A Novel Small-Molecule Inhibitor Targeting the IL-6 Receptor β Subunit, Glycoprotein 130

Hong, Soon-Sun,Choi, Jung Ho,Lee, Sung Yoon,Park, Yeon-Hwa,Park, Kyung-Yeon,Lee, Joo Young,Kim, Juyoung,Gajulapati, Veeraswamy,Goo, Ja-Il,Singh, Sarbjit,Lee, Kyeong,Kim, Young-Kook,Im, So Hee,Ahn, Sun The American Association of Immunologists, Inc. 2015 JOURNAL OF IMMUNOLOGY Vol.195 No.1

<P>IL-6 is a major causative factor of inflammatory disease. Although IL-6 and its signaling pathways are promising targets, orally available small-molecule drugs specific for IL-6 have not been developed. To discover IL-6 antagonists, we screened our in-house chemical library and identified-LMT-28, a novel synthetic compound, as a candidate IL-6 blocker. The activity, mechanism of action, and direct molecular target of LMT-28 were investigated. A reporter gene assay showed that LMT-28 suppressed activation of STAT3 induced by IL-6, but not activation induced by leukemia inhibitory factor. In addition, LMT-28 downregulated IL-6-stimulated phosphorylation of STAT3, gp130, and JAK2 protein and substantially inhibited IL-6-dependent TF-1 cell proliferation. LMT-28 antagonized IL-6-induced TNF-alpha production in vivo. In pathologic models, oral administration of LMT-28 alleviated collagen-induced arthritis and acute pancreatitis in mice. Based on the observation of upstream IL-6 signal inhibition by LMT-28, we hypothesized IL-6, IL-6R alpha, or gp130 to be putative molecular targets. We subsequently demonstrated direct interaction of LMT-28 with gp130 and specific reduction of IL-6/IL-6R alpha complex binding to gp130 in the presence of LMT-28, which was measured by surface plasmon resonance analysis. Taken together, our data suggest that LMT-28 is a novel synthetic IL-6 inhibitor that functions through direct binding to gp130.</P>

Interleukin 17 (IL-17) Increases the Expression of Toll-like Receptor-2, 4, and 9 by Increasing IL-1β and IL-6 Production in Autoimmune Arthritis

LEE, JUN-HEE,CHO, MI-LA,KIM, JU-IN,MOON, YOUNG-MEE,OH, HYE-JWA,KIM, GEUN-TAE,RYU, SUN,BAEK, SEUNG-HOON,LEE, SUN-HEE,KIM, HO-YOUN,KIM, SUNG-IL The Journal of Rheumatology 2009 The Journal of rheumatology Vol.36 No.4

<B>Objective.</B><P>To examine the effect of interleukin 17 (IL-17) on the expression of Toll-like receptor (TLR)-2, 4, and 9 in collagen-induced arthritis (CIA) in mice.</P><B>Methods.</B><P>On Days 28 and 32 after induction of CIA in mice, phosphate-buffered saline (PBS group) or IL-17 (IL-17 group) was injected into both knee joints. On Day 35, mice were sacrificed. The severity of knee joint arthritis, synovial inflammation, and bone destruction was measured by a scoring system using macrography and histological analysis. Synovial expression of TLR-2, 4, 9, IL-17, IL-1ß, tumor necrosis factor-α (TNF-α), and IL-6 was determined by real-time PCR and immunohistochemistry. Synoviocytes of CIA mice were cultured with IL-17 and with neutralizing antibodies to cytokine, and the expression of TLR-2, 4, 9, IL-1ß, TNF-α, and IL-6 was determined by real-time RT-PCR.</P><B>Results.</B><P>In CIA mice, knee arthritis scores, synovial inflammation, bone destruction scores, and expression of synovial TLR-2, 4, and 9, IL-17, IL-1ß, TNF-α and IL-6 were higher in the IL-17 and PBS groups than in normal DBA1 mice. These variables were also significantly higher in the IL-17 group than in the PBS group. In CIA synoviocytes, IL-17 increased the expression of TLR-2, 4, and 9, and this effect was significantly alleviated by neutralizing antibodies to IL-17, IL-1ß, and IL-6.</P><B>Conclusion.</B><P>IL-17 aggravates joint inflammation and destruction, and increases the synovial expression of TLR-2, 4, and 9 by increasing IL-1ß and IL-6. These results imply that the IL-17-induced increase in expression of TLR-2, 4, and 9, and IL-1ß and IL-6 production are involved in the IL-17-induced aggravation of arthritis.</P>

