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of June 18, 2017.
Outside-to-Inside Signal Through the
Membrane TNF- α Induces E-Selectin
(CD62E) Expression on Activated Human
CD4+ T Cells
Shin-ichi Harashima, Takahiko Horiuchi, Nobuaki Hatta,
Chika Morita, Masanori Higuchi, Takuya Sawabe, Hiroshi
Tsukamoto, Tomoko Tahira, Kenshi Hayashi, Shigeru Fujita
and Yoshiyuki Niho
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 166:130-136; ;
doi: 10.4049/jimmunol.166.1.130
http://www.jimmunol.org/content/166/1/130
Outside-to-Inside Signal Through the Membrane TNF-␣
Induces E-Selectin (CD62E) Expression on Activated Human
CD4ⴙ T Cells1
Shin-ichi Harashima,* Takahiko Horiuchi,2* Nobuaki Hatta,‡ Chika Morita,*
Masanori Higuchi,* Takuya Sawabe,* Hiroshi Tsukamoto,* Tomoko Tahira,† Kenshi Hayashi,†
Shigeru Fujita,‡ and Yoshiyuki Niho*
umor necrosis factor-␣ is a multifunctional cytokine produced by activated macrophages as well as by several
other cell types, such as lymphocytes and neutrophils (1–
3). The precursor form of TNF-␣, called membrane TNF-␣, is the
26-kDa cell surface transmembrane type II polypeptide, consists of
N-terminal 30-aa residues of cytoplasmic domain, followed by 26
aa of transmembrane domain and 177 aa of extracellular domain.
The C-terminal portion with 157 aa is cleaved from the membrane
TNF-␣ by TNF-␣-converting enzyme and acts as a soluble form of
17-kDa polypeptide (4, 5). Most of the TNF-␣-mediated biological
effects, such as cell proliferation, apoptosis, and cytokine production, are attributed to the soluble form of TNF-␣, whereas the
function of membrane TNF-␣ is much less understood. The biological effects of TNF-␣ are mediated through two distinct cell
surface receptors, TNF-RI (p55) and TNF-RII (p75). Although
TNF-RI is supposed to be responsible for most of the TNF-␣mediated biological effects, the function of TNF-RII is not well
understood.
T
*First Department of Internal Medicine, Faculty of Medicine, and †Institute of Genetic Information, Kyushu University, Fukuoka, Japan; and ‡First Department of Internal Medicine, School of Medicine, Ehime University, Ehime, Japan
Received for publication December 1, 1999. Accepted for publication September
29, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by grants-in-aid from the Ministry of Education,
Science, Sports, and Culture of Japan (10670417) and the Yokoyama Foundation for
Clinical Pharmacology.
2
Address correspondence and reprint requests to Dr. Takahiko Horiuchi, First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka
812-8582, Japan. E-mail address: [email protected]
Copyright © 2001 by The American Association of Immunologists
The biological function of membrane TNF-␣ was first reported
as cytotoxic activity (6 – 8), which was followed by the identification of several immunological functions. The membrane TNF-␣ is
involved in the polyclonal B cell activation induced by HIV-infected CD4⫹ T cells (9) and by human T cell leukemia virus
(HTLV)3-I-infected CD4⫹ T cells (10, 11), IL-10 production from
monocytes (12), and ICAM-1 expression from endothelial cells (13).
Costimulatory signals for IL-4-dependent Ig synthesis are also provided by membrane TNF-␣ (14). All of these biological effects by
membrane TNF-␣ are mediated through a cell-to-cell contact fashion.
The signal transmitted from membrane TNF-␣ to the target cells is
likely to be mediated through both TNF receptors; however, it is more
potent on TNF-RII than soluble TNF-␣ (15).
In contrast to the above functions of membrane TNF-␣ as a
ligand, the reverse (outside-to-inside) signal via membrane TNF-␣
into the cells expressing membrane TNF-␣ is poorly understood.
Considering its extreme amino acid conservation (nearly 90%) between different animal species (16) and its phosphorylation in
some monocytic cells and in 26-kDa precursor TNF-␣-transfected
HeLa cells (17), the cytoplasmic domain of membrane TNF-␣
should play some critical role in the cellular functions. Recently,
our group first demonstrated the signaling through the membrane
TNF-␣ into HTLV-I-infected human T cells that express this molecule on their surface. Upon activation of membrane TNF-␣ with
anti-TNF-␣ Ab, IL-2 and IFN-␥ were induced with concurrent
elevation of intracellular calcium concentration (10, 11). This finding of calcium mobilization by membrane TNF-␣ was confirmed
3
Abbreviations used in this paper: HTLV, human T cell leukemia virus; FasL, Fas
ligand; CD40L, CD40 ligand; /FITC, FITC-conjugated; /PE, PE-conjugated; VLA,
very late Ag; CD30L, CD30 ligand; UTR, untranslated region.
0022-1767/01/$02.00
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The membrane TNF-␣ is known to serve as a precursor of the soluble form of TNF-␣. Although it has been reported the biological
functions of the membrane TNF-␣ as a ligand, the outside-to-inside (reverse) signal transmitted through membrane TNF-␣ is
poorly understood. Here we report a novel function mediated by outside-to-inside signal via membrane TNF-␣ into the cells
expressing membrane TNF-␣. Activation by anti-TNF-␣ Ab against membrane TNF-␣ on human T cell leukemia virus (HTLV)
I-infected T cell line, MT-2, or PHA-activated normal human CD4ⴙ T cells resulted in the induction of an adhesion molecule,
E-selectin (CD62E), on the cells with the peak of 12–24 h, which completely disappeared by 48 h. When wild-type or mutant
membrane TNF-␣ (R78T/S79T) resistant to proteolytic cleavage was introduced into Jurkat or HeLa cells, E-selectin was induced
by the treatment with anti-TNF-␣ Ab with the similar kinetics. Membrane TNF-␣-expressing Jurkat cells also up-regulated
E-selectin when brought into cell-to-cell contact with TNF receptor-expressing HeLa cells. Northern blot analysis and RT-PCR
analysis showed that the membrane TNF-␣-mediated E-selectin expression was up-regulated at the level of transcription. These
results not only confirmed our previous findings of reverse signaling through membrane TNF-␣, but also presented evidence that
E-selectin was inducible in cell types different from endothelial cells. It is strongly suggested that membrane TNF-␣ is a novel
proinflammatory cell surface molecule that transmits bipolar signals in local inflammation. The Journal of Immunology, 2001,
166: 130 –136.
