Activation of Lymphotoxin-Beta Receptor Enhances the LPS-Induced

Immunology Letters 165 (2015) 63–69

Contents lists available at ScienceDirect

Immunology Letters

j ournal homepage: www.elsevier.com/locate/immlet

Activation of lymphotoxin-beta receptor enhances the LPS-induced

expression of IL-8 through NF-␬B and IRF-1

a a b a,∗

Seok-Won Jang , Su-Geun Lim , Kyoungho Suk , Won-Ha Lee

School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu 702-701, Republic of Korea

Department of Pharmacology, Brain Science & Engineering Institute, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University

School of Medicine, Daegu 700-422, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Lymphotoxin-beta receptor (LT␤R), a receptor for LIGHT and LT␣1␤2, is expressed on the epithelial,

Received 3 February 2015

stromal, and myeloid cells. LT␤R is known to affect the lymphoid organ development and immune

Received in revised form 19 March 2015

homeostasis. However, its role in macrophage function has not been sufﬁciently elucidated. The effect

Accepted 6 April 2015 ␤

of LT R stimulation in the inﬂammatory activation of macrophages was investigated by treating the

Available online 15 April 2015

human macrophage-like cell line THP-1 with LT␤R-speciﬁc monoclonal antibody. Interestingly, com-

bined treatment with anti-LT␤R antibody and LPS caused the synergistic induction of IL-8 expression

Keywords:

at the transcriptional level. Analysis indicated that nuclear factor (NF)-␬B activity was enhanced via

MARCKS

␤

Macrophages the mitogen-activated protein kinase (MAPK) and glycogen synthase kinase (GSK)-3 /cAMP response

␤

Cytokine element binding protein (CREB) pathways. In addition, LT R stimulation induced the expression of inter-

Signaling feron regulatory factor (IRF)-1, one of the major transcription factors of IL-8 gene. Down-regulation of

LPS IRF-1 expression reduced the enhancing effect caused by LT␤R stimulation. This indicates that the LT␤R

␤

LT R stimulation enhances the LPS-induced expression of IL-8 via the combined action of NF-␬B and IRF-1.

␤

1. Introduction who reported LT R to be an inducer of cross-tolerance to the Toll-

like receptor (TLR) ligands.

Lymphotoxin-beta receptor (LT␤R) is a cellular receptor for The cytokine IL-8 was initially identiﬁed for its strong

LIGHT (tumor necrosis factor superfamily 14) and LT␣1␤2 which chemotatic activity toward neutrophils and T-lymphocytes. How-

are expressed on the surface of the cellular counterparts. The LT␤R ever, it is currently believed to be multifunctional, as it has

expression can be detected on the stroma of secondary lymphoid been determined to induce migration, invasion, proliferation, and

organs and of thymus, and in cells of myeloid lineage (reviewed angiogenesis. Aberrant expression of IL-8 is associated with the

in [1]). LT␤R has diverse functionalities; its acts as the regulator of pathogenesis of various diseases. IL-8 is the major chemoattrac-

lymphoid organ development [2,3], mediator of cancer-associated tant for neutrophils in chronic inﬂammatory diseases [14,15]. In

inﬂammation [4,5], and homeostatic stimulator of dendritic cell atherogenesis, IL-8 contributes to plaque formation as a potent

expansion [6,7]. During inﬂammatory activation, ligand binding angiogenic factor [16,17] and induces the migration and prolifera-

induces trimerization of LT␤R, which leads to the recruitment of tion of T-lymphocytes, macrophages, endothelial cells, and smooth

TNF receptor-associated factor (TRAF) for the stimulation of the muscle cells [18–20]. During cancer development, IL-8 expres-

classical NF-␬B pathway via TRAF2/5 [8,9] or the non-canonical sion is elevated in the tumor microenvironment; this leads to the

␬

NF- B pathway via TRAF3 [10]. These inﬂammatory activities were enhancement of cancer-associated activities, such as angiogenesis,

observed in non-immune cells, such as the human embryonic kid- proliferation, invasion and metastasis [21–26]. Because of this, IL-8

ney (HEK)293 cells and the mouse embryonic ﬁbroblasts. Although is considered to be a major therapeutic target for many diseases,

macrophages express high levels of LT␤R, its role in macrophage including cystic ﬁbrosis [27], ocular inﬂammation [28], melanoma

function has not been analyzed except by Wimmer et al. [11–13], [29], breast cancer [30], and colorectal cancer [31].