The Correlation of Serum IL-12B Expression With Disease Activity in Patients With Inflammatory Bowel Disease

Lee, Hye Won,Chung, Sook Hee,Moon, Chang Mo,Che, Xiumei,Kim, Seung Won,Park, Soo Jung,Hong, Sung Pil,Kim, Tae Il,Kim, Won Ho,Cheon, Jae Hee Williams & Wilkins Co 2016 Medicine Vol.95 No.23

<▼1><P>Supplemental Digital Content is available in the text</P></▼1><▼2><P><B>Abstract</B></P><P>Genetic variants in <I>IL12B</I>, encoding the p40 subunit common in interleukin-12 (IL-12) and interleukin-23, were identified as the susceptibility loci for inflammatory bowel disease (IBD). This study aimed to identify the correlation of serum IL-12B expression with disease activity in patients with IBD and evaluate the possibility of IL-12B as a biomarker for assessing inflammatory status in IBD.</P><P>A total of 102 patients with IBD, including 38, 32, and 32 patients with Crohn's disease (CD), ulcerative colitis (UC), and intestinal Behçet's disease (intestinal BD), respectively, were included. The clinical and laboratory data from the patients were collected at the time of serum IL-12B measurement. Serum IL-12B levels were measured using an enzyme-linked immunosorbent assay.</P><P>The median IL-12B levels in patients with CD, UC, and intestinal BD were significantly higher than those in controls (1.87, 2.74, and 2.73 pg/mL, respectively, vs. 1.42 pg/mL, all <I>P</I> <0.05). IL-12B concentrations were associated with disease activity in patients with UC and intestinal BD but not in those with CD. IL-12B levels were increased with increasing disease activity in patients with UC (<I>P</I> <0.001). Likewise, patients with active intestinal BD had higher IL-12B levels than those without active disease (<I>P</I> = 0.008). IL-12B levels were correlated with the endoscopic disease activity of UC (<I>P</I> = 0.002) and intestinal BD (<I>P</I> = 0.001) but not that of CD.</P><P>Serum IL-12B levels were significantly correlated with clinical and endoscopic disease activity in patients with UC and intestinal BD, suggesting its potential use as a biomarker for assessing disease activity in these patients.</P></▼2>

류마티스 관절염 동물 모델에서 활막의 RANKL/OPG mRNA 발현 비율 및 IL-17의 효과

이준희 ( Jun Hee Lee ),김근태 ( Geun Tae Kim ),류선 ( Sun Ryu ),김주인 ( Ju In Kim ),백승훈 ( Seung Hoon Baek ),김성일 ( Sung Il Kim ) 대한류마티스학회 2006 대한류마티스학회지 Vol.13 No.2

