Microarray analysis of immediate-type allergy in KU812 cells in response to fulvic acid
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- Motojima, H., O. Villareal, M., Han, J. et al. Cytotechnology (2011) 63: 181. doi:10.1007/s10616-010-9333-6
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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.
KeywordsImmediate-type allergyHistamine releaseMicroarray analysisFulvic acidSolubilized excess sludge
Dinitrophenylated bovine serum albumin
Fetal bovine serum
Human leukemia basophilic
Phorbol 12-myristate 13-acetate
PMA plus calcium ionophore A23187
Rat basophilic leukemia
Systemic lupus erythematosus
Fulvic acid extracted from solubilized sludge
Fulvic acid (FA) is a class of compounds of humic substances together with humic acid and humin, and is formed through the degradation of organic substances such as dead plants, microbes and animals by chemical and biological processes. Recently, the physiological action of FA on the human body has been studied. For example, the possible application of coal-derived FA as antimicrobials (van Rensburg et al. 2000), and the anti-inflammatory property of coal-derived FA have been reported (van Rensburg et al. 2001). In addition, the FA extracted from peat (Tachibana et al. 2004) had an antioxidative activity and the FA extracted from Canadian Sphagnum Peat (Yamada et al. 2007) had an inhibitory effect on chemical mediator release in rat basophilic leukemia cells (RBL-2H3).
Basophil activation has been implicated in allergic reaction and immune modulation. These events trigger the secretions of chemical mediators such as cytokines. Among the cytokines, IL4 and IL13 are the major cytokines for IgE production, facilitating Th2 differentiation and allergic responses (Gauchat et al. 1993). Basophils could rapidly secrete large amount of IL4 and IL13, and have been detected in organs affected by allergic reactions, such as bronchial and airway biopsies from asthma patients, nasal lavage fluids from allergic rhinitis patients, as well as skin biopsies from atopic and contact dermatitis. Human basophils, therefore, play a crucial role as a high priority source of IL4, IL13, and histamine that polarize the early type 2 immune response leading to allergy (Chen et al. 2009; Zhu et al. 1998; Yanagihara et al. 1997).
In our previous study, we extracted FA from solubilized excess sludge (SS-FA) and determined the structural properties of SS-FA using elemental analysis, fourier transform infrared spectroscopy and 1H-nuclear magnetic resonance spectroscopy (Motojima et al. 2009). Excess sludge is one of the components of sewage sludge, and Japan generates 1.7 million tons of excess sludge every year. Recently, there is a demand for the use of this excess sludge. Under the circumstances, we developed a pretreatment technique for the improvement of the extraction rate of FA using solubilization and carried out extraction following the standard method of the International Humic Substances Society. Moreover, to search for a utility for SS-FA, we examined its inhibitory effect on the chemical mediator release in rat basophilic leukemia (RBL-2H3) cells (Motojima et al. 2009). Our experiment showed that SS-FA had β-hexosaminidase release inhibitory effect on the antigen–antibody binding stage and the antigen-receptor binding stage in RBL-2H3 cells.
In the present study, in order to gain more information regarding the mechanism of the suppression of the degranulation following SS-FA treatment, we studied the effect of SS-FA on the immediate-type allergic reaction in human leukemia basophilic (KU812) cells. The inhibitory effect of SS-FA on the degranulation in PMA (20 nmol L−1) plus calcium ionophore A23187 (1 μmol L−1) (PMACI)-stimulated KU812 cells was examined using histamine release assay. The cytotoxicity of SS-FA at the concentration used for the histamine release assay was assessed by MTT assay. Microarray analysis is a technique that has been shown to be useful for the simultaneous profiling of global gene expression and uncovering new genes or new functions of known genes (Thornton et al. 2002). For these reasons, microarray assay was conducted to determine which genes were differentially expressed in response to SS-FA in PMACI-activated KU812 cells.
Materials and methods
Extraction and purification of SS-FA
Excess sludge was sampled from the return line of an aeration reactor at a municipal wastewater treatment plant in Japan. Extraction and isolation procedures were carried out following the protocol of the International Humic Substances Society. The procedure for the structural features analysis of SS-FA was described in the previous report (Motojima et al. 2009).
Reagent, cell and cell culture
KU812 cells were purchased from the Riken Cell Bank, Japan and were maintained in RPMI1640 medium (Gibco BRL, Grand island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone Co. Japan) containing 50 U mL−1 of penicillin and 50 μg mL−1 of streptomycin, and were cultured at 37 °C in a humidified atmosphere with 5% CO2. RBL-2H3 cells were purchased from JCRB Bank, Japan, and were cultivated in Eagle’s Minimum Essential Medium with 10% FBS and 2 mmol L−1 of l-glutamine at 37 °C in 5% CO2 incubator. Calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma, Japan (Tokyo) and dissolved in DMSO at a concentration of 10 mM. Dinitrophenylated bovine serum albumin (DNP-BSA) was obtained from Cosmo Biotechnology Co. (Japan), and anti-DNP-IgE was from Sigma (Japan).