Although macrophages, along with endothelial cells, are a major

source of IL-8, the regulation of IL-8 expression in macrophages

has not been sufﬁciently elucidated. Interestingly, lipopolysac-

∗ charide (LPS)-induced expression of IL-8 was observed to be

Corresponding author. Tel.: +82 53 950 5388; fax: +82 53 943 6925.

␤

E-mail address: [email protected] (W.-H. Lee). synergistically enhanced by the stimulation of LT R in the human

http://dx.doi.org/10.1016/j.imlet.2015.04.001

64 S.-W. Jang et al. / Immunology Letters 165 (2015) 63–69

macrophage-like cell line THP-1. An analysis of the molecular 2.4. Western blot analysis

mechanisms responsible for these effects revealed the involvement

of NF-␬B, the major transcription factor involved in the regula- Cell lysates were obtained for Western blot at various time

tion of inﬂammatory mediators, and IRF-1, a transcription factor points after activation and analysis was performed as described

involved in the host defense against pathogens, tumor prevention, previously [32,33]. Brieﬂy, cell lysates were collected using NP-40

and development of the immune system. lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris, pH 8.0) containing

1 protease inhibitor cocktail (Calbiochem). Proteins were resolved

by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

2. Materials and methods (SDS-PAGE) and transferred to a nitrocellulose membrane (Mil-

lipore, USA), the membrane was incubated in blocking solution

2.1. Reagents (TBS-T: 5% nonfat dried milk in TBS containing 0.1% Tween-20),

◦

washed with TBS-T, incubated at 4 C with primary antibodies in

The mouse IgG1 isotype control and mouse monoclonal anti- blocking solution for 1–13 h, washed with TBS-T, incubated with

bodies (mAbs) to LT␤R and IRF-1 were obtained from R&D horseradish peroxidase (HRP)-conjugated secondary antibodies at

◦

Systems (Minneapolis, MN, USA). SB203580 was obtained from 4 C for 1 h and then washed. Bands were visualized by using

Calbiochem International Inc. (La Jolla, CA, USA). U0126, rab- enhanced chemiluminescence detection reagents (Pierce, Rock-

bit polyclonal antibodies to extracellular signal-regulated kinase ford, USA) and by exposure to X-ray ﬁlms. For the densitometric

(ERK)-1/2 (p42/44 mitogen-activated protein kinase (MAPK)), analysis of Western blot data, band intensities were measured by

phospho-ERK-1/2 (Thr202/Tyr204), I␬B-␣, p38 MAPK, phosphor- densitometer and normalized with band intensities of correspond-

p38 MAPK (Thr180/Tyr182), and Jun amino-terminal kinase (JNK) ing actin loading control.

MAPK, rabbit mAbs to cAMP response element-binding protein

(CREB) and phospho-NF-␬B p65 (ser536) (93H1), and mouse

2.5. Reverse transcription polymerase chain reaction (RT-PCR)

mAb to phospho-JNK MAPK (Thr183/Tyr185) (G9), phopho-I␬B-␣

(Ser32/36) (5A5), and phospho-CREB (ser-133) (1B6) were acquired

Conventional and real time RT-PCR was performed as described

from Cell Signaling (Danvers, MA, USA). Mouse mAb to NF-␬B

previously [34,35]. Brieﬂy, 5 ␮g of total RNAs isolated from cells

p65 and goat polyclonal antibody to ␤-actin were obtained from

were treated with RNase-free DNase (BD-Pharmingen, San Jose, CA,

Santa Cruz (Santa Cruz, CA, USA). Bacterial LPS and SP600125 were

USA). The RNA was used as a template for the generation of ﬁrst-

purchased from Sigma–Aldrich (St. Louis, MO, USA). TWS119 was TM

strand complementary DNAs (cDNAs) using a RevertAid ﬁrst

acquired from Selleck Chemicals (Houston, TX, USA).