Objective: To investigate the synovial mRNA expression of receptor activator of NFκB (RANK), RANK ligand (RANKL), osteoprotegerin (OPG) and RANKL/OPG mRNA expression ratio, and to evaluate the effects of IL-17 in experimental rheumatoid arthritis (RA) model. Methods: After induction of collagen-induced arthritis (CIA) by type II collagen in DBA1 mice, mice were anesthetized at day 28 and a small aperture in the skin of the knee was performed. Mice, in which arthritis of knee was present, were selected and divided into 3 groups, and phosphate-buffered saline (PBS group), IL-17 (IL-17 group) or anti-IL-17 monoclonal antibody (anti-IL-17 group) was injected to both knee joint at day 28 and 32. At day 35, mice were sacrificed and synovium of knee joints were isolated. Synovial mRNA expression of RANKL, RANK and OPG was assessed by real-time RT-PCR and immunohistochemical stain. Results: Synovial RANKL and RANK mRNA expressions were significantly different among IL-17, PBS, anti-IL-17 and normal group (IL-17>PBS>anti-IL-17>normal group), and synovial OPG mRNA expressions in PBS, IL-17 and anti-IL-17 group were significantly high than those in normal group, however, there was no significant difference among IL-17, PBS and anti-IL-17 group. RANKL/OPG mRNA ratio was significantly different among these groups (IL-17>PBS>anti-IL-17>normal group). In immunohistochemical stain, RANKL, RANK and OPG-positive cells were expressed at synovium. Conclusion: Synovial RANKL/OPG mRNA ratio was enhanced in CIA, and IL-17 induced higher RANKL/OPG ratio in the synovium of CIA, which was blocked by anti-IL-17 antibody. These results suggest that RANKL/OPG mRNA ratio play an important roles on bone destruction, and IL-17 may be actively involved in bone destruction by enhancing RANKL/OPG ratio in CIA model.

Intracelular Signaling Pathways for Type II lgE Receptor (CD23) Induction by Interleukin-4 and Anti-CD40 Antibody

Lee,Choong-Eun,Kim,Hyun-il,Park,Hee-Jeoung The Korea Science and Technology Center 1997 BMB Reports Vol.30 No.6

Since the role of CD40 on the interleukin-4(IL-4)-induced B cell activation has been strongly implicated in the agumentation of lgE production and response, we have investigated the intracelluar signaling pathways utilized by IL-4 and CD40 for type Ⅱ lgE receptor (CD23) expression. IL-4 and anti-CD40 antibody treatment of human B cells, independently caused a rapid induction of CD23 gene activation within 2 h. There was a noticeable synergism between the action of the two agents inducing CD23 expression: the addition of anti-CD40 to the IL-4-treated culture significantly agumented the IL-4-induced CD23 on both mRNA and surface protein levels, and the inclusion of IL-4 in the anti-CD40-treated cells caused a further increase of CD23 expression far above the maximal level induced by anti-CD40. Protein tyrosine kinase (PTK) inhibitors effectively suppressed the both IL-4- and anti-CD40-induced CD23 expression, whereas protein kinase C (PKC) inhibotors had no effects. Electrophoretic mobility shift assays (EMSA) have shown that IL-4 and anti-CD40 induce the activation of NF-IL-4 and NF-?B, respectively, binding to the CD23 promoter, both in a PKC-independent and PTK-dependent manner. These data suggest that the synergistic activation of CD23 gene expression by IL-4 and anti-CD40 is mediated by co-operative action of distinct nuclear factors, each of which is rapidly activated via PKC-independent and PTK-dependent process.

Intracellular Signaling Pathways for Type Ⅱ IgE Receptor ( CD23 ) Induction by Interleukin - 4 and Anti - CD40 Antibody

Lee, Choong Eun,Kim, Hyun Il,Park, Hee Jeoung 생화학분자생물학회 1999 BMB Reports Vol.30 No.6

Since the role of CD40 on the interleukin-4(IL-4) -induced B cell activation has been strongly implicated in the agumentation of IgE production and response, we have investigated the intracelluar signaling pathways utilized by IL-4 and CD40 for type II IgE receptor (CD23) expression. IL-4 and anti-CD40 antibody treatment of human B cells, independently caused a rapid induction of CD23 gene activation within 2 h. There was a noticeable synergism between the action of the two agents inducing CD23 expression: the addition of anti-CD40 to the IL-4-treated culture significantly agumented the IL-4-induced CD23 on both mRNA and surface protein levels, and the inclusion of IL-4 in the anti-CD40-treated cells caused a further increase of CD23 expression far above the maximal level induced by anti-CD40. Protein tyrosine kinase (PTK) inhibitors effectively suppressed the both IL-4- and anti -CD40-induced CD23 expression. whereas protein kinase C (PKC) inhibitors had no effects. Electrophoretic mobility shift assays (EMSA) have shown that IL-4 and anti-CD40 induce the activation of NF-IL-4 and NF-_kB, respectively, binding to the CD23 promoter, both in a PKC-independent and PTK-dependent manner. These data suggest that the synergistic activation of CD23 gene expression by IL-4 and anti-CD40 is mediated by co-operative action of distinct nuclear factors. each of which is rapidly activated via PKC-independent and PTK-dependent process.