Measurement of intracellular calcium [Ca2+]i level
[Ca2+]i measurement was performed using the method of Aase and Arna (1991) with some modifications. Cells (5 × 105 cells mL−1 in 100 μL of the medium) were preincubated at 37 °C for 24 h in 96-well plates in a medium containing 10% FBS and anti-DNP IgE (final concentration 0.3 μg mL−1). The cells were washed twice with 200 μl of PBS to eliminate the free IgE. The cells were then incubated with 100 μL per well of a loading buffer containing Fluo3-AM (Caladium Kit-Fluo3, Dojindo Co.) at 37 °C for 1 h in a 5% CO2 incubator. After washing the dye from the cell surface, cells were incubated with 60 μl per well of a recording medium (Caladium Kit-Fluo3, Dojindo Co.) containing the sample (0.1, 10.0 μg mL−1 SS-FA) for 15 min. The fluorescence intensity was determined 50 s, after adding 5 μL of the DNP-BSA antigen using a microplate reader at an excitation wavelength of 490 nm and emission wavelength of 530 nm.
Histamine release assay
KU812 cells at a density of 1 × 106 cells mL−1 were preincubated for 24 h in RPMI1640 medium supplemented with 10% FBS. The cells were treated with or without 25 μL per well of various concentrations of SS-FA (0.1–10 μg mL−1) for 15 min, and followed by stimulation with PMACI for 30 min at 37 °C. The cells were separated from the released histamine by centrifugation at 190×g for 3 min at 4 °C. The supernatant (200 μL) was transferred to a 96-well ELISA system. The fluorescence intensity was measured at 414 nm using a multidetection microplate reader (Power Scan HT. Dainippon Pharmaceutical Co.), and the histamine concentration determined by ELISA kit according to the manufactures’ instruction.
Cell proliferation was assessed by MTT assay which is a measure of the mitochondrial respiration of the cells (Mossam 1983). KU812 cells were seeded onto 96-well plates at 1 × 106 cells mL−1 in 80 μL of medium, respectively. After an overnight incubation, 10 μL of the SS-FA dissolved in the medium was added to obtain the final concentrations of 0.1, 1.0 and 10.0 μg mL−1. The cells were then incubated for 24 h before 10 μL of 5 mg mL−1 of MTT was added. After 24 h of incubation, 100 μL of 10% sodium dodecyl sulfate was added, followed by another 24 h of incubation to completely dissolve the formazan produced by the cells. The absorbance was then determined at 570 nm with a microplate reader. Blanks were prepared at the same time to correct for the absorbance caused by sample color and by the inherent ability of a sample to reduce MTT in the absence of cells. The optical density of the formazan produced by the untreated control cells represented 100% proliferation.
KU812 cells at concentration of 1 × 106 cells mL−1 were treated with or without 0.1 μg mL−1 of SS-FA for 15 min and incubated at 37 °C. After exposure to PMACI for 30 min, total RNA was extracted from the cells using 1 mL of Isogen (Nippon Gene Co., Tokyo, Japan) following the manufactures instruction.
Synthetic DNA oligonucleotide probes were installed as probes onto Genopal (Mitsubishi Rayon, Tokyo, Japan), which is composed of plastic hollow fibers. With this system oligonucleotide DNA probes are attached to a gel within the three-dimensional space of each hollow fiber (Hohjoh and Fukushima 2007).
First, RNA was amplified using the MessageAmpII biotin-enhanced amplification kit (Applied Biosystems Japan, Tokyo, Japan), according to the manufacturer’s instructions, and column purified. Biotinylated aRNA (5 μg) was fragmented using fragmentation reagents (Applied Biosystems Japan) and then incubated at 94 °C for 7.5 min. The fragmentation reaction was terminated by the addition of stop solution. Hybridization was carried out with a DNA microarray (Genopal, Mitsubishi Rayon) in 180 μL of hybridization buffer (0.12 mol L−1 Tris–HCl/0.12 mol L−1 NaCl, 0.05% Tween-20) and 5 μg of fragmented biotinylated aRNA at 65 °C overnight. After hybridization, the DNA microarray was washed twice in wash solution A (0.12 mol L−1 Tris–HCl/0.12 mol L−1 NaCl, 0.05% Tween-20) followed by washing in wash solution B (0.12 mol L−1 Tris–HCl/0.12 mol L−1 NaCl). The DNA microarray was then labeled with streptavidin-Cy5 (GE Healthcare Bio-Science KK, Tokyo, Japan). The fluorescent labeled-DNA microarray was washed for 5 min four times in wash solution B at room temperature. Hybridization signal acquisition was performed using a DNA microarray reader adopting multibeam excitation technology (Yokogawa Electric Co., Tokyo, Japan) (Isozaki et al. 2006). The DNA microarrays were scanned at multiple exposure times ranging from 0.1 to 40 s and the intensity values with the best exposure condition for each spot were selected.
Statistical significance (p < 0.05) was evaluated by one-way ANOVA, and if significant, group means were compared using Bonferroni’s post hoc. Homogeneous subsets of significance were determined by Duncan’s post hoc test.