strand cDNA synthesis kit (Fermentas, Hanover, MD, USA) with

500 ng oligo (dT)12–18 primers. PCR primers were designed using

the ABI PRISM Primer Express 2.0 (Applied Biosystems), and pre-

2.2. Cell culture and activation

pared by Geno Tech Corp (Daejeon, Korea). Primer sequences were

5 -CAA ATC CCG GGG CTC ATC TGG-3 (forward) and 5 -CTG GCT

The human macrophage-like cell line THP-1 was cultured in

CCT TTT CCC CTG CTT TGT-3 (reverse) for IRF-1 and 5 -TGC AGT

RPMI 1640 (WelGENE Inc, Korea), supplemented with 10% FBS,

GGC AAA GTG GAG ATT-3 (forward) and 5 -TTG AAT TTG CCG

0.05 mM ␤-mercaptoethanol, and 1× streptomycin–penicillin at

◦ 5 TGA GTG GA-3 (reverse) for GAPDH. The real time PCR reaction

37 C in 5% CO2. THP-1 cells (1.0 × 10 cells/well in 100 ␮l of

was performed in the ABI PRISM 7300 sequence detector (Applied

RPMI 1640 medium in 96 well plate) were pretreated for 30 min

Biosystems) using the SYBR green PCR mix (Applied Biosystems)

with 0.5–5 ␮g/ml of anti-LT␤R mAb (clone 71319, R&D Systems)

with cDNA corresponding to 125 ng of original RNA and 400 nM

and then stimulated with 100 ng/ml LPS for 2–24 h. For cross-

primers in a volume of 20 ␮l. The threshold cycle (Ct) values

tolerance experiment, the pretreatment period was extended to

obtained for each reaction were normalized using the GAPDH Ct

30 h. If required, cells were treated with various inhibitors for

value. Primers corresponding to IL-8 and GAPDH for conventional

30 min before the mAb pretreatment: 10 ␮M U0126 for ERK

RT-PCR were designed using the national center for biotechnol-

MAPK, 5 ␮M SB203580 for p38 MAPK, 10 ␮M SP600125 for JNK

ogy information (NCBI) primer-basic local alignment search tool

MAPK, and 10 ␮M TWS119 for GSK3␤. All the inhibitors were

(BLAST), and synthesized by Geno Tech Corp (Daejeon, Korea).

dissolved DMSO. Triplicate samples were prepared for ELISA

Primer sequences were 5 -CAA GGA AAA CTG GGT GCA GA-3 (for-

measurements.

ward) and 5 -TCG GAT ATT CTC TTG GCC CT-3 (reverse) for IL-8

and 5 -ATC ACT GCC ACC CAG AAG AC-3 (forward) and 5 -TGA

GCT TGA CAA AGT GGT CG-3 (reverse) for GAPDH. After heating

2.3. Enzyme-linked immunosorbent assay (ELISA ◦

at 94 C for 5 min, PCR was performed under following conditions:

◦ ◦ ◦

35 cycles of 94 C for 1 min, 58 C for 1 min, 72 C for 1 min. After

The quantity of cytokines present in the supernatant was mea- ◦

second step, PCR was performed 1 cycle of 72 C for 7 min. PCR

sured using a sandwich ELISA (R&D Systems). Brieﬂy, ﬂat-bottom

products were resolved on a 1.2% agarose gel stained with ethid-

96 well plates were coated with capture antibodies (diluted to

◦ ium bromide. For the densitometric analysis of RT-PCR data, band

1/250 in coating buffer) and incubated overnight at 4 C. Wells were

intensities were measured by densitometer and normalized with

washed three times with 0.05% Tween-20 diluted in PBS (wash

intensities of corresponding GAPDH bands.

buffer) and blocked with 200 ␮l of 1× Assay diluent for 1 h at RT.

After washing, 100 ␮l of samples/well were added and incubated

for 2 h at RT. The wells were sequentially incubated 1 h at RT with 2.6. RNA interference

100 ␮l of detection antibodies (diluted to 1/250 in 1× Assay dilu-

ent), streptavidin-HRP (diluted to 1/250 in 1× Assay diluent) for The IRF-1 expression was suppressed using a mixture containing

30 min at RT, 100 ␮l of substrate solution (eBioscience) for 30 min three speciﬁc small interfering RNA (siRNA) coding for IRF-1, pur-

at RT, and 50 ␮l 2N H2SO4 to stop the reaction. The colorimetric chased from Santa Cruz Biotechnology. The siRNA were transfected

change was detected by microplate reader set to 450 nm (corrected into cell lines as described in a previous study [36] using Dharma-

by absorption at 540 nm). The measurement was performed in trip- FECT (Dharmacon Inc., Lafayette, CO, USA), as per the manufacturer

licate and the detection limit was <10 pg/ml. protocols.

