Establishment of a novel method of screening anti-allergic lactic acid bacteria

In this study, we attempted to establish a novel method of screening anti-allergic lactic acid bacteria (LAB). We cloned the human histidine decarboxylase (HDC) promoter into the promoter-less pPhi-Yellow-RPL-dest1 vector and established KU812F cells transduced with this vector (pHDCp-Phi-Yellow/KU812F). After adding LAB to these cells, the change in fluorescence intensity was monitored by flow cytometry. After screening, we identified several LAB strains that downregulated HDC promoter activity. Functional analysis of these LAB strains indicated that two LAB strains inhibited histamine release from KU812F cells, indicating that this assay system can be used to screen for anti-allergic LAB in a high-throughput manner.     

Autocrine regulation of terminal differentiation by interleukin-6 in the pluripotent KU812 cell line

The human KU812 leukemic cell line is a model for studying cell commitment towards different hematopoietic lineages. Indeed, this cell line is characterized by both a capacity for self-renewal and the ability to differentiate spontaneously along erythroid and basophilic cell lineages. In this study we show that interleukin-6 (IL-6) and its specific receptor (IL-6-R) are spontaneously expressed in the human KU812 cell line. Addition of antibody against IL-6 weakly inhibited its cell proliferation (20 to 30%) suggesting that the endogenous production of IL-6 was partially responsible for the growth of the cell line. In contrast, the spontaneous terminal differentiation of this cell line towards the erythroid and basophilic lineages was inhibited by an antibody against IL-6 and this effect was reversed by addition of recombinant human IL-6 (rIL-6). These results suggest that IL-6 is involved more in differentiation than in the proliferation of KU812 cells. After several passages, KU812 cells lose their capacity to differentiate spontaneously. In these cells, the IL-6-R was no more detectable. We therefore suggest that this loss of spontaneous differentiation is associated with an interruption of the IL-6 autocrine loop.     

IL-3 promotes basophilic differentiation of KU812 cells through high affinity binding sites

The myeloid precursor cell line KU812 exhibits a constitutive potential to differentiate into basophilic cells. In the present study, the influence of recombinant human (rh)IL-2, rhIL-3, and recombinant human granulocyte-macrophage-CSF on basophilic differentiation of KU812-F cells was studied. Of all cytokines tested, rhIL-3 induced a significant increase in formation of metachromatically granulated cells (from 10% in control cultures up to 30% in cultures supplemented with 100 U/ml of rhIL-3) as well as dose-dependent (1.5- to 3 fold) increase in cellular histamine in KU812-F cell cultures. In addition, KU812-F cells exposed to rhIL-3 bound more IgE antibody than cells cultured in control medium with up to 3.3-fold increases in the mean fluorescence intensity on days 2 and/or 5 compared with control (p less than 0.001). RhIL-3 failed to induce significant changes in expression of the Tac-reactive subunit of the IL-2R (CD25), surface aminopeptidase N (CD13), ICAM-1 Ag (CD54), or CD40 Ag on KU812-F cells. To investigate the mechanism of IL-3 action on KU812-F cells, receptor analyses were performed by using 125I-radiolabeled rhIL-3. Quantitative binding studies and Scatchard plot analyses revealed the presence of a single class of 1910 to 2460 high affinity IL-3-binding sites per KU812-F cell with an apparent dissociation constant of 1.22 to 2.35 x 10(-9) M. Together, these results show that rhIL-3 promotes basophilic differentiation of KU812-F cells through a specific receptor.     

Immunoglobulin D enhances interleukin-6 release from the KU812 human prebasophil cell line

Despite the role of secreted immunoglobulin D (IgD) remains still largely unknown, previous studies have suggested that secreted IgD could induce basophils degranulation in some allergic asthma patients. In the present study we have searched direct evidence of the action of IgD on KU812 cells, generally classified as an immature basophilic cell line. We analyzed by flow cytometry the capacity of IgD, purified from IgD myeloma sera, to bind KU812 cells. Biotinylated monomeric IgD (mIgD) and biotinylated oligomeric IgD (oIgD) could bind KU812 cells. Blocking experiments with others immunoglobulin isotypes showed that KU812 cells expressed an unspecific receptor for IgD. However, oIgD but not mIgD enhances the release of interleukin-6 (IL-6) from KU812 cells. On the other hand, mIgD and oIgD failed to induce histamine release from KU812 cells or from cord blood derived basophils. Since IL-6 is known to induce basophil differentiation, we proposed that IgD could be implicated in allergic disorders by stimulating IL-6 release by prebasophil cells, then IL-6 could further induce an autocrine maturation of the cells.     

