and passive cutaneous anaphylaxis in normal or DJ-1 knockout (KO) mice. The mechanism of DJ-1 action was examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biological approaches. RESULTS Patients with atopic dermatitis had increased levels of ROS and diminished levels of DJ-1. DJ-1 KO mice exhibited enhanced passive cutaneous anaphylaxis and augmented ROS levels in sera and bone marrow-derived mast cells (BMMCs). Furthermore, antigen-induced degranulation and production of TNF-α and IL-4 were significantly amplified in DJ-1 KO and anti-DJ-1 small interfering RNA-transfected BMMCs compared with that seen in wild-type (WT) BMMCs. Studies with these cells and BMMCs transfected with small interfering RNAs against the phosphatases Src homology domain 2-containing protein tyrosine phosphatase (SHP) 1 and SHP-2 revealed that the DJ-1 KO phenotype could be attributed to suppression of SHP-1 activity and enhancement of SHP-2 activity, leading to strengthened signaling through linker for activation of T cells, phospholipase Cγ, and mitogen-activated protein kinases. CONCLUSIONS A deficiency or constitutive activation of DJ-1 can have implications in mast cell-driven allergic diseases, such as asthma and anaphylaxis.

Allergen-specific B cell subset responses in cow’s milk allergy of late eczematous reactions in atopic dermatitis

B cells have regulatory functions in immune responses. Antigen-specific responses of B cell subsets by allergen stimulation ex vivo were examined in milk allergy of late eczematous reactions. Eight milk allergy subjects and 13 milk tolerant subjects were selected by DBPCFC. PBMCs were stimulated by casein ex vivo and stained for B cell subsets using monoclonal antibodies. CD19+ B cells unchanged from 8.7+/-3.8% to 8.0+/-5.1% (p=0.504, n=8) in the milk allergy group and decreased in the milk tolerant group from 8.5+/-3.2% to 5.0+/-1.6% (p=0.001, n=13). The fraction of apoptotic B cells in B cells significantly decreased 4.4+/-3.1% to 1.3+/-0.4% (p=0.027, n=4) in the allergy group and insignificantly increased from 2.8+/-0.6% to 5.4+/-2.6% (p=0.059, n=6) in the milk tolerant group. CD5+ regulatory B1 cell% in B cells decreased in milk allergy subjects from 36.2+/-5.0% to 31.0+/-5.7% (p=0.010) and unchanged in milk tolerant subjects from 41.6+/-10.2% to 43.8+/-10.0% (p=0.413). IL-10 producing CD19+CD5+ regulatory B cell% in CD19+CD5+ regulatory B cells significantly decreased from 24.9+/-6.5% to 13.8+/-5.6% (p=0.002, n=5) by casein stimulation in milk allergy group and unchanged from 44.8+/-11.3% to 43.9+/-10.0% (p=0.297, n=5) in the milk tolerant group. B cell subset responses to IL-4 and IL-5 were also similar in both groups. B cell subset changes seemed to have diagnostic value. Exact immunologic roles of regulatory CD5+ B1 cells need further investigation.

Tolerogenic effects of interferon-gamma with induction of allergen-specific interleukin-10-producing regulatory B cell (Br1) changes in non-IgE-mediated food allergy

In this study, specific oral tolerance induction using interferon-gamma (IFN-γ) could successfully treat food allergies. Allergen-specific IL-10-producing regulatory B cell (Br1) responses are characteristic in immune tolerance of food allergies. The in-vivo effects of IFN-γ on allergen-induced changes in Br1 proportion and numbers in food allergies were investigated. Oral food challenges were conducted and 20 allergic patients to cows milk were selected. Of these 20 patients, five were treated with IFN-γ and milk (SOTI group), five were treated with only milk, five were treated with only IFN-γ, and five did not receive any treatment. In addition, 10 milk-tolerant subjects were involved in this study. Peripheral blood mononuclear cells (PBMCs) were stimulated using casein and stained for CD5, CD19, annexin V, and IL-10 before and after treatment. Allergy tolerance was induced only in the SOTI group along with induction of allergen-induced Br1 changes. Thus, IFN-γ can show tolerogenic effects in vivo when introduced with an allergen, which may be at least partly due to its effect on allergen-induced Br1 responses.

Mesenteric IL-10-producing CD5+ regulatory B cells suppress cow’s milk casein-induced allergic responses in mice

Food allergy is a hypersensitive immune reaction to food proteins. We have previously demonstrated the presence of IL-10-producing CD5+ B cells and suggested their potential role in regulating cow’s milk casein allergy in humans and IgE-mediated anaphylaxis in mice. In this study, we determined whether IL-10-producing CD5+ regulatory B cells control casein-induced food allergic responses in mice and, if so, the underlying mechanisms. The induction of oral tolerance (OT) by casein suppressed casein-induced allergic responses including the decrease of body temperature, symptom score, diarrhea, recruitment of mast cells and eosinophils into jejunum, and other biological parameters in mice. Notably, the population of IL-10-producing CD5+ B cells was increased in mesenteric lymph node (MLN), but not in spleen or peritoneal cavity (PeC) in OT mice. The adoptive transfer of CD5+ B cells from MLN, but not those from spleen and PeC, suppressed the casein-induced allergic responses in an allergen-specific and IL-10-dependent manner. The inhibitory effect of IL-10-producing CD5+ B cells on casein-induced allergic response was dependent on Foxp3+ regulatory T cells. Taken together, mesenteric IL-10-producing regulatory B cells control food allergy via Foxp3+ regulatory T cells and could potentially act as a therapeutic regulator for food allergy.

Interleukin-32α production is regulated by MyD88-dependent and independent pathways in IL-1β-stimulated human alveolar epithelial cells

Interleukin (IL)-32 is a recently described cytokine that appears to play a critical role in a variety of inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, thus far, the regulation of IL-32 production has not been fully established. Here, we report on signaling pathways that regulate the production of IL-32α, the most abundant isoform, in the human alveolar epithelial cell line, A549. IL-32α was expressed and secreted by IL-1β. The IL-32 expression was attenuated by PP2 (a Src-family kinase [SFK] inhibitor), rottlerin (a protein kinase [PK] Cδ inhibitor), and LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor). Furthermore, the overexpression of Fgr rather than other SFKs upregulated IL-32α expression, while Fgr small interfering RNA (siRNA) transfection downregulated it. The suppression of Fgr with PP2 and Fgr siRNA inhibited activating phosphorylation of PKCδ and PI3K/Akt, but not IL-1 receptor-associated kinase (IRAK)1, a well-known MyD88-dependent signaling molecule, and Erk1/2, p38, and JNK. Rottlerin and PKCδ siRNA also inhibited expression of IL-32α and activation of PI3K/Akt, but not of IRAK1 and mitogen activation protein (MAP) kinases. MyD88 siRNA suppressed the expression of IL-32α and the phosphorylation of IRAK1, PI3K, and MAP kinases, but not of PKCδ. Of interest, both Fgr/PKCδ and MyD88-dependent signals regulated PI3K/Akt, suggesting that it is a crosstalk molecule. Among MyD88-dependent MAP kinases, only p38 regulated IL-32α expression and PI3K/Akt activation. With these results, we demonstrated that the expression and secretion of IL-32α are regulated by MyD88-dependent IRAK1/p38/PI3K and independent Fgr/PKCδ/PI3K pathways, and that Fgr and PKCδ are critical for the MyD88-independent IL-32α production.