S.-W. Jang et al. / Immunology Letters 165 (2015) 63–69 65

2.7. ELISA-based measurement of NF-B binding activity that of p38 MAPK was not affected. In agreement with this obser-

vation, the inhibitors of ERK or JNK MAPK efﬁciently blocked IL-8

NF-␬B binding activity was measured as per a previously production in cells treated with the antibody and LPS (Fig. 2B).

described method [37]. Brieﬂy, biotin-labeled double-stranded MAPK activation is known to activate NF-␬B [38–40], the major

oligonucleotides containing a consensus NF-␬B binding site (5 - transcription factor involved in inﬂammatory activation in the

CAC AGT TGA GGG GAC TTT CCC AGG C-3 ) (0.02 nm/well) was macrophages. In order to test the functional activity of NF-␬B, the

immobilized in streptavidin-coated 96-well culture plates. Whole NF-␬B binding sequence elements were immobilized in a culture

cell or nuclear lysates were then added to the NF-␬B oligo plates plate, and the binding activity was tested in cells stimulated with

(100 ␮l/well in phosphate buffered saline), and incubated at room the antibody and LPS. As seen in Fig. 3A, LPS-induced NF-␬B binding

temperature for 1 h. The whole cell and nuclear lysates were was enhanced by antibody pretreatment (with statistical signiﬁ-

observed to produce the same results. The plates were then sequen- cance), indicating that LT␤R-mediated enhancement of ERK and

tially incubated with antibodies speciﬁc to NF-␬B p65 subunit, JNK MAPKs leads to the enhancement of NF-␬B activity.

horseradish peroxidase (HPR)-labeled secondary antibody and CREB is a ubiquitous transcription factor involved in pro-

tetramethylbenzidine (chromogen). The absorbance (450–540 nm) liferation, survival, development, and tumorigenesis. During

of the solution was then measured, and the values normalized by inﬂammatory response, phospho-CREB antagonizes NF-␬B activ-

subtracting the background values. For blocking, cell lysates were ity (reviewed in [41]). In order to determine if LT␤R activation

pre-incubated with 1.0 nm/sample of double-stranded oligonu- affects CREB activity, CREB phosphorylation was examined. As seen

cleotides containing the wild type NF-␬B binding sequence or a in Fig. 3B, pretreatment with anti-LR␤R mAb reduced LPS-induced

mutant sequence (5 -CAC AGT TGA GGC CAC TTT CCC AGG C-3 ) phosphorylation of CREB.

before addition to the NF-␬B oligo plates. Recent studies have revealed GSK3 to be a pivotal regulator of

inﬂammation [42] and an upstream regulator of CREB [41]. GSK3␤

inactivates CREB via the direct phosphorylation of ser-129 [43], as

2.8. Statistical analysis

well as the indirect de-phosphorylation of ser-133 [44]. As the CREB

phosphorylation levels were altered as a result of LT␤R and TLR4

All data were presented as mean ± standard error of mean

stimulation, GSK3␤ was believed to regulate this process. In order

(SEM), with the number of independent experiments indicated in

to examine this possibility, THP-1 cells were stimulated with anti-

the ﬁgure legends. All analyses were performed using the SPSS soft-

␤

LT R and LPS in the presence of a GSK3␤ inhibitor. As seen in Fig. 3C,

ware (IBM, Armonk, NY, USA) with one-way ANOVA or the paired

␤

GSK3 inhibition resulted in the increased phosphorylation of CREB

or unpaired Student’s t-test, as appropriate. Differences were con-

(ser-133). This indicated that LT␤R affects NF-␬B activity via the

sidered signiﬁcant at p < 0.05.

␤

GSK3 /CREB pathway (in addition to the MPK pathway). In agree-

ment with this observation, the IL-8 enhancing effect of LT␤R was

3. Results

greatly suppressed in the presence of the GSK3␤ inhibitor (Fig. 3D).

