Mast cell-mediated allergic response is suppressed by sophorae flos: inhibition of SRC-family kinase.

Lee, Jun Ho,Kim, Jie Wan,Ko, Na Young,Mun, Se Hwan,Kim, Do Kyun,Kim, Ju Dong,Won, Hyung Sik,Shin, Hwa Sup,Kim, Hyung Sik,Her, Erk,Kim, Young Mi,Choi, Wahn Soo The Society 2008 Experimental biology and medicine Vol.233 No.10

<P>Complementary and alternative medicines are considered as a promising direction for the development of anti-allergic therapies in oriental countries. We screened approximately 100 oriental herbal medicines for anti-allergic activity. Sophorae flos exhibited the most potent effect on degranulation in antigen-stimulated mast cells. We further investigated the effect of Sophorae flos on the IgE-mediated allergic response in vivo and its mechanism of action in mast cells. Sophorae flos exhibited a significant inhibitory effect on degranulation in antigen-stimulated mast cells with IC(50) values of approximately 31.6 microg/mL (RBL-2H3 mast cells) and approximately 47.8 microg/mL (bone marrow-derived mast cells). Sophorae flos also suppressed the expression and secretion of TNF-alpha and IL-4 in the cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Sophorae flos inhibited the activating phosphorylation of Syk and LAT in mast cells. Further downstream, activating phosphorylation of Akt and the prototypic MAP kinases, namely, p38, ERK1/2, and JNK, were also inhibited. These results suggest that Sophorae flos inhibits the Src family kinase-dependent signaling cascades in mast cells and may thus exert anti-allergic activity.</P>

Polygoni cuspidati radix inhibits the activation of Syk kinase in mast cells for antiallergic activity.

Lim, Beong Ou,Lee, Jun Ho,Ko, Na Young,Mun, Se Hwan,Kim, Jie Wan,Kim, Do Kyun,Kim, Ju Dong,Kim, Bo Kyung,Kim, Hyung Sik,Her, Erk,Lee, Hoi Young,Choi, Wahn Soo The Society 2007 Experimental biology and medicine Vol.232 No.11

<P>The antiallergic activity of Polygoni cuspidati radix (PR) and the mechanism of action by which it functions were investigated in this study. The extract of PR exhibited potent inhibitory activity in mast cells; its IC50 values were 62 +/- 2.1 microg/ml for RBL-2H3 mast cells and 46 +/- 3.2 microg/m for bone marrow-derived mast cells by antigen stimulation, and it also suppressed the expression of tumor necrosis factor-alpha and interleukin-4 in RBL-2H3 cells. According to the in vivo animal allergy model, it inhibited a local allergic reaction, passive cutaneous anaphylaxis, in a dose-dependent manner. With regard to its mechanism of action, PR inhibited the activating phosphorylation of Syk, a key signaling protein for the activation of mast cells. It also suppressed Akt and the mitogen-activated protein kinases ERK1/2, p38, and JNK, which are critical for the production of various inflammatory cytokines in mast cells. The results of the study indicate that the antiallergic activity of PR is mediated through the inhibition of histamine release and allergic cytokine production by the inhibition of Syk activating phosphorylation in mast cells.</P>

The Interaction of Cytokine and Glucocorticoid in the Programmed Cell Death of Eosinophils