The Journal of Immunology
Materials and Methods
Cells
A HTLV-I-infected T cell clone MT-2 was derived from a patient with
adult T cell leukemia. MT-2 cells, normal human CD4⫹ T cells, and human
lymphoblastoid T cell line Jurkat cells were maintained in RPMI 1640
supplemented with 10% heat-inactivated FBS, 100 IU/ml of penicillin, and
100 mg/ml of streptomycin at 37°C in a 5% CO2-humidified atmosphere.
Human epithelioid cervical carcinoma cell line HeLa cells were cultured in
the same condition described above except for using DMEM instead of
RPMI 1640. Normal human CD4⫹ T cells were purified from the PBMC
of healthy volunteers negative for serum Abs to HTLV-I using anti-CD4
mouse mAb with magnetic beads (Dynal, Oslo, Norway). HUVEC were
harvested from the human umbilical veins and cultured as described previously (22).
Cell stimulation
Flasks (25 ml; Becton Dickinson Labware, Lincoln Park, NJ) were coated
with 1 ␮g/ml of rabbit polyclonal anti-human TNF-␣ Ab (Genzyme, Cambridge, MA), anti-human TNF-␣ mouse mAb (Genzyme), rabbit polyclonal anti-human Fas ligand (FasL) Ab (Santa Cruz Biotechnology, Santa
Cruz, CA), or rabbit polyclonal anti-human CD40 ligand (CD40L) Ab
(Santa Cruz Biotechnology) in carbonate buffer (35 mM NaHCO3, 15 mM
Na2CO3, pH 9.6) and were incubated overnight at 4°C. Normal CD4⫹ T
cells (2 ⫻ 105/ml) were stimulated with 100 ng/ml of PHA (Wako Chemical, Osaka, Japan) for 12 h, followed by washing in PBS. The activated
CD4⫹ T cells (2 ⫻ 105/ml) or MT-2 cells (2 ⫻ 105/ml), both of which
expressed membrane TNF-␣ on the cell surface, were incubated in a 25-ml
flask immobilized with anti-TNF-␣ Ab for 12–24 h in the presence or
absence of various inhibitors, 100 ng/ml of herbimycin A (Wako Chemical), 40 ␮g/ml of genistein (Wako Chemical), 100 ␮M of N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7; Wako Chemical), 100 ␮M of
manumycin, 100 ng/ml of wortmannin (Wako Chemical), 100 ␮M of 1-(5isoquinolinylsulfonyl)-2-methylpiperazine (H-7; Wako Chemical), 100
␮g/ml of cycloheximide (Wako Chemical), and 2 mM of EGTA (Wako
Chemical). Jurkat cells (2 ⫻ 105/ml) were incubated in a 25-ml flask immobilized with anti-CD40L or FasL for 12–24 h. Rat anti-human TNF-␣
receptor (p75) mAb (Genzyme) and anti-human TNF-␣ receptor (p55) Ab
(Bender MedSystems, Vienna, Austria) were used for blocking the function of TNF-␣. These blocking Abs were added to the culture media concurrently with the stimulation of membrane TNF-␣ by immobilized antiTNF-␣ Ab. The presence of soluble TNF-␣ in the supernatant was
quantified by a commercial ELISA kit (Endogen, Woburn, MA). Recombinant human TNF-␣ (17 kDa) was provided by Dainippon Pharmaceutical
(Osaka, Japan).
FACS analysis
The experimental procedure has been described previously (10). Briefly,
cells were washed three times with staining medium, PBS containing 1%
FCS and 0.1% NaN3. Cells (5 ⫻ 105 per sample) were stained on ice. Cell
surface molecules including TNF-␣ and adhesion molecules were detected
by FACScan (Becton Dickinson) using the following FITC-conjugated
(/FITC) and PE-conjugated (/PE) Abs. For TNF-␣, anti-human TNF-␣/
FITC mAb (R&D Systems, Minneapolis, MN) was used. For the detection
of adhesion molecules, anti-human E-selectin (CD62E)/FITC mAb, antihuman P-selectin (CD62P)/FITC (Southern Biotechnology Associates,
Birmingham, AL), anti-human very late Ag (VLA)-4/PE mAb (Ancell,
Bayport, MN), anti-human ICAM/FITC mAb, anti-human LFA-1/PE
mAb, anti-human L-selectin (CD62L)/PE mAb, and anti-human VCAM1/FITC mAb (Immunotech, Marseilles, France) were used. Anti-human
CD30 ligand (CD30L)/FITC mAb (Santa Cruz Biotechnology), antihuman CD40L/FITC (MBL, Nagoya, Japan), and rabbit polyclonal antihuman FasL Ab followed by FITC-conjugated goat anti-rabbit Ig (Southern Biotechnology Associates) were used to detect the expression of the
members of TNF ligand family other than TNF-␣. Mouse IgG1/FITC,
IgG1/PE (Dako, Glostrup, Denmark), and rabbit Ig (Cappel, Durham, NC)
followed by goat anti-rabbit Ig/FITC (Southern Biotechnology Associates)
were used as negative control. Expression of TNF-RI and TNF-RII was
studied using anti-human TNF-RI mAb/FITC (Genzyme) and anti-human
TNF-RII mAb/PE (Genzyme). To confirm the expression of membrane
TNF-␣, soluble TNF-␣ that might be bound to the cell surface was removed by washing with acidified buffer. PHA-activated CD4⫹ T cells (1 ⫻
106 cells) were suspended in 1 ml of 50 mM glycine HCl buffer containing
150 mM NaCl, pH 3.0, for 3 min at 4°C. After extensive washing with PBS
several times, the expression of membrane TNF-␣ on the cells was studied
by FACS analysis.