Intracellular IL-4, IL-10, and IFN-gamma levels of leukemic cells and bone marrow T cells in acute leukemia.

Park, Hun Hee,Kim, Myungshin,Lee, Bong-Hee,Lim, Jihyang,Kim, Yonggoo,Lee, Eun Jung,Min, Woo Sung,Kang, Chang Suk,Kim, Won Il,Shim, Sang In,Han, Kyungja Institute for Clinical Science] 2006 Annals of clinical and laboratory science Vol.36 No.1

<P>The quantitative levels of intracellular cytokines IL-4, IL-10, and IFN-gamma (ie, the number of bound PE-conjugated antibody molecules/cell) of leukemic cells and bone marrow T cells (bmT cells) of acute leukemia patients were analyzed by flow cytometry. One hundred, thirty-one (95 AML, 25 ALL, 11 ABL) patients were studied. The leukemic cell IL-4 level was highest in the monocytic AML group (1735 +/- 1056) and lowest in the dysplastic AML group (960 +/- 545). The IFN-gamma level was highest in the acute promyelocytic leukemia (APL) group (495 +/- 159), and lowest in the ALL group (252 +/- 119). The IL-10 level was not significantly different among the diagnosis groups. In bmT cells, the IL-10 level was highest in the dysplastic AML group (972 +/- 1049) and lowest in the APL group (397 +/- 352). The leukemic cell cytokine levels were lowest and bmT cell cytokine levels were highest in the dysplastic AML group. There were no significant correlations of these cytokine levels with 2-yr survival rate, complete remission (CR) rate, or relapse rate. The cytokine levels of bmT cells at the time of CR became normal and were not different among the diagnosis groups. In summary, leukemic cell and bmT cell cytoplasmic expression profiles of IL-4, IL-10, and IFN-gamma are characteristic for each diagnostic group of acute leukemia patients and the profiles of bmT cells are normal at the time of CR.</P>

Intracellular Signaling Pathways for Type II IgE Receptor (CD23) Induction by Interleukin - 4 and Anti - CD40 Antibody

Kim, Hyun-Il,Park, Hee-Jeoung,Lee, Choong-Eun Korean Society for Biochemistry and Molecular Biol 1997 Journal of biochemistry and molecular biology Vol.30 No.6

Since the role of CD40 on the interleukin-4(IL-4) -induced B cell activation has been strongly implicated in the agumentation of IgE production and response, we have investigated the intracelluar signaling pathways utilized by IL-4 and CD40 for type II IgE receptor (CD23) expression. IL-4 and anti-CD40 antibody treatment of human B cells, independently caused a rapid induction of CD23 gene activation within 2 h. There was a noticeable synergism between the action of the two agents inducing CD23 expression: the addition of anti-CD40 to the IL-4-treated culture significantly agumented the IL-4-induced CD23 on both mRNA and surface protein levels, and the inclusion of IL-4 in the anti-CD40-treated cells caused a further increase of CD23 expression far above the maximal level induced by anti-CD40. Protein tyrosine kinase (PTK) inhibitors effectively suppressed the both IL-4- and anti -CD40-induced CD23 expression. whereas protein kinase C (PKC) inhibitors had no effects. Electrophoretic mobility shift assays (EMSA) have shown that IL-4 and anti-CD40 induce the activation of NF-IL-4 and $NF-_{K}B$, respectively, binding to the CD23 promoter, both in a PKC-independent and PTK-dependent manner. These data suggest that the synergistic activation of CD23 gene expression by IL-4 and anti-CD40 is mediated by co-operative action of distinct nuclear factors. each of which is rapidly activated via PKC-independent and PTK-dependent process.