Her, Erk,Austen, K. Frank 中央醫學社 1996 中央醫學 Vol.61 No.12

The in vitro viability and the physical and functional phenotype of human peripheral blood eosinophils are regulated by the action of granulocyte-macrophage colony-stimulating factor(GM -CSF), interleukin-3(IL-3), and IL-5. In the idiopathic hypereosinophils syndrome and the tryptophan-associated eosinophilia/myalgia syndrome, the presence of hypodence eosinophils in the blood is associated with excessive serum levels of IL-5. The aim of study was to characterize, and to utilize this model cell system to determine the endothelial cell-mediated interactions of IL-1 and dexamethasone on eosinophil viability. GMCSF was established as the constitutive and elicited human umbilical vein endothelial cell-derived eosinophil viability-sustaining factor. Stimulation of endothelium cell monolayers with interleukin-1 (IL-1 ; 5 U/ml) increased the 48-h elaboration of GM-CSF from a mean of 3.2 to a mean of 8.2 pM(p <0.05). Dexamethasone(100 nM) decreased the constitutive GM-CSF elaboration by 49%(p<0.001) but did not diminish production by IL-1-stimulated endothelium. However, eosinophil viability decreased by 21% in dexamethasone-pretreated IL-l-stimulated endothelial cell-conditioned medium(p <0.05), which suggested viability antagonism by glucocorticoids. After 24h of culture, eosinoph1 l viability for replicate cells in enriched medium alone or with l pm GM -CSF decreased from means of 43 and 75 % to means of 21 and 54%, respectively, when dexamethasone was induced(p <0.05). However, 10 pM GM-CSF, IL-3, or IL-5 protected the cells against dexamethasone and against endonucleasesspecific DNA fragmentation. In this model system of eosinophil-tissue interactions, dexamethasone prevents the endothelial cells from inducing a pathologic phenotypic change in the eosinophil by suppression of GM-CSF elaboration to concentration that are not cytoprotective. Cytokine priming by GM-CSF, IL-3, or IL-5 may account for the differential responsiveness of eosinophillic disorders to glucocorticoids.

A Dual Effect of Hydrocortisone on the Inhibition of intracellular Free Calcium Level Which are Highly Correlated with Leukotrien C₄ Production in Rat Basophilic Leukemia Cells : 세포내 칼슘량과 LEUKOTRIENE C₄ 생산은 不可無의 相關關係