RNA preparation
MT-2 cells were stimulated by immobilized rabbit anti-human TNF-␣
polyclonal Ab for 1, 2, or 12 h. HUVEC were stimulated with LPS as
described (22). Total RNA was prepared from the MT-2 cells and HUVEC
as described (23).
RT-PCR analysis
Total RNA was subjected to RT-PCR by using a GeneAmp RNA PCR kit
(Perkin-Elmer, Foster City, CA) as described (24). Briefly, 1 ␮g of total
RNA was reverse transcribed in the presence of Moloney murine leukemia
virus (MoLV) reverse transcriptase, oligo dT, and 25 ␮M dNTP at 25°C for
60 min in a total reaction mixture of 25 ␮l. The primers for E-selectin were
designed based on the published cDNA sequence (25). The sense primer
was 5⬘-CCAGTGCTTATTGTCAGC-3⬘, and the anti-sense primer was 5⬘CACATTGCAGGCTGGAAT-3⬘ with the expected size of 610 bp, corresponding to the extracellular domain of E-selectin (amino acid residues
15–219). The nucleotide sequence for the ␤-actin was described previously
(22), and the expected size of the PCR product was 314 bp. PCR were
performed using 1 ␮l of the cDNA product as template, 0.2 ␮M of each
primer, 25 ␮M of dNTP, 2 ␮Ci [␣-32P]dCTP (Amersham, Arlington
Heights, IL), 0.125 U of Taq polymerase, and the standard buffer supplied
by the manufacturer in a total reaction volume of 5 ␮l. Reactions were
conducted for 30 cycles consisting of 30 s at 94°C, 30 s at 60°C, and 1 min
at 72°C. The PCR products were then subjected to electrophoresis on 1%
agarose gels, and the DNA bands were visualized with ethidium bromide.
Northern blot analysis
A total of 10 ␮g RNA was denatured with formaldehyde, subjected to
electrophoresis in a formaldehyde/1% agarose gel, transferred to Hybond-N⫹ membrane (Amersham), and covalently linked by UV irradiation
using a Stratalinker UV cross-linker. Hybridizations were performed at
65°C overnight in buffer (0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA).
The cDNA for E-selectin (610 bp) and GAPDH (340 bp) was labeled with
[␣-32P]dATP using Multiprime DNA labeling systems (Amersham) and
was used as probes. After hybridization, filters were washed with 0.2 ⫻
SSC at 65°C for 20 min with four changes. Autoradiography was performed with an intensifying screen at ⫺70°C for 18 h.
Expression of membrane TNF-␣ on Jurkat and HeLa cells
Wild-type membrane TNF-␣ cDNA was obtained by using RT-PCR of the
total RNA from normal human PBMC. The primer sequences were 5⬘ATGAGCACTGAAAGCATGATCCGGGACGTG-3⬘ and 5⬘-TCACAG
GGCAATGATCCCAAAGTAGACCTG-3⬘ and the product size was 702
bp, encompassing the entire coding region of the membrane TNF-␣. The
uncleavable mutant form of membrane TNF-␣ was generated by using the
oligodeoxyribonucleotide-directed amber method according to the manufacturer’s instructions (Mutan-Super Express Km kit; Takara Shuzo,
Kyoto, Japan). Briefly, the membrane TNF-␣ was subcloned into pKF19.
Both the codon AGA for Arg77 at position ⫹2 (2 aa downstream from the
TNF-␣-converting enzyme cleavage site) and the codon TCA for Ser78 at
position ⫹3 were substituted to the codon ACA for Thr and the codon
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subsequently by others (18). Membrane TNF-␣ has also been
shown to modulate anti-CD3-triggered T cell cytokine expression
in mice (19). Thus it is suggested that membrane TNF-␣ expressed
on T cells is a bipolar positive regulator of inflammation either
transmitting signals as a ligand to the target cells or receiving
signals through membrane TNF-␣ itself into T cells.
These lines of evidence prompted us to extend our search for
novel proinflammatory functions mediated through membrane
TNF-␣ into T cells expressing this molecule. Here we report that
activation of membrane TNF-␣ on human T cells induced expression of E-selectin, an adhesion molecule of the selectin family that
is known to be exclusively expressed on activated endothelial cells
(20), except in the case of astrocytes that expresses E-selectin after
TNF-␣ stimulation (21). Moreover, expression of E-selectin was
also demonstrated by the treatment with anti-TNF-␣ Ab of the
membrane TNF-␣-transfected Jurkat and HeLa cells. Thus we confirmed the reverse signaling from membrane TNF-␣ into T cells
and, importantly as well, we showed further evidence that E-selectin was inducible from cell types that are different from endothelial cells.