Microarray analysis of immediate-type allergy in KU812 cells in response to fulvic acid

Fulvic acid (FA) is class of compounds of humic substances formed through the degradation of organic substances by chemical and biological processes. FA has been utilized in traditional Chinese medicine and possesses various pharmacological properties. Previously, we reported that FA extracted from solubilized excess sludge (SS-FA) had an inhibitory effect on β-hexosaminidase release in human leukemia basophilic (KU812) cells. In this study, we investigated the effects of SS-FA on the immediate-type allergic reaction and studied its possible mechanisms of action in KU812 cells following activation with phorbol myristate acetate (20 nmol L⁻¹) plus calcium ionophore A23187 (1 μmol L⁻¹) (PMACI). The inhibitory effect of SS-FA on degranulation in PMACI-stimulated KU812 cells was examined using histamine release assay. SS-FA significantly decreased the histamine release in KU812 cells at concentrations of 0.1–10.0 μg mL⁻¹. To gain more information regarding the mechanism of the suppression of degranulation following SS-FA treatment, microarray was conducted to determine which genes were differentially expressed in response to SS-FA in PMACI-activated KU812 cells. From a total of 201 genes in the DNA chip, 28 genes were up-regulated and 173 genes were down-regulated in cells pretreated with SS-FA for 15 min and stimulated with PMACI. From the 71 genes that showed more than two fold change in expression, 16 genes were significantly down-regulated that were subjected to hierarchical clustering. SS-FA affected the expression of genes that were involved in the following pathways: signal transduction, cytokine–cytokine receptor interaction, immune response, cell adhesion molecules and IgE receptor β subunit response.     

Increase in histamine content and enhancement of high affinity IgE receptor FcεRI expression in the human leukemia KU812 cells upon treatment with hydrocortisone

Hydrocortisone was investigated for its ability todifferentiate human leukemia KU812 cells into maturehematopoietic cells including basophils. Hydrocortisonetreatment increased the amount of intracellular histamine byup-regulation of L-histidine decarboxylase (HDC) mRNA andenhanced cell surface expression of the high affinity IgEreceptor FcRI. Histamine is catalyzed from L-histidine byHDC, which in blood cell types is only expressed in basophilsand mast cells. Cells, on which the FcRI expression wasenhanced by hydrocortisone, were shown to release histaminewhen stimulated with anti-IgE antibody after sensitizationwith myeloma IgE, implying that the induced FcRI moleculeswere able to transduce a signal for degranulation. Theseresults suggest that hydrocortisone promotes differentiationof KU812 cells into functionally mature basophilic cells.     

JNK-dependent NFATc1 pathway positively regulates IL-13 gene expression induced by (–)-epigallocatechin-3-gallate in human basophilic KU812 cells

(–)-Epigallocatechin-3-gallate (EGCG) has been reported to possess a wide range of biological and pharmacological properties. In this study, we investigated the effects of EGCG on IL-13 gene expression in human basophilic KU812 cells. The IL-13 mRNA expression level was dose-dependently increased by treatment with EGCG (5–20 μM) for 1 h and additional incubation in a medium for 23 h. EGCG significantly increased the intracellular peroxide level as detected by the peroxide-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate. A pharmacological experiment using catalase and a structure–activity relationship study revealed that the exogenously produced H₂O₂ significantly, but partially, contributed to the IL-13 expression as well as the intracellular oxidative status. Furthermore, EGCG at the concentration required for IL-13 up-regulation activated c-Jun NH₂-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in KU812 cells. Transfection of a JNK-specific siRNA as well as treatment with a JNK-specific inhibitor, SP600125, significantly reduced the EGCG-induced IL-13 mRNA expression, by 47.1 and 44.6%, respectively. In addition, we observed the nuclear translocation, mRNA up-regulation, and activation of DNA binding with the IL-13 promoter of nuclear factor of activated T cells (NFATc1) in the EGCG-treated cells. These data provide biological evidence that EGCG induces IL-13 mRNA expression via the JNK-dependent NFATc1 pathway in KU812 cells.     

The 67kDa laminin receptor as a primary determinant of anti-allergic effects of O-methylated EGCG

Previously we have reported that the O-methylated derivative of (−)-epigallocatechin-3-O-gallate (EGCG), (−)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3”Me), possesses anti-allergic activities such as inhibition of histamine release and suppression of the high-affinity IgE receptor (FcεRI) expression. However, the underlying mechanism is still unclear. Recently we have identified the 67 kDa laminin receptor (67LR) as a cell-surface receptor that can mediate biological activities of EGCG. Here we show that the suppression of myosin II regulatory light chain (MRLC) phosphorylation through the cell-surface binding to the 67 LR contributes to the inhibitory effect of EGCG3”Me on the histamine release from the human basophilic KU812 cells. The 67LR also mediated the EGCG3”Me-induced suppression of FcεRI expression by reducing ERK1/2 phosphorylation. These results suggest that anti-allergic effects of EGCG3”Me may be triggered by the inhibition of MRLC or ERK1/2 phosphorylation mediated through the cell-surface 67LR.     