3.1. LTˇR stimulation enhances the LPS-induced expression of

IL-8 3.3. LTˇR stimulation enhances the expression of IRF-1, a

transcription factor for IL-8

THP-1 is a human macrophage-like cell line that expresses LT␤R,

␬

as determined by ﬂow cytometry (data not shown). Monoclonal The enhancement of LPS-induced activation of NF- B by anti-

␤

antibody (mAb) against LT␤R was used for the speciﬁc stimula- LT R mAb pretreatment appeared to be responsible for the slight

␣

␤

tion of LT R since utilization of natural ligands such as LIGHT or increase in TNF- and MCP-1. However, the extent of increase in

␣

lymphotoxin may cross activate other receptors of TNF receptor IL-8 production was much greater that of TNF- or MCP-1. This indi-

␤

superfamily. As seen in Fig. 1A, anti-LT␤R mAb alone failed to cated that LT R stimulation activated some additional IL-8 speciﬁc

induce cytokine production. The application of mAb for a 30 min factors. Interestingly, the expression IRF-1, a transcription factor

␣

pretreatment caused a strong increase in LPS-induced production involved in the expression of IL-8, but not that of TNF- or MCP-1,

␤

of IL-8 (Fig. 1A and B). This result indicates that the mAb has ago- was induced in the THP-1 cells by anti-LT R mAb or LPS treatment,

nistic activity. On the other hand, the isotype-matching mouse IgG as determined by real time RT-PCR (Fig. 4A). Accordingly, West-

did not enhance the LPS response, indicating that this enhancement ern blot analysis revealed that the cells sequentially treated with

␤

did not result from non-speciﬁc interactions of the antibody with anti-LT R and LPS displayed higher overall levels of IRF-1 when

the FcR present on the macrophage surface. Other cytokines such compared to the cells treated with LPS alone (Fig. 4B).

as TNF-␣, and MCP-1 was similarly increased by the mAb but the In order to conﬁrm the role of IRF-1 in the enhancement of

enhancing effect was less than that of IL-8. Therefore, further stud- LPS-induced expression of IL-8, the expression of IRF-1 was down-

␤

ies were focused on IL-8. In order to determine if the enhancement regulated by RNA interference. A mixture of three LT R-speciﬁc

of IL-8 production occurred at the transcriptional level, RT-PCR siRNA was used to transfect the THP-1 cells. IRF-1 expression of

analysis of the cytokine mRNA was performed. As seen in Fig. 1C, was successfully down-regulated by the IRF-1 speciﬁc siRNAs, but

LPS-induced IL-8 mRNA levels were increased by antibody pre- not by the control siRNA (Fig. 4C). These transfectants were pre-

␤

treatment indicating that the pretreatment with anti-LT␤R mAb treated with anti-LT R mAb and stimulated with LPS. As seen in

␤

resulted in the enhancement of LPS-induced signaling pathway, Fig. 4D, the enhancing effect of anti-LT R mAb was decreased in

which in turn lead to transcription activation of the IL-8 gene. cells transfected with IRF-1 speciﬁc siRNA compared to the cells

transfected with the control siRNA.

3.2. LTˇR induces NF-B activation via the MAPK and glycogen

synthase kinase (GSK)3ˇ/CREB pathways. 3.4. LTˇR-induced cross tolerance applies to TNF-˛, but not to

IL-8

As the MAPK activation is induced by LPS, MAPK activation was

investigated in THP-1 cells pretreated with anti-LT␤R mAb and A previous analysis of LT␤R expression in macrophages revealed

stimulated with LPS. As seen in Fig. 2A, ERK and JNK MAPK phos- that the stimulation of LT␤R for an extended period of time induces

phorylation was enhanced by the antibody pretreatment, while tolerance against later stimulation by TLR ligands. Wimmer et al.