131
132
REVERSE SIGNAL BY MEMBRANE TNF-␣ INDUCES E-SELECTIN ON HUMAN T CELLS
ACA for Thr, respectively, which has already been shown to result in the
effective reduction in the cleavage of membrane TNF-␣ (17). The oligonucleotide sequence to introduce those mutations in the membrane TNF-␣
was 5⬘-CAGGCAGTCACAACATCTTCTC-3⬘. The oligonucleotide sequences of the wild-type and mutant forms of membrane TNF-␣ were
confirmed by direct sequencing using Amplicycle sequencing kit (PerkinElmer) (26). Wild-type and uncleavable mutant forms of membrane TNF-␣
were cloned into pCXN2 expression vector (27) and were transfected into
human lymphoblastoid T cell line, Jurkat, or epithelioid cervical carcinoma
cell line, HeLa, by using LIPOFECTAMINE (Life Technologies, Grand
Island, NY). The transfected Jurkat and HeLa cells were selected in the
presence of 1.5 or 1.0 mg/ml of G418 (Sigma, St. Louis, MO) in RPMI
1640 supplemented with 10% FCS, respectively. The transfected Jurkat
cells (2 ⫻ 105 cells/ml) and HeLa cells were stimulated with rabbit antihuman TNF-␣ polyclonal Ab (diluted to 1:1000) to see the up-regulation of
E-selectin. The competition by an excess amount of soluble TNF-␣ was
studied by adding 1 ␮g/ml of recombinant soluble TNF-␣ to the culture
medium.
Expression of TNF-RII on HeLa cells
Coculture of membrane TNF-␣-expressing T cells with TNF-RIIexpressing HeLa cells
TNF-RII-expressing or untransfected HeLa cells were cultured in a 12.5-ml
flask in DMEM plus 10% FCS until ⬃80% confluent. Then, the culture
medium was discarded, and Jurkat cells were transfected with uncleavable
mutant form of membrane TNF-␣ (5 ⫻ 105 cells/ml of RPMI 1640 plus
10% FCS; 5 ml in a total volume) were added to the culture flask. Coculture of membrane TNF-␣-expressing Jurkat cells with TNF-RII-expressing
HeLa cells was continued for up to 72 h with or without
TNF-RII-neutralizing Ab.
Western blot analysis
MT-2 cells (2 ⫻ 105 cells/ml) or Jurkat cells transfected with uncleavable
mutant form of membrane TNF-␣ (2 ⫻ 105 cells/ml) were incubated in a
25-ml flask immobilized by rabbit anti-human TNF-␣ polyclonal Ab for 1,
2, 5, 10, 20, and 40 min. Then 10 ␮l of the MT-2 cells were mixed with
an equal amount of 2 ⫻ SDS sample buffer (0.125 M Tris, 4% SDS, 20%
v/v glycerol, 0.01% bromophenol blue, 10% 2-ME, pH 6.8) and boiled for
5 min. The lysates were applied to a 12% polyacrylamide gel and were
electrophoresed for 3 h at 30 mA. After electrophoresis, the gel was equilibrated in transfer buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol)
for 1 h and then transferred to nitrocellulose filter (Schleicher & Schuell,
Keene, NH) for 1 h at 80 mA in transfer buffer. Proteins were visualized on
the filter by using a peroxidase-conjugated anti-phosphotyrosine Ab
(4G10) (Upstate Biotechnology, Waltham, MA). Membrane TNF-␣-transfected Jurkat or HeLa cells were treated in the same manner except that the
detection of the protein was performed by the reaction of rabbit anti-human
TNF-␣ polyclonal Ab (Genzyme) followed by the visualization with HRPconjugated anti-rabbit Ig Ab (Amersham).
Phosphorylation of serine residues of membrane TNF-␣ was studied in
membrane TNF-␣-transfected Jurkat cells. Western blot was performed as
described above except that rabbit polyclonal anti-phosphoserine Ab
(Zymed, South San Francisco, CA) was used for detection Ab.
Results
Membrane TNF-␣-mediated expression of E-selectin (CD62E)
on the HTLV-I-infected T cell line, MT-2
To study the effects of membrane TNF-␣ on the expression of
adhesion molecules, we first used the HTLV-I-infected T cell line,
MT-2, which has been shown to constitutively express membrane
TNF-␣ on the surface by FACS analysis as well as Western blot
analysis (10). As shown in Fig. 1a, the expression of membrane
TNF-␣ on the surface of MT-2 cells was confirmed by FACS
analysis. After the stimulation of membrane TNF-␣ with immobilized rabbit anti-human TNF-␣ polyclonal Ab, the expression of
various adhesion molecules (ICAM-1, LFA-1, VCAM-1, VLA-4,
L-selectin, P-selectin, and E-selectin) was studied using FACS
FIGURE 1. a, Expression of membrane TNF-␣ on the surface of the
HTLV-I-infected T cell line, MT-2. Cells are stained with FITC-conjugated mouse anti-human TNF-␣ mAb (filled peak) and analyzed by flow
cytometry. Background staining is shown by a black line. b, Time course
of the E-selectin (CD62E) expression on MT-2 cells after the stimulation
of membrane TNF-␣ with immobilized rabbit polyclonal anti-TNF-␣ Ab
for 0, 12, 24, and 48 h (filled peaks). Background staining is shown by
black lines. c, Membrane TNF-␣-mediated E-selectin expression in the
presence of blocking Abs against TNF-RI (p55) and TNF-RII (p75) on
MT-2 cells. MT-2 cells were incubated in the presence of Abs against
TNF-RI (p55) and TNF-RII (p75) in the flask immobilized with rabbit
polyclonal anti-TNF-␣ Ab (filled peak). Cells are stained with FITC-conjugated mouse anti-human E-selectin mAb, and the result obtained after
12 h of incubation is shown as a representative. A thin black line denotes
background staining (mouse IgG1/FITC).
