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Departments of
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Hematology and Oncology and
Dermatology, Graduate School of Medicine, and
Laboratory of Virus Immunology, Research Center for Acquired Immunodeficiency Syndrome, The Institute for Virus Research, Kyoto University, Kyoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apart from the role to deliver costimulatory signals to activated OX40+ T cells, gp34 has been reported to have some biological functions. In mouse, stimulation of OX40 ligand on B cells resulted in B cell proliferation and Ig secretion, under the condition that the B cells were cocultured with CD40 ligand (CD40L)-transfected L cells (9). In human, stimulation of activated dendritic cells through gp34, when their cell surface molecule CD40 was prestimulated to induce the expression of gp34, has been reported to enhance their maturation and increase the production of several inflammatory cytokines (8). However, the mechanism by which intracellular signaling pathways are activated through gp34 to elicit these biological effects in gp34-expressing cells still remains unknown.
This is the case with other members of the TNF family, such as FasL, CD30L, CD40L, 4-1BBL, which have been reported to elicit certain biological effects (13, 14, 15, 16, 17), while intracellular signaling of members of the TNF receptor family has been studied in detail (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). In the present study, we analyzed intracellular events triggered by gp34 stimulation. To avoid the possibility that other pathways in addition to OX40/gp34 are activated in examining the intracellular signaling events of gp34, we prepared a recombinant soluble form of human OX40 to stimulate a human gp34-transfected mouse epithelial cell line, MMCE, and demonstrated that the gp34 stimulation increased c-jun and c-fos mRNA levels in this transfectant. Additional studies using human gp34 deletion mutants determined the critical cytoplasmic portion of gp34, which is required to mediate these intracellular signals. Moreover, we demonstrated that the recombinant soluble form of OX40 stimulates HUVECs to express c-jun mRNA transcript, suggesting the possibility that interaction between activated T cells and vascular endothelial cells elicits some biological effects not only in T cells but also in endothelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVECs were purchased from Kurabo (Osaka, Japan) and were cultured on gelatin-coated culture flasks in M199 medium (Life Technologies, Gaithersburg, MD) supplemented with 15% FCS (BioWittaker, Verviers, Belgium), 90 µg/ml porcine intestinal heparin (Sigma, St. Louis, MO), 30 µg/ml endothelial cell growth supplement (Nakalai Tesque, Kyoto, Japan).
A murine epithelial cell line, MMCE (a gift of Dr. W. Ostertag, Hamburg University, Hamburg, Germany), was cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS, 30 µg/ml tobramycin (Shionogi Pharmaceutical, Osaka, Japan), and 2 mM L-glutamine. COS-7 cells were cultured in DMEM (Life Technologies) supplemented with 10% FCS, 30 µg/ml tobramycin, and 2 mM L-glutamine.
Monoclonal antibodies
The mAb against human gp34, ik-1, was produced in our laboratory as follows. An expression vector of human gp34, pMKITneo-gp34, was prepared as described (32), linearized and introduced into WR19L, a murine T lymphoma cell line, by electroporation. Cells were subjected to G418 selection, and a stable transfectant clone (WR19L/gp34) expressing high levels of gp34 was selected by FACS analysis using anti-gp34 mAb 5A8 (a gift of Dr. Y. Tanaka, Kitasato University, Sagamihara, Japan) and expanded. A BALB/c mouse was immunized with WR19L/gp34 cells emulsified with CFA (Sigma) on day 0 and immunized with WR19L/gp34 cells suspended in PBS on day 7 and day 14; then, on day 15, the regional lymph node cells from the mouse were fused with PAI, a partner myeloma cell line (34). Hybridomas were subjected to hypoxanthine, aminopterin, and thymidine selection, and the culture supernatants were screened based on difference in reactivity with transfectant and parental WR19L. An mAb, named ik-1, was found to react with transfectant but not parental cells. The gp34-specific reactivity of ik-1 was confirmed by FACS analysis of COS-7 cells transiently transfected with gp34 cDNA or other irrelevant cDNAs and also by examining its inhibitory effects on the binding between OX40-transfected human T cell line HSB-2/OX40 and MMCE/gp34. ik-1 was typed as IgG1 by a mouse mAb typing kit (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Isotype-and subclass-matched negative control Ab MOPC-21 was purchased from Sigma. An mAb against OX40, 315 (IgG2a), was described elsewhere (7).