66 S.-W. Jang et al. / Immunology Letters 165 (2015) 63–69

Fig. 1. Stimulation of LT␤R in combination with LPS treatment resulted in the synergistic expression of IL-8 at the transcription levels. (A) THP-1 cells were pretreated with

the indicated concentrations of anti-LT␤R mAb of mIgG for 30 min. These were then stimulated with LPS (100 ng/ml) for 24 h (8 h for TNF-␣). The IL-8, TNF-␣, and MCP-1

secretion levels were measured by ELISA (*p < 0.05, **p < 0.01, and ***p < 0.001, when compared to the LPS-treated positive control). (B) Cells were prepared as described in (A)

for indicated times. Culture supernatants were then collected for the measurement of IL-8 concentrations (*p < 0.05 and ***p < 0.001, when compared to the corresponding

LPS-treated samples). (C) Cells were prepared as described in (A) for indicated times and RT-PCR analysis was performed to measure IL-8 and GAPDH mRNA levels. Band

intensities were then measured using densitometer and normalized with band intensities of corresponding GAPDH control. Values indicate relative mRNA levels to zero time

control sample (*p < 0.05, when compared to the corresponding LPS-treated samples).

Fig. 2. Stimulation of LT␤R enhances LPS-induced activation of ERK and JNK MAPKs. (A) THP-1 cells were pretreated with 5 ␮g/ml anti-LT␤R for 30 min and then stimulated

with 100 ng/ml LPS for the indicated times. The activation status of p38, ERK, and JNK MAPKs were analyzed by measuring their phosphorylation levels using Western blot.

(B) Cells were treated as in (A) for 24 h in the presence of MAPK inhibitors (10 ␮M U0126 for ERK, 5 ␮M SB203580 for p38, and 10 ␮M SP600125 for JNK). Culture supernatants

were then collected and the concentrations of IL-8 measured using ELISA. Vehicle control (VC), 0.2% DMSO.

[12] reported that the pretreatment of macrophages with anti- cells, the stimulation of LT␤R with LIGHT had a major effect on

␤

LT R mAb for 30 h (instead of 30 min, as in our analysis) induced the IL-6 and IL-8 expression via the MAPK/NF-␬B pathway [45].

cross-tolerance to TLR4 and TLR9 ligands which was manifested by Overexpression of LT␤R in HEK293 cells led to an NF-␬B- and

the decrease in the expression of TNF-␣ or IL-6. In order to examine AP-1-dependent increase in IL-8 promoter activity [46]. Our data

if this type of LT␤R-induced cross tolerance applied to IL-8, THP-1 demonstrated that the stimulation of LT␤R enhances LPS-induced

cells were treated with anti-LT␤R mAb for 30 h and stimulated with activation of NF-␬B and IRF-1 in macrophages. This is the ﬁrst

LPS. As seen in Fig. 5, the LPS-induced production of TNF-␣ was sig- report that linked LT␤R activity to IRF-1 activation. The macrophage

niﬁcantly blocked, while the production of IL-8 was unaffected by speciﬁcity of this event remains unclear, and must be investigated

LT␤R pretreatment. further. The synergistic action of NF-␬B and IRF-1 is responsible for

the enhanced expression of IL-8 in cells treated with anti-LT␤R and

␬ ␣

4. Discussion LPS. As only NF- B (and not IRF-1) is involved in TNF- or MCP-1

expression, the effect of LT␤R stimulation on these cytokines would

Our data demonstrate that LT␤R has an enhancing effect on LPS- be less, compared to that on IL-8 expression (Fig. 1A). As both IRF-

␬

induced IL-8 expression in the human macrophage-like cell line 1 and NF- B are involved in IL-8 expression, the blocking effect

THP-1. The effect of LT␤R on IL-8 expression was also observed of IRF-1-speciﬁc siRNA was determined to be incomplete in IL-8

in cell types other than macrophages. In bronchial epithelial expression (Fig. 4).

S.-W. Jang et al. / Immunology Letters 165 (2015) 63–69 67

Fig. 3. Stimulation of LT␤R enhances NF-␬B activation through the suppression of CREB activity. (A) THP-1 cells were pretreated with 5 ␮g/ml anti-LT␤R mAb for 30 min and

then stimulated with 100 ng/ml LPS for 30 min. The functional activation of NF-␬B was then measured using an ELISA-based NF-␬B binding assay as described in Section 2.