analysis. Of the molecules studied, only E-selectin showed a pronounced increase in the expression. Fig. 1b shows the kinetics of
E-selectin expression. Although, in the absence of stimulation of
membrane TNF-␣, only a trace amount of E-selectin was expressed, E-selectin was significantly induced on MT-2 cells 12–24
h after the stimulation of membrane TNF-␣. The E-selectin expression on MT-2 cells was decreased to the undetectable level by
48 h after the stimulation of membrane TNF-␣. In contrast, the
stimulation of membrane TNF-␣ with immobilized anti-human
TNF-␣ mouse mAb did not have any effect on E-selectin up-regulation. To rule out the possibility that the expression of E-selectin
by anti-TNF-␣ Ab treatment was due to neutralization of soluble
TNF-␣, we next studied the expression of E-selectin in the presence of the blocking Abs against TNF-RI (p55) and TNF-RII (p75)
(Fig. 1c). The kinetics of E-selectin expression by the stimulation
of membrane TNF-␣ by anti-TNF-␣ Ab was not altered in the
presence of blocking Abs against TNF-RI and TNF-RII. Moreover, the addition of human recombinant soluble TNF-␣ did not
affect the expression of E-selectin on MT-2 cells up to the concentration of 100 ng/ml, which exceeded the steady-state concentration (50 –100 pg/ml) of soluble TNF-␣ in the culture medium
(data not shown). Thus it is concluded that E-selectin expression
by anti-TNF-␣ Ab is mediated by direct stimulation through membrane TNF-␣, not by the indirect effect on soluble TNF-␣.
The expression of L-selectin (CD62L), P-selectin (CD62P),
LFA-1, and VLA-4 was not observed either before or after the
stimulation of membrane TNF-␣ with immobilized rabbit antiTNF-␣ Ab. In contrast, ICAM-1 and VCAM-1 were constitutively
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The procedure will be described elsewhere. Briefly, wild-type TNF-RII
cDNA was obtained by using RT-PCR of the total RNA from normal
human PBMC cloned into pCXN2 expression vector and was transfected
into HeLa cells, followed by the selection with G418 and by the sorting of
cells strongly positive for TNF-RII using FACScan.
The Journal of Immunology
expressed on MT-2 cells, which were slightly down-regulated by
the treatment of immobilized anti-TNF-␣ Ab (data not shown).
Induction of E-selectin on activated normal human
CD4⫹ T cells
FIGURE 2. E-selectin expression on normal human CD4⫹T cells stimulated with anti-TNF-␣ Ab. CD4⫹ T cells are purified from normal human
PBMC with magnetic beads and activated with PHA for 12 h, which resulted
in the expression of membrane TNF-␣ (filled peak in a) but not expression of
E-selectin (filled peaks in b) on FACS analysis. These cells are further incubated in the flask immobilized with rabbit polyclonal anti-TNF-␣ Ab for 12 h
in the absence (c) or presence (d) of blocking Abs against TNF-RI and TNFRII. Filled peaks again represent E-selectin mAb-specific staining. Background staining is shown by black lines (mouse IgG1/FITC).
Induction of E-selectin on Jurkat and HeLa cells transfected
with membrane TNF-␣
To investigate E-selectin expression in the cells that are stably and
abundantly expressing membrane TNF-␣, we transfected the wildtype or uncleavable mutant form of membrane TNF-␣ in human
lymphoblastoid T cell line, Jurkat, and human epithelioid cervical
carcinoma cell line, HeLa, and studied the effect of stimulation
with polyclonal anti-TNF-␣ Ab. Although membrane TNF-␣ was
not expressed on the surface of steady-state Jurkat or HeLa cells,
transfection with the uncleavable mutant form of TNF-␣ resulted
in the constitutive expression of membrane TNF-␣ when assessed
by FACS analysis and Western blot analysis (Fig. 3, a and b). In
Jurkat cells, although E-selectin was not expressed without stimulation, E-selectin was induced with the maximum expression between 4 and 12 h after the treatment with polyclonal anti-TNF-␣
Ab, which reduced at 48 h after the treatment. The expression of
E-selectin at 4 h after the stimulation was shown as a representative (Fig. 3c). This kinetics of E-selectin expression in transfected
Jurkat cells was almost similar with those of CD4⫹ T cells. An
excess amount of human recombinant soluble TNF-␣ (1 ␮g/ml)
almost completely inhibited this E-selectin up-regulation by antiTNF-␣ Ab on membrane TNF-␣-expressing Jurkat cells, suggesting again the specificity of anti-TNF-␣ effect (data not shown).
Although less pronounced, almost identical kinetics of E-selectin
expression was obtained in the HeLa cells transfected with the
uncleavable mutant form of membrane TNF-␣. Fig. 3d shows a
representative result in the transfected HeLa cells at 4 h after the
treatment with polyclonal anti-TNF-␣ Ab. The Jurkat or HeLa
cells transfected with the wild-type membrane TNF-␣ expressed a
considerable amount of membrane TNF-␣ on their surface. However, soluble TNF-␣ was more significantly secreted in the culture
medium of the wild-type membrane TNF-␣ transfectants compared with that of uncleavable mutant transfectants as assessed by
Western blot analysis. Upon activation with polyclonal antiTNF-␣ Ab, E-selectin was similarly induced on the Jurkat or HeLa
FIGURE 3. FACS analysis and Western blot analysis of the expression of
membrane TNF-␣ on Jurkat (a) or HeLa (b) cells after the transfection of the
uncleavable form of membrane TNF-␣ (R78T/S79T) using expression vector
pCXN2. Stable expression of membrane TNF-␣ was obtained after the selection with G418 (filled peaks). In both cells lines, Western blot analysis also
confirmed the expression of the uncleavable form of membrane TNF-␣ (Mt).
c, Untransfected cells. E-selectin expression 4 h after the stimulation of rabbit
polyclonal anti-human TNF-␣ Ab is demonstrated in c for transfected Jurkat
cells and in d for transfected HeLa cells. E-selectin was significantly induced
4 h after the stimulation of membrane TNF-␣. The maximum expression of
E-selectin was seen at 4 –12 h, which was decreased at 48 h. Filled peaks
indicate stainings with FITC-conjugated mouse anti-human E-selectin mAb,
and black lines are backgrounds (mouse IgG1/FITC).