Her, Erk 건국대학교 1992 學術誌 Vol.36 No.2

人造 부신피질 호르몬중의 하나인 HYDROCORTISONE(HC)은 가장 강한 抗 Allergy 抗염증약이다. 이 약은 Allergy와 염증을 일으키는 물질들의 생산과 세포의 분비를 억제하는 것으로 알려져 있다. Leukotriene(LTC4)는 이러한 Allergy와 염증을 일으키는 주요한 물질중의 하나이다. LTC4는 5-lipoxygenase(5-LOX)라는 효소에 의해 세포막 지질에서 생성된 Arachdonic acid라는 지방산으로부터 생산된다. 이 효소의 촉매작용은 칼슘의 농도에 크게 의존한다. 이러한 학문 바탕 위에서, 호중구 암세포(RBL-2H3)에 있어 항원에 의해 유기된 LTC4 생산을 HC가 어떠한 mechanism속에서 억제하는지 알고자 함이 실험연구의 목적이다. 기본실험에 있어, LTC4 생산을 의한 DNP10BSA 항체 IgE와 항원 DNP10BSA의 최고 효과적인 농도는 각각 2㎍/106 cells, 18.8ng/106 cells이었다. HC가 항원이 유기 생산한 LTC4량을 억제하는 것은 배양시간과 농도에 의존했었다. 최고 효과적인 항원 농도에 의해 유기 생산된 LTC4량을 50% 억제하는데 최고 효과적인 HC농도(1㎍/ml)로 2.5 시간의 사전 배양이 요구되었다. HC 사전 배양시간이 증가함에 따라 항원에 유기생산된 LTC4량이 감소하는데, 4시간 사전 배양은 control에 비해 75%을 감소시켰다(p < 0.005). 4시간 HC 사전 배양후, LTC4 생산을 억제하는데 HC의 IC50價는 0.3μM이였고, 최대 억제농도는 27μM였다. LTC4량을 측정하는데 Radioimmunoassay(RIA)법의 신뢰 여부를 알기 위해, 다른 방법중의 하나인 Thin layer chromatography(TLC)법을 이용하여 비교해 봤다. 비교 결과, RIA법의 신뢰도가 높았고, 上同 2개의 방법에 의한 값이 거의 相同했었으며, RBL-2H3 암세포는 LTC4를 거의 생산치 않고 주로 LTC4를 생산한다는 것이 발견되었다(LTC4 vs LTD4 p<0.005). 세포내 방사선 동위원소 칼슘(45Ca2+)의 침투량을 측정하는 실험에 있어서는 세포내의 칼슘의 이온화와 불이 온화를 구별할 수 없는 단점이 있어 이온화된 칼슘만을 측정할 수 있는 방법을 모색했었다. 세포내 이온화된 칼슘 측정이 중요한 것은 세포학에서 말하는 주요한 second messenger가 이온화된 칼슘이기 때문이다. 그래서 이온화된 칼슘의 양의 증감을 측정하기 위해 Quin2/AM법을 사용했다. 항 DNP10 BSA 항체 IgE가 감작된 RBL-2H3 암세포에 항원 DNP10BSA로 유기했을 때 세포내 이온화된 칼슘([Ca2+]i,)의 양이 비교구보다 3.5배나 증가했었다(p<0.01). 항원 DNP10 BSA로 RBL-2H3를 유기했을 때 [Ca2+]i량이 최고치에 도달하는 데 소요되는 시간은 1.5∼2분 정도였다. 그런데 HC가 사전 처리한 세포에서는 [Ca2+]i, 최고치가 현저히 억제되었다(p<0.01). 항원을 투입한 뒤 12분후, HC를 사전 처리하지 않는 세포에 있어서는 최고치량에서 7 ± 3% 정도 감소했었다. 이에 반해 HC를 사전 처리한 세포에서는 75 ± 7% 감소했었다(p<0.005). HC를 6시간 사전 처리한 세포들은 현저히 LTC4 생산뿐만 아니라 [Ca2+], 증가를 현저히 감소시켰다(p<0.05). 이러한 HC 효라는 세포내 칼슘 침투를 억제하는 藥 Nifedipine과 세포내 칼슘을 세포외로 pumping하는 藥 TPA의 효과와 흡사했다. 그래서 HC는 세포내 칼슘량을 억제하는 데 양면성이 보인다고 예측되어진다. 그리고 [Ca2+]i량의 증감과 LTC4 생산 증감과 밀접한 상관관계가 있다는 것이 통계적으로 입증되었다(r = 0.94, p<0.01). 결론적으로 호염기구에서 HC이 LTC4를 억제하는 mechanism은 일차적으로 미지의 단백질을 생산하고 이러한 단백질이 세포내 칼슘의 침투를 억제하거나 세포내 칼슘을 세포외로 pumping함으로써 [Ca2+]i량을 억제하는 것 같다. Hydrocortisone(HC) is extremely potent anti-allergic and antiinflammalory druge which suppress the biosynthesis and/or release of inflammatory mediators. Leukotriene C4 (LTC4) is one of the maim mediators, which is produced through the 5-lipoxygenase. This enzyme activity is dependent on high Ca2+ concentrations. The aim of this study was to insight the mechanism of HC actions on antigen-induced LTC4 formation in rat basophilic leukemia(RBL-2H3) cells. The maximal effective doses IgE and its antigen DNP10BSA for stimulation of the LTC4 formation were 2㎍.106 cells and 18.8ng/106 cells, respectively, The inhibitory effect of HC on the antigen-induced LTC4 formation was time-and dele-dependent. The half maximal effective time for the HC action was about 2.5 hr(hours) of preincubation. This time-dependent inhibition increased slowly to approximately 75% inhibition(p < 0.005) after 4 hr of pretreatment. Under 4 hr pretreatment with HC, IC50 values of HC on LTC4 production was 0.3μM, and maximal inhibitory dose was 2μM. In validation of the radiommunoassay(RIA) for the measurement of LTC4 production using a thin layer chromatography(TLC), I found that RBL-2H3 produced mainly LTC4, while only slightly LTD4 (LTC4 vs LTD4 p,0.005). The results obtained with TLC with regard to LTC4 formation were similar to that obtained by RIA. Since radioisotope labelled calcium(45Ca2+) studios have limited resolution to distinguish intracellular free calcium([Ca2+]i) from the whole cellular Ca2+, 1 measured [Ca2+]i, using a Quin2/AM probe. In the cells stimulated by maximal effective dose of antigen, HC markedly suppressed [Ca2+]i elevation (p 01). Half maximal time for elevation of [Ca2+]i induced by antigen was less than 1 min, and maximal elevation of [Ca2+]i (3 fold increase) was reached within 1.5∼2 minutes(min). This peak of [Ca2+]i level in the control group decreased gradually to 7 ± 3% during 12 min of incubation. Following pretreatment with HC, the antigen-stimulated increase of [Ca2+], was stunted by 41 ± 8%(p<0.01) after 2∼3 min and drastically reduced by 75 ± 7%(p<0.005) at 12 min. Preincubation for 6 hr of RBL-2H3 cells with HC(1㎍/ml) abolished the anti DNP10BSA antibody IgE and antigen DNP10BSA complex(Ag-Ab) induced [Ca2+]i increment and LTC4 production(p <0.05). This HC effect on two parameters mimicked the direct effects of Ca2+ efflux enhancer, TPA, and a Ca2+ channel blocks, nifedipine. Moreover, there was high correlation between [Ca2+]i elevation and LTC4 formation(4 = 0.94, p < 0.01), suggesting a causal relation between the two parameters. These data indicate that the inhibitory effect of HC on LTC4 formation requires a DNA-dependent protein synthesis and reduction of [Ca2+]i.