Preparation of soluble OX40
The extracellular portion of OX40 (nucleotide sequence, 6–644)
was amplified by PCR and ligated into the expression vector pME18S
(7) (a gift of Dr. K. Maruyama, Tokyo Medical and Dental
University, School of Medicine, Tokyo, Japan). Ten micrograms of
pME18S-soluble OX40 was transfected into COS-7 cells by the
DEAE-dextran method (35). The transfected cells were
cultured with DMEM with 10% FCS for 24 h and with 2% FCS for
another 72 h. The culture supernatants (soluble OX40 supernatants)
were collected and concentrated 10-fold with Centriprep 30 (Amicon,
Beverly, MA) before the assay. The supernatants of COS-7 cells
transfected with empty vector (mock supernatants) were also prepared at
the same time by the same protocol. Both supernatants were subjected to
SDS-PAGE, and the presence of the soluble form of OX40 was detected by
Western blotting with anti-OX40 Ab (315; 20 µg/ml) (Fig. 1
). The supernatants were added to the
cell culture at 50% v/v for each assay.
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The construct for gp34 cytoplasmic deletion mutants (see Fig. 3A) were generated by PCR method using ATG (start
codon)-tagged oligonucleotides as the primer and pMKITneo-gp34 as the
template. The PCR products were ligated into pMKITneo to construct
pMKITneo-gp34-del 1, -del 2, and -del 3. The partial DNA sequences of
all the gp34 deletion mutants were determined using an automated
sequencer, ABI Prism310 (Perkin-Elmer, Foster City, CA). The plasmids
were linearized by digestion with CpoI and introduced into MMCE cells
by the electroporation. The stably transfected cells were dispensed in
96-well flat-bottom plates for clonal selection and cultured in RPMI
1640 medium containing 1 mg/ml G418 (Sigma) for 3 wk. The empty vector
was transfected into MMCE cells, and the cells were cultured in a 10-cm
dish with RPMI 1640 containing 1 mg/ml G418, which were used as control
cells (MMCE/mock). The expression of human gp34 on the transfected
cells was examined with a FACScan (Becton Dickinson, San Jose, CA)
using anti-human gp34 Ab (ik-1) (Fig. 2A and
3B).
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Total RNA from cultured cells was isolated by using RNeasy Mini
Kit (Qiagen, Hilden, Germany). Total RNA was fractionated in
formaldehyde-containing 1.0% agarose gels, washed gently in 200 ml of
20x SSC (3 M NaCl, 0.3 M trisodium citrate) for 30 min, transferred to
a nylon membrane, Hybond N (Amersham Pharmacia Biotech) in 20x SSC,
and UV cross-linked (Stratagene, La Jolla, CA). Mouse c-fos
(covering 2473–2939), human c-fos 1400–3500(1400–3500), mouse
c-jun 931–1245(931–1245), and human c-jun 1275–1590(1275–1590)
cDNA fragments were used as probes. The probe for ß-actin was
purchased from Clontech (Palo Alto, CA). Probes were labeled with
[
-32P]dCTP (Amersham Pharmacia Biotech)
using Rediprime II (Amersham Pharmacia Biotech). Prehybridization and
hybridization were performed at 42°C in 50% formamide, 5x SSPE, 5x
Denhardt solution, 0.5% SDS, and 100 µg/ml heat-denatured salmon
sperm DNA (Sigma). After prehybridization, heat-denatured probes were
added, and the filter was incubated at 42°C for 18 h. The filter
was washed once in 2x SSC, 0.1% SDS at room temperature and
subsequently washed twice in 0.1x SSC, 0.1% SDS at 55°C and
subjected to autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze whether the intracellular signaling pathways are
activated through gp34 stimulation, we examined the levels of
c-jun and c-fos mRNA in a human gp34-transfected
cell line with or without gp34 stimulation. First of all, we prepared
soluble OX40 supernatants to stimulate gp34+
cells. The crude and unpurified supernatants were used