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We next investigated the expression of E-selectin in normal CD4⫹ T
cells because HTLV-I usually infects CD4⫹ T cells, and MT-2 cells
are, in fact, positive for CD4 (10). FACS analysis using anti-TNF-␣
Ab showed that unstimulated CD4⫹ T cells from normal individuals
did not express membrane TNF-␣, whereas treatment of normal
CD4⫹ T cells with 100 ng/ml of PHA for 12 h resulted in the expression of membrane TNF-␣ (Fig. 2a). The treatment with acidified
buffer to dissociate the cell surface-soluble form of TNF-␣ did not
alter the intensity of this staining by anti-TNF-␣ Ab, which confirmed
that the membrane TNF-␣ was expressed on PHA-activated CD4⫹ T
cells (data not shown). E-selectin was not expressed in either the
PHA-unstimulated or -stimulated conditions (Fig. 2b). Then, the
membrane TNF-␣ expressed on the activated human CD4⫹ T cells
was stimulated with immobilized rabbit anti-human TNF-␣ Ab. The
expression of E-selectin was induced with a similar kinetics to that of
MT-2 with the maximum induction at 12–24 h. The expression of
E-selectin after 12 h of stimulation of membrane TNF-␣ is shown as
a representative in Fig. 2c. This membrane TNF-␣-induced E-selectin
expression was again unaffected by the treatment of blocking Abs
against TNF-RI and TNF-RII as in the case of MT-2 cells (Fig. 2d).
We next analyzed the expression of E-selectin in normal human
CD8⫹ T cells. Although membrane TNF-␣ was expressed in response
to 100 ng/ml of PHA in the same manner as that of CD4⫹ T cells,
E-selectin was not expressed by the treatment of immobilized antiTNF-␣ Ab (data not shown).
133
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REVERSE SIGNAL BY MEMBRANE TNF-␣ INDUCES E-SELECTIN ON HUMAN T CELLS
cells transfected with wild-type membrane TNF-␣ (data not
shown).
Up-regulation of E-selectin in cell-to-cell contact condition
To see the effect of cell-to-cell contact on E-selectin expression,
Jurkat cells transfected with uncleavable TNF-␣ (membrane TNF␣-expressing T cells) (Fig. 4a) were cocultured with HeLa cells
transfected with full-length TNF-RII cDNA. Although wild-type
HeLa cells expressed almost undetectable level of TNF-RI and
TNF-RII, transfected HeLa cells were strongly and stably positive
for TNF-RII on their surface (Fig. 4b). The expression of E-selectin was induced as early as 6 h after the start of coculture on
membrane TNF-␣-expressing Jurkat cells. The induction of E-selectin reached a maximum at 24 h, which continued at least up to
72 h after the initiation of coculture (Fig. 4c). This up-regulation of
E-selectin was abolished in the presence of TNF-RII-specific neutralizing Ab. In addition, membrane TNF-␣-expressing Jurkat cells
did not show any induction of E-selectin by the coculture with
untransfected HeLa cells (data not shown).
The message of the E-selectin gene was studied by using RT-PCR
and Northern blot analysis. RT-PCR analysis showed a faint
610-bp band of partial E-selectin cDNA before the stimulation of
membrane TNF-␣, which was increased upon activation of the
membrane TNF-␣ as early as 1 h after the stimulation of membrane TNF-␣ (Fig. 5a). The E-selectin message was stably increased until 12 h after the stimulation. The amount of control
␤-actin product was similar between the samples. Northern blot
analysis displayed similar results as those of RT-PCR analysis.
The message size of E-selectin induced in the MT-2 cells was
identical with that of E-selectin in control HUVEC (Fig. 5b).
Intracellular signal transduction mechanism mediated by
membrane TNF-␣
FIGURE 4. Up-regulation of E-selectin on TNF-␣-expressing Jurkat cells
cocultured with TNF-RII-expressing HeLa cells. a, Membrane TNF-␣ expression on Jurkat cells. The uncleavable mutant form of membrane TNF-␣ was
stably expressed on Jurkat cells as described in Fig. 3a. Background staining
is shown by a black line (mouse IgG1/FITC). b, TNF-RII expression on transfected HeLa cells. TNF-RII cDNA was cloned into expression vector pCXN2
and was stably expressed in HeLa cells. After sorting by FACS based on
anti-TNF-RII Ab, HeLa cells strongly expressing TNF-RII on their surface
were selected. Background staining is shown by a black line (mouse IgG1/PE).
c, Up-regulation of E-selectin on membrane TNF-␣-expressing Jurkat cells
when brought into cell-to-cell contact with TNF-RII-expressing HeLa cells.
When membrane TNF-␣-expressing Jurkat cells were cocultured with TNFRII-expressing HeLa cells, E-selectin was up-regulated as early as 6 h after the
initiation of coculture, which reached a maximum at 24 h and lasted up to 72 h.
Expression of E-selectin on membrane TNF-␣-expressing Jurkat cells after
24 h of coculture was shown as a representative. Background staining is shown
by black lines (mouse IgG1/FITC).
To understand the intracellular signals essential for the membrane
TNF-␣-mediated E-selectin expression on T cells, the effects of
various inhibitors for intracellular signal transduction were studied
in normal human CD4⫹ T cells. Up to the concentrations of 100
␮M of H-7 (protein kinase C inhibitor), 100 ng/ml of herbimycin
A (tyrosine kinase inhibitor), 40 ␮g/ml of genistein (tyrosine kinase inhibitor), 100 ng/ml of wortmannin (myosine light chain
kinase inhibitor), 100 ␮M of manumycin (farnesyl transferase inhibitor), 100 ␮M of W-7 (calmodulin inhibitor), and 2 mM of
EGTA, the membrane TNF-␣-mediated E-selectin expression was
not affected (data not shown). We next studied the tyrosine phosphorylation induced by membrane TNF-␣ stimulation in MT-2 and
Jurkat cells transfected with membrane TNF-␣. Upon stimulation
of membrane TNF-␣ with immobilized anti-TNF-␣ Ab, altered
tyrosine phosphorylation patterns were not clearly observed. In
addition, membrane TNF-␣, but not soluble TNF-␣, was constitutively phosphorylated at the serine residues, which confirmed the
data reported by Pócsik (17) (data not shown). Treatment of membrane TNF-␣-expressing Jurkat cells with 100 ng/ml of cycloheximide resulted in the significant, but not complete, inhibition of
anti-TNF-␣-mediated E-selectin up-regulation. It is suggested that
E-selectin up-regulation is at least in part independent of novel
protein synthesis (data not shown).