Results and discussion
Effect of SS-FA on histamine release in PMACI-stimulated KU812 cells
Cytotoxicity effect of SS-FA on KU812 cells
Gene expression profile of SS-FA treated cells
Down-regulated genes in KU812 cells that were treated with SS-FA plus PMACI
SS-FA plus PMACI ((SS-FA plus PMACI)/PMACI)a
Cytokine- Cytokine receptor interaction
Homo sapiens interleukin 13
Homo sapiens interleukin 12 receptor, beta 1
Homo sapiens interleukin 13 receptor, alpha 1
Homo sapiens chemokine(C–C motif) ligand 11
Homo sapiens inhibin, beta C
Homo sapiens bone morphogenetic protein 2
Homo sapiens bone morphogenetic protein 6
Homo sapiens fms-related tyrosine kinase 3
Homo sapiens mitogen-activated protein kinase 8
Homo sapiens Fas (TNF receptor superfamily, member 6
Homo sapiens interferon regulatory factor 3
Homo sapiens guanylate binding protein 3
Cell adhesion molecules
Homo sapiens integrin, alpha 2
Homo sapiens integrin, alpha M
Homo sapiens selectin L
IgE receptor β subunit
Homo sapiens membrane-spanning 4-domains, subfamily A, member 2
Mitogen-activated protein kinase 8 (MAPK8) was up-regulated following PMACI stimulation but SS-FA treatment suppressed its expression. The encoded protein is a member of the serine/threonine protein kinase family. This kinase can activate both the MAP kinase and JNK kinase pathways. Moreover, MAP3K8 promotes the production of tumor necrosis factor (TNF)-α. Rasheed et al. (2009) reported that activation of MAPKs was intimately associated with the expression of pro-inflammatory cytokines, reduction of pro-inflammatory cytokines expression, and inhibited PMACI-induced phosphorylation of JNKp54/p46- and ERKp44/p42 in KU812 cell. In our experiment, pro-inflammatory cytokines TNF (63% SS-FA plus PMACI/PMACI alone) and the cytokine receptors such as tumor necrosis factor receptor superfamily, member 6 (TNFRSF6) (FAS) might be down-regulated for suppression of MAPK8 gene expression following SS-FA treatment.
The IgE-receptor, a tetramer composed of an alpha, beta and 2 disulfide-linked gamma chains, is found on the surface of mast cells and basophils. MS4A2 gene encodes the beta subunit of the high affinity IgE receptor which is a member of the membrane-spanning 4A gene family. In this study, MS4A2 expression was highly increased by PMACI treatment, but decreased by SS-FA treatment. Moreover, the expression of the gene for Fc fragment of IgE, high affinity I, receptor for gamma polypeptide (FCER1G) which was increased two-fold by PMACI-treatment for 30 min when compared to the control, was down-regulated by SS-FA treatment by 84%. In our previous study, SS-FA showed an inhibitory effect on β-hexosaminidase release at the antibody-receptor binding stage (Motojima et al. 2009). SS-FA affected not only the binding of IgE and FcεRI receptor but also the expression of the IgE-receptor.
Interferon regulatory factor (IRF) and integrin alpha M (ITGAM) are systemic lupus erythematosus (SLE) candidate genes (Hom et al. 2008; Isaac et al. 2009). SLE is a potentially deadly systemic illness, and is sometimes considered a model for systemic humoral autoimmune diseases. Autoantibodies play an important role in the pathogenesis of SLE, and the diverse clinical manifestations of the disease are caused by the deposition of antibody-containing immune complexes in blood vessels, leading to inflammation in the kidney, brain and skin. In this study, IRF and ITGAM gene expression were increased two fold by PMACI-treatment but decreased by SS-FA treatment.
Bone morphogenetic proteins (BMPs) are closely correlated with tumor differentiation and skeletal metastasis. It has been reported that BMP-2 and -6 gene expression can activate breast cancer and prostate cancer (Zhang et al. 2007; Yamamoto et al. 2002; Alarmo et al. 2008). Generally, FA and humic acid has been known as material that has anti-oxidative and anti-cancer effect (Tachibana et al. 2004; Wang et al. 1996; Yang et al. 2004). In our previous study, SS-FA had anti-oxidative activity as determined by the results of the DPPH radical scavenging assay. These results may indicate that SS-FA has an anti-cancer effect.
In conclusion, SS-FA decreased the histamine release and the expression of various genes related to the allergy responses which occur in PMACI-stimulated KU812 cells. Our study suggests that FA obtained from excess sludge may be useful as a new resource of FA production. SS-FA may be of therapeutic use as a treatment for immediate-type allergy by suppressing basophils activation, and this effect may occur as a result of the decreased expressions of the following genes-BMP2, BMP6, CCL11, FLT3, GBP3, IL13, IL12RB1, L13RA1, INHBC, ITGA2/CD49b, ITGAM, IRF8, MAPK8, MS4A2, SELL and TNFRSF6/Fas. However, the precise mechanisms by which the anti-inflammatory effects occur, as well as the major bioactive component remains to be elucidated.
This research was partially supported by the Mitsui & Co., Ltd. Environment Fund.