류마티스 관절염 동물 모델에서 Toll-Like Receptors의 발현

이준희 ( Jun Hee Lee ),이수봉 ( Soo Bong Lee ),김근태 ( Geun Tae Kim ),류선 ( Sun Ryu ),김주인 ( Ju In Kim ),이선희 ( Sun Hee Lee ),김성일 ( Sung Il Kim ) 대한류마티스학회 2006 대한류마티스학회지 Vol.13 No.2

Objective: To evaluate the expression of Toll-like receptor (TLR) 2, 4 and 9 and investigate the effects of IL-17 on the expression of TLRs in experimental rheumatoid arthritis (RA) model. Methods: After induction of collagen-induced arthritis (CIA) by type II collagen in DBA1 mice, phosphate-buffered saline (PBS, PBS group) or IL-17 (IL-17 group) was injected to both knee joint at day 28 and 32. At day 35, mice were sacrificed and knee joints were isolated. Synovial mRNA expressions of TLR-2, 4 and 9 determined by real-time RT-PCR were compared among normal DBA1 mice (normal group), PBS and IL-17 group. Results: Synovial TLR-2, 4, and 9 mRNA expressions of IL-17 and PBS group were significantly higher than normal group, and those of IL-17 group were higher than PBS group. Conclusion: Synovial TLR-2, 4 and 9 expression was enhanced in CIA and up-regulated by local overexpression of IL-17. These results suggest that TLRs play a roles on CIA and IL-17 induced aggravation of arthritis in CIA.

Ahnak-knockout mice show susceptibility to Bartonella henselae infection because of CD4+ T cell inactivation and decreased cytokine secretion

( Eun Wha Choi ),( Hee Woo Lee ),( Jun Sik Lee ),( Il Yong Kim ),( Jae Hoon Shin ),( Je Kyung Se ) 생화학분자생물학회(구 한국생화학분자생물학회) 2019 BMB Reports Vol.52 No.4

The present study evaluated the role of AHNAK in Bartonella henselae infection. Mice were intraperitoneally inoculated with 2 × 10⁸ colony-forming units of B. henselae Houston-1 on day 0 and subsequently on day 10. Blood and tissue samples of the mice were collected 8 days after the final B. henselae injection. B. henselae infection in the liver of Ahnak-knockout and wild-type mice was confirmed by performing polymerase chain reaction, with Bartonella adhesion A as a marker. The proportion of B. henselaeinfected cells increased in the liver of the Ahnak-knockout mice. Granulomatous lesions, inflammatory cytokine levels, and liver enzyme levels were also higher in the liver of the Ahnak-knockout mice than in the liver of the wild-type mice, indicating that Ahnak deletion accelerated B. henselae infection. The proportion of CD4+interferon-y(IFN-y)⁺ and CD4⁺interleukin (IL)-4⁺ cells was significantly lower in the B. henselae-infected Ahnak-knockout mice than in the B. henselae-infected wild-type mice. In vitro stimulation with B. henselae significantly increased IFN-y and IL-4 secretion in the splenocytes obtained from the B. henselae-infected wild-type mice, but did not increase IFN-y and IL-4 secretion in the splenocytes obtained from the B. henselae-infected Ahnak-KO mice. In contrast, IL-1α, IL-1β, IL-6, IL-10, RANTES, and tumor necrosis factor-α secretion was significantly elevated in the splenocytes obtained from both B. henselae-infected wild-type and Ahnak-knockout mice. These results indicate that Ahnak deletion promotes B. henselae infection. Impaired IFN-y and IL-4 secretion in the Ahnak-knockout mice suggests the impairment of Th1 and Th2 immunity in these mice. [BMB Reports 2019; 52(4): 289-294]