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Up-regulation of E-selectin message after the stimulation of
membrane TNF-␣
FIGURE 5. Up-regulation of E-selectin message in MT-2 cells after the
stimulation of membrane TNF-␣. a, RT-PCR analysis. Total RNA purified
from MT-2 cells after the incubation in the immobilized anti-TNF-␣ Ab for
the indicated time or from control HUVEC were reverse transcribed and
amplified with E-selectin-specific primers (expected size of 610 bp). After
the stimulation of membrane TNF-␣ (1, 2, and 12 h), the E-selectin message was significantly increased compared with that of the trace amount of
steady-state expression without stimulation (0 h). The amount of control
␤-actin expression was similar at these conditions. The positive control of
the E-selectin message was induced upon activation with LPS in HUVEC.
M denotes the marker of 1 kb ladder. b, Northern blot analysis. Total RNA
(10 ␮g) was applied to each lane. The radiolabeled 610-bp E-selectin
cDNA and 340-bp GAPDH cDNA were hybridized and autoradiographed.
Similar results with those of RT-PCR analysis were obtained. E-selectin
message expression corresponding to ⬃3.9 kb was observed in the positive
control HUVEC. In MT-2 cells, E-selectin expression was gradually induced until 12 h after the stimulation of membrane TNF-␣. The size of the
messages in MT-2 cells was identical with that of HUVEC.
The Journal of Immunology
E-selectin was not up-regulated by CD40L and FasL
We next investigated whether E-selectin up-regulation is mediated
by other members of the TNF ligand family. FACS analysis
showed that FasL and CD40L were significantly expressed on
wild-type Jurkat cells, whereas only a trace amount of CD30L was
detected (Fig. 6a). Stimulation with immobilized rabbit polyclonal
anti-CD40L or anti-FasL did not result in the up-regulation of
E-selectin in Jurkat cells (Fig. 6b).
Discussion
FIGURE 6. a, FACS analysis of CD40L, FasL, and CD30L expression
in Jurkat cells. Expression of the TNF ligand family proteins was studied
using anti-human CD40L/FITC mAb, anti-human CD30L/FITC mAb, and
rabbit polyclonal anti-human FasL Ab, followed by FITC-conjugated goat
anti-rabbit Ig. Mouse IgG1/FITC was used for background staining for
CD40L and CD30L. Rabbit Ig was used for background staining for FasL.
b, Lack of E-selectin up-regulation by the stimulation of CD40L and FasL
in Jurkat cells. Jurkat cells (2 ⫻ 105 cells/ml) were incubated in 25-ml
flasks immobilized with rabbit polyclonal anti-human CD40L or FasL up
to 24 h and were studied for E-selectin expression by FACS analysis. The
results after 12 h of incubation are shown as representatives.
ing through the molecules of the TNF-ligand family has been accumulated. CD40L is essential for the development of T cell helper
functions (31). It is also shown that CD40L costimulation is important
in the regulation of IL-4 production from the ligand-bearing T cells
(32). CD30L transduces signals to the neutrophil on which it is expressed and induces IL-8 expression and a rapid respiratory burst (33).
CD27L, also known as CD70, costimulates the proliferation of T cells
when cross-linked with CD70-specific Ab (34). These lines of evidence, along with our findings on the membrane TNF-␣, strongly
suggest that TNF-␣ family proteins are bipolar molecules that transduce signals either from or into the ligand-bearing cells. The reverse
signaling on E-selectin up-regulation was specific for membrane
TNF-␣, as stimulation of FasL or CD40L did not cause E-selectin
up-regulation in Jurkat cells (Fig. 6).
It is important as well that E-selectin, an adhesion molecule that
is believed to be almost exclusively expressed on endothelial cells,
is induced on CD4⫹ T cells and on Jurkat and HeLa cells. Eselectin, also known as CD62E and ELAM-1 (endothelial-leukocyte adhesion molecule-1), is a member of selectin family that
plays a pivotal role in the first step of adhesion (rolling) of leukocytes to endothelial cells (35), which is followed by integrin-mediated leukocyte arrest and endothelial transmigration. The selectin
family of proteins, E-, P-, and L-selectin, have common mosaic
structures, consisting of N-terminal lectin domains, an epidermal
growth factor (EGF)-like motif, varying numbers of short consensus repeats homologous to complement regulatory domains, a
transmembrane domain, and a short cytoplasmic tail. Expression
of E-selectin is under the control of a number of cytokines and
bacterial endotoxin at the level of transcription. TNF-␣, IL-1, as
well as LPS up-regulate the expression of E-selectin (25), whereas
TGF-␤ acts as a suppressor (36). The time course of the E-selectin
expression is different between CD4⫹ T cells induced by membrane TNF-␣ (Fig. 2c) and HUVEC induced by the soluble form
of TNF-␣, as the peak of E selectin expression of the former is
between 12 and 24 h and that of the latter is between 3 and 6 h after
the stimulation. Among three forms of E-selectin mRNA generated
by differential use of the 3⬘-untranslated region (UTR), the shortest
transcript (⬃1 kb of the 3⬘-UTR was deleted) has a longer half-life
than that with longer 3⬘-UTR, which is supposed to explain the
sustained expression of E-selectin in the human dermal microvascular endothelial cells (HDMEC) (37). However, the involvement
of alternative E-selectin transcript is not the case in T cells, as there
were no differences in the size of E-selectin messages between
HUVEC and MT-2 cells assessed by Northern blot analysis (Fig.