Inhibitory effects of phloroglucinol derivatives isolated from Ecklonia stolonifera on FcεRI expression

Two bioactive phloroglucinol derivatives, dioxinodehydroeckol (DHE) and phlorofucofuroeckol A (PFF-A) were isolated from edible marine brown alga, Ecklonia stolonifera, and evaluated for effects on cell surface FcεRI expression in KU812F cells. DHE and PFF-A were found to reduce the cell surface expression, and total cellular protein and mRNA levels for the FcεRI α chain. Moreover, both compounds exerted inhibitory effects against the elevation of intracellular calcium concentration [Ca²⁺]_i and histamine release from anti-FcεRI α chain antibody (CRA-1)-stimulated cells. These inhibitory effects were stronger for PFF-A than for DHE. These results show that two phloroglucinol derivatives, DHE and PFF-A, may exert anti-allergic effects via the inhibition of FcεRI expression, calcium influx, and degranulation in basophils, and contributes to the pharmacological activities of marine brown alga, including E. stolonifera.     

Autophagy Facilitates Antibody-Enhanced Dengue Virus Infection in Human Pre-Basophil/Mast Cells

Background

Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.

Methodology/Principal Findings

We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4B^C74A mutant.

Conclusions/Significance

Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.     

MyD88 but not TLR2, 4 or 9 is essential for IL-12 induction by lactic acid bacteria

Although lactic acid bacteria (LAB) affect the immune system, for example, having an anti-allergic effect, little is known about the actual mechanisms of immune modulation. Toll-like receptors (TLRs) recognize conserved microbial molecular patterns, and are presumed to be involved in the recognition of LAB. However, there are few detailed reports examining the relationships between TLR and LAB. We measured here production of IL-12, a cytokine considered to play an important role in anti-allergic effects, induced by Lactobacillus paracasei strain KW3110 and other typical LAB by cells from TLR2-, TLR4-, TLR9- and myeloid differentiation factor 88 (MyD88)-deficient mice. Unexpectedly, similar cytokine production from wild-type and TLR2-, 4- and 9-deficient mice was observed. In contrast, cells from MyD88-deficient mice failed to respond to stimulation with LAB. It is therefore concluded that although LAB, including strain KW3110, are not likely to be recognized by TLR2, 4 or 9, MyD88 is essential for the response to these bacteria.     

MyD88 but Not TLR2, 4 or 9 Is Essential for IL-12 Induction by Lactic Acid Bacteria

Although lactic acid bacteria (LAB) affect the immune system, for example, having an anti-allergic effect, little is known about the actual mechanisms of immune modulation. Toll-like receptors (TLRs) recognize conserved microbial molecular patterns, and are presumed to be involved in the recognition of LAB. However, there are few detailed reports examining the relationships between TLR and LAB. We measured here production of IL-12, a cytokine considered to play an important role in anti-allergic effects, induced by Lactobacillus paracasei strain KW3110 and other typical LAB by cells from TLR2-, TLR4-, TLR9- and myeloid differentiation factor 88 (MyD88)-deficient mice. Unexpectedly, similar cytokine production from wild-type and TLR2-, 4- and 9-deficient mice was observed. In contrast, cells from MyD88-deficient mice failed to respond to stimulation with LAB. It is therefore concluded that although LAB, including strain KW3110, are not likely to be recognized by TLR2, 4 or 9, MyD88 is essential for the response to these bacteria.     

Identification of a methylated tea catechin as an inhibitor of degranulation in human basophilic KU812 cells

We examined the constituents of tea that had an inhibitory effect on the degranulation process in the human basophilic cell line, KU812. Among the constituents purified from a extract of 'Benihomare' oolong tea by column chromatography, a methylated (-)-epigallocatechin gallate ((-)-epigallocatechin 3-O-(3-O-methyl) gallate) was found to inhibit the degranulation of KU812 cells that had been stimulated with calcium ionophore A23187. The inhibitory effect of this methylated (-)-epigallocatechin gallate on degranulation was greater than that of (-)-epigallocatechin gallate. This result indicates that methylation of (-)-epigallocatechin gallate may be an effective modification for the catechin to inhibit degranulation from human basophils.