***p < 0.001. (B) Cells were treated as in (A) for the indicated times. The protein levels of phospho-CREB, CREB and actin were analyzed using Western blot. Band intensities

were then measured using densitometer and normalized with band intensities of corresponding actin control. Values indicate measurements relative to the zero time no

treatment control (***p < 0.001 when compared to the zero time control). (C) Cells were pretreated with 10 ␮M TWS119 (GSK3␤ inhibitor) for 30 min, and with 5 ␮g/ml

anti-LT␤R mAb for 30 min. The cells were then stimulated with 100 ng/ml LPS for the indicated times for the analysis of phospho-CREB and actin using Western blot. Band

intensities were then measured using densitometer and normalized with band intensities of corresponding actin control. Values indicate measurements relative to the zero

time no treatment control (*p < 0.05 and **p < 0.01 when compared to the zero time control). (D) Cells were treated as in (C) for 24 h. Culture supernatants were then collected

␣

for TNF- and IL-8 concentration measurement using ELISA (***p < 0.001 when compared to the corresponding LPS-treated positive control). VC, 0.04% DMSO.

Pretreatment with anti-LT␤R antibody for a longer period of TRM30␣ subsequently suppressed the expression of TNF-␣ and

time caused a reduction in the LPS-induced production of TNF-␣, IL-6 in cells stimulated with TLR ligands [11,13]. As the extended

while that of IL-8 was unaffected. This indicated that LT␤R-induced stimulation of LT␤R did not affect IL-8 expression, it appeared that

cross-tolerance may apply to some, but not all, of the proin- the IRF-1 activity induced by anti-LT␤R treatment persisted during

ﬂammatory mediators. During the extended pretreatment period, the 30 h treatment period and this was strong enough to overcome

␤ ␬

LT R-mediated signaling activated the expression of the tripar- the NF- B inactivation. This further indicated that IRF-1 activity

tite motif protein (TRIM)30␣, a negative regulator of NF-␬B [47], could sustain and induce IL-8 expression, even when the NF-␬B

in a TNF receptor-associated factor (TRAF)3-dependent manner. activity was suppressed.

Fig. 4. Stimulation of LT␤R induces the expression of IRF-1, which in turn is responsible for the activation of IL-8 gene expression. (A) THP-1 cells were treated with 5 ␮g/ml

␤

anti-LT R mAb or LPS for 2 h, and the IRF-1 gene expression measured using real-time RT-PCR (***p < 0.001 when compared to the control). (B) Cells were pretreated with

5 ␮g/ml anti-LT␤R for 30 min and stimulated with 100 ng/ml LPS for the indicated times. The IRF-1 protein levels were analyzed using Western blot. Band intensities were

then measured using densitometer and normalized with band intensities of corresponding actin control. Values indicate measurements relative to the zero time no treatment

control (*p < 0.05 and **p < 0.01 when compared to the zero time no treatment control). (C) Cells were transfected with control siRNA (siCON) or IRF-1-speciﬁc siRNA (siIRF1).

The IRF-1 and actin expression were analyzed using Western blot at 48 h after treansfection. (D) Control or siIRF1 transfectants were treated as in (B) for 24 h. Culture

supernatants were then collected to measure the IL-8 concentration (ELISA). *p < 0.05. NS, no signiﬁcance.

68 S.-W. Jang et al. / Immunology Letters 165 (2015) 63–69

␤

Fig. 5. LT R-induced cross-tolerance to LPS affects TNF-␣ but not IL-8. THP-1 cells were pretreated with 5 ␮g/ml anti-LT␤R mAb for 30 h, and subsequently stimulated with

100 ng/ml LPS for 9 h. Culture supernatants were then collected to measure TNF-␣ and IL-8 concentrations using ELISA (***p < 0.001).

Current data indicate that LT␤R enhanced the LPS-induced NF- Acknowledgements

␬

B activity through two separate mechanisms; the MAPK and

␤

GSK3 /CREB pathways. MAPKs play a pivotal role in LPS-induced This research was supported by the Basic Science Research

activation of pro-inﬂammatory cytokines and MAPK inhibition Program of the National Research Foundation of Korea (NRF),

blocks the LPS-induced activation of NF-␬B [38–40] and TNF-␣ funded by the Ministry of Education, Science and Technology

expression [48–50]. The NF-␬B activity is manifested through its (2012R1A1A4A01005809 and 2014R1A1A2053381).

interaction with the transcription coactivator CREB-binding pro-

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