5b). It has been reported that TNF-␣-induced E-selectin expression
was shown both at the mRNA level and at the protein level in
nonendothelial vascular smooth muscle cells pretreated with the
protein synthesis inhibitor cycloheximide (38), which suggests the
presence of a tissue-specific repressor protein for the transcription
of E-selectin. Thus it is suggested that E-selectin expression in
human CD4⫹ T cells by membrane TNF-␣ is partly due to the
attenuation of the function of repressor protein. In this study, we
were unable to identify an intracellular signal transduction mechanism that is essential for the expression of E-selectin of the reverse signal of membrane TNF-␣. However, it is possible that
some Ser/Thr kinase might be important for its effect on E-selectin
expression, as membrane TNF-␣ was phosphorylated at the cytoplasmic Ser residues (17), which was confirmed in our study as
well (data not shown). The identification of the membrane TNF␣-associated proteins would contribute to further understanding of
the biological functions of membrane TNF-␣. The cytoplasmic
domain of the membrane TNF-␣ should be essential for the binding of membrane TNF-␣-associated proteins because substitution
of a particular amino acid residue by site-directed mutagenesis
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
We here presented the evidence that membrane TNF-␣ expressed
on activated CD4⫹ T cells or stably expressed on Jurkat or HeLa
cells, when treated with polyclonal anti-TNF-␣, induced the expression of an adhesion molecule, E-selectin. E-selectin up-regulation was also observed in membrane TNF-␣-expressing Jurkat
cells when brought into cell-to-cell contact with TNF-RII-expressing HeLa transfectant cells. Although E-selectin expression has
been considered to be almost exclusively confined to endothelial
cells, our data provided strong evidence that E-selectin is expressed on the cell types different from endothelial cells. We have
already demonstrated that calcium mobilization and production of
cytokines such as IFN-␥ and IL-2 was induced in HTLV-I-infected
human T cells by the stimulation of membrane TNF-␣ (10, 11).
Moreover, ligation of soluble TNF receptor to membrane TNF-␣
has recently been shown to cause calcium mobilization in the
mouse macrophage cell line, RAW267.4, as well (18). This study
further confirmed these previous findings on the reverse signaling
through membrane TNF-␣.
The members of TNF-␣/TNF-␣ receptor superfamily are rapidly
increasing in recent years. The TNF-␣ (TNF-ligand) family comprises over 10 proteins, such as lymphotoxin-␣, CD40L, CD30L,
CD27L, FasL, TNF-related apoptosis-inducing ligand (TRAIL),
Ox40L, DR3L/Apo3L/TWEAK, and OPGL/RANKL/TRANCE,
all of which display significant homology with TNF-␣ in the carboxyl-terminal receptor-binding region (⬃145-aa residues) and are
believed to act as trimers (28 –30). The N-terminal cytoplasmic
domain is conserved across different species, but not between family members. Although lymphotoxin-␣ is entirely secreted and
TNF-␣ is mostly secreted, the other members of the TNF-ligand
family usually stay on the cell surface and exert their biological
activities in a cell-to-cell contact manner. In addition to the wellcharacterized function as ligands, evidence for the reverse signal-
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15.
16.
17.
18.
19.
20.
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22.
23.
24.
25.
26.
27.
28.
29.
30.
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resulted in the complete loss of E-selectin expression without affecting the expression of membrane TNF-␣ on the transfected Jurkat cells (N. Hatta, et al., manuscript in preparation).
To understand the function of E-selectin induced on CD4⫹ T
cells, further investigation is needed. Many immunohistochemical
studies have failed to detect the expression of E-selectin on leukocytes; however, in the colonic biopsies from patients with ulcerative colitis, inflammatory cells (primarily mononuclear cells)
in the lamina propria were E-selectin positive (39). Considering its
well characterized function as an adhesion molecule, it is likely
that E-selectin expressed not only on endothelial cells but also on
T cells coordinately function in the adhesion of T cells to endothelial cells in at least some of the conditions of inflammation.
Alternatively, E-selectin on CD4⫹ T cells might generate transmembrane signals for unknown novel functions through its cytoplasmic binding proteins, such as cytoskeletal proteins and focal
adhesion kinase (FAK, pp125FAK) (40, 41). Such an outside-toinside signal is demonstrated in the case of L-selectin (42).
In conclusion, we presented the evidence that membrane TNF-␣
upon activation with anti-TNF-␣ Ab can induce E-selectin on
HTLV-I-infected T cell line, MT-2, and activated normal human
CD4⫹ T cells that are expressing the ligand on their surface.
Transfection of wild-type or uncleavable mutant membrane
TNF-␣ in Jurkat or HeLa cells followed by the stimulation with
polyclonal anti-TNF-␣ Ab also resulted in the expression of Eselectin. In addition, stimulation with TNF-RII-expressing HeLa
cells also induced E-selectin expression in membrane TNF-␣-expressing Jurkat cells in cell-to-cell contact manner. Taking account
of our previous findings that membrane TNF-␣ mediates Th1 type
cytokine production (10, 11), the function of membrane TNF-␣
was confirmed as a novel cell surface molecule that transmits signals to activate the cells expressing it on their surface. It is also
important that E-selectin expression was not limited to endothelial
cells. E-selectin might be inducible in the nonendothelial cell types
expressing membrane TNF-␣, which will contribute to the cell-tocell adhesion and interaction. Thus it is suggested that membrane
TNF-␣ is a bipolar molecule that transmits signals positively, regulating local inflammation both as a ligand and as an acceptor.