Antigen-induced Cytosolic free Calcium and Leukotriene C₄Elevations are Inhibited by Glucocorticoid Receptor-mediated Protein Synthesis in Rat Basophilic Leukemia (2H3) Cells^#

Her, Erk,Zor, Uriel 大韓免疫學會 1995 大韓免疫學會誌 Vol.17 No.3

부신피질 호르몬인 gucecorticoid(GC)는 아주 강한 항 알러지 및 항 염증약이다. 이 약은 알러지와 염증을 일으키는 물질들의 생산과 분비를 억제하는 것으로 알려져 있다. 염증유발물질 중의 하나인leukotrieneC4(LTC4)는 5-lipoxigenase(5-LOX)라는 효소에 의해 생산되어진다. 이 촉매작용은 높은 칼슘이온 농도에 크게 의존한다. 인조 부신피갈호르몬 증의 하나인hydrocortisone(HC)에 의해 억제되어진 호템기구 암세포내 칼슘이온 농도(a"fi) 증가와 LT 생산은 배양 시간에 의존했었다. 항원에의해 음기 증가된 세포네 (Ci')i과 LT 생산이 HC사전배양에 의해 억제되어지는 데에 필요한 소요시간은 적어도 t시간 정도였다. 약 2-5시간정도의 사전배양은 항원에 의해 유기 생산된 3i과LT량을 50% 억제했다. 4시간 사전 배양은 세포내 a"li과 Lf 생산을 비교구에 비해 각각 45%(p<0.01),75%(p<0.005) 감소시켰다-GC 수용체 길항제연 RU486과 cortexolone은 이 두 pararneter들을 억제하는 HC효과를 완전히 억제했다(p<0.01). 또한 단배질 합성 저해효소인 cycloheximide와 RNA중합효소 억제재인 actinomycin-D도HC의 억제효과를 차단했다 短제.01). 이러한 HC의 억제효파가 sex-steroid에서나 mineralocorticoid에는 나타나지 않았는데 반해 gluocorticoid 일종인 dexamethasone에는 나타났다(p<0.01). 이상의 결과에서 나타난 것처럼 HC의 억제효과는 일반 다른 steroid와는 달리 틴ucocorticosteroid만 가지는 특수성에 기인했다. 이러한 결과들은 GC는 수용체 매개로 생합성한 단백질로서 항 알러지 및 항 염증작을에 중요한 역할를 한다는 것을 제시하고 있다.

The interaction of cytokine and glucocorticoid in the programmed cell death of esinophils

Erk Her,Frank Austen 대한천식 및 알레르기학회 ( 구 대한알레르기학회 ) 1995 춘계학술대회 초록집 Vol.1995 No.-

Modulation of the Synthesis of Nine Proteins by Glucocorticoid in Rat Basophilic Leukemia (2H3) Cells^($)

Her, Erk,Zor, Uriel 大韓免疫學會 1995 大韓免疫學會誌 Vol.17 No.3

항원에 자극된 호염기구에 있어, 인조 glucocorticoid 중의 하나인 hydrocortisone (HC)에 의한leukotriene C4(LTC4)생산억제 호과는 적어토 2-5시간정도의 지연시간이 필요했다. 이러한 HC의 지연효과는 cycloheximide와 eC recepfo. 길항제에 의해 억 제되 었다 ffer and Zor, submitted). 이 러 한 실험결과들은 班C는 단백질생산에 의한 간접작용을 한다는 것을 암시했다. 그래서 HC에 의해 조절생산펀 단백질이 어떠한 단백질인지 알기위해 호염기구 암세포를 등위원소 i놀eoni緖을 례벨했다. 1'높Tmethionine을 레벨하는 동안,다행이도 HC가 이 동위원소의 축적이나 아미노산에 결합하는것을 억제하지는 않았다. One-dimensional SDS-PAGE을 이용한 단백질분리법에서는 HC유무에 아무런 차이가 없었다. 그래서 단백질분리가 쉬운 two곯mensional gel기법을 사용했다- 이 실험기법에서, HC에 의해 9개의 단백질생살 증감이 있었는데 이 중 8개의 단백질 acidic prole구 1 basicole조) 생산이 증가했으며, 한개의 acidic protein 생산이 감소했다. %5이 9개 단백질 중 5개 단백질(363kDa/pI 5.6; 38kDa/pI 5.9; 40kDa/p16.0; 42kDa/p16.1; 43kD리pl 5.2)은불용성 단백질이 였고,4개의 단백질(51kDa/pl 6.0; 52kDa/pI pl 6.8; 53kDa/pI 4.5: 71kDa/pl 8.4)은 불용성단백 질이 였다. 수용성,불용성 단백질 구분은 시료를 1關,Oeoxg속도하에서 1시간동안 원심분리한 후 상총액부위에 있는 단백질을 수용성 단백질이라 하고,절랫부위에 있는 단백질을 불용성 단백질이라 했다. 이 9게 단백질 중 분자량이 36-42tDa단백질이 phospholipaseA,억제물질이라고 밝혀진 lipocortin의분자량과 유사하다 .

Glucocorticoid inhibition of antigen - induced inositolphosphate formation by basophilic leukemia cells : possible involvement of phosphatases and other several proteins.

허억(Erk Her) 건국대학교 유전공학연구소 1993 제1회 건국대학교-KIST유전공학연구소 공동심포지움 Vol.- No.-

Human Neutrophil의 수명연장과 Superoxide 생산에 관여하는 미지의 Monocyte 생성물질

허억,배진우 大韓免疫學會 1993 大韓免疫學會誌 Vol.15 No.-

It has long been known that neutrophils are quickly infilterated and recruited to infected sites and then kill invaders by phagocytic action. Unfortunatly it is not yet revealed which molecule or cytokine is involved in the phagocytic action and viability sustaining activity of neutrophils. The aim of this study was whether lipopolysaccharide (LPS)-stimulated monocyte may control those neutrophil actions. Human peripheral blood monocytes and neutrophils were isolated by Ficollpaque density sedimentation from heparin anti-coagulated blood of healthy adult donors. After preparation of these cells, the purity of both was more than 90%. Monocytes stimulated in various dose(0.1-10pug/ml) of LPS for various times of incubation(0-3 days). and then LPS-stimulated monocyte conditioned medium was collected in order to find an optimal dose and incubation time for the neutrophil viability. It was found out that 3,ug/ml of LPS in 24 hours incubation was maximal effective condition for the activity of neutrophil sustaining viability. Monocyte conditioned medium (MCM) under this condition was used for the comparison with LPS-nonstimulated monocyte conditioned medium or enriched medium alone. When neutrophils were stimulated with each medium for 1-3 days, the activity of neutrophil sustaining viability with MCM was significantly higher than the activity with other medium (in 1 day of culture, 72-1:8 vs 4311:7 vs 17 ±10; p <O. 01). The superoxide production of neutrophil stimulated with MCM for 24 hours incubation was significantly higher than that with other medium under fMLP doses of 0.1-100,uM (p <0.01). Under fMLP l,uM, the superoxide production is predominantly different between them(23.8±2 vs 10.3±3 vs 7.8±1.6). The maximal effective dose of GM-CSF(granulocyte/macrophage colony stimulating factor ; 10pM) enhanced the neutrophil viability in 1 day of culture (50 ± 4%) . In the study to assess whether MCM contains GM-CSF, anti-GM-CSF antibody slightly blocked the MCM-dependent neutrophil viability(73±9 vs 50±12; p <0.07), indicating that MCM might not contain GM-CSF. These data indicate that LPS-stimulated monocyte surely product a factor for the neutrophil sustaining viability and the enhancement of superoxide production, suggesting that a factor is not GMCSF. If more than one factor were producted form LPS-stimulated monocyte, one minor factor might be GM-CSF.

PHA로 자극된 T cell에서 분비된 미지의 물질이 지니는 Human Neutrophil의 수명연장, Superoxide 및 Leukotriene C₄생산증가 작용효과

허억,양영목 大韓免疫學會 1993 大韓免疫學會誌 Vol.15 No.-

There are several direct and indirect ways in which T cells could enhance the anti-bacterial capabilities of neutrophils. However it is not yet clear out which molecule or cytokine produced by T cells is involved in the phagocytic action and viability sustaining activity of neutrophils. The aim of this study was about a factor, which produced by the phytohaemagglutinin(PHA)-stimulated T cells, may control those neutrophil actions. Human peripheral blood T cells and neutrophils were isolated by Ficoll-paque density sedimentation from heparinized blood of healthy adult donors. The purity of these cells were more than 90%. T cells were stimulated in various dose(0.1-10gg/ml) of PHA for various times of incubation(0-3 days), and then PHA-stimulated T cell conditioned medium was collected in order to find an optimal dose and incubation time for the neutrophil viability. It was found out that 1,ug/ml of PHA in 12 hours incubation was maximal effective condition for the neutrophil sustaining viability. The effects of PHA-stimulated T cell conditioned medium(TCM) on the neutrophils were used for the comparison with PHA-nonstimulated TCM or enriched medium alone. Neutrophil sustaining viability with PHA-stimulated TCM for 24 hours incubation was significantly higher than other groups (80 ± 10 vs 25 ±15 vs 13 ± 9; p <0.01). The superoxide prodution from neutrophils with PHA-stimulated TCM for 24 hours incubation were also significantly higher than other groups(25±4 vs 11±4 vs 7±5; p <0.01). In the leukotriene C4 (LTC4) release, neutrophils with PHA-stimulated TCM for 24 hours incubation were different from other group (105 ± 20 vs 65 ±1O vs 25 ± 32 ; p <0.01), and unlikely other parameters the cells with PHA-nonstimulated TCM was different from the cells with enriched medium alone(65 -1:10 vs 25-± 32; p<0.05). In two dimension electrophoresis it was shown that PHA-stimulated T cells enhanced three proteins(66kD, 60kD, 45kD) and diminished one(40kD) in the production and/or release of proteins in comparison with PHA-nonstimulated T cells. These data suggest that these proteins from the PHA-stimulated T cells might be involved in the phagocytic actions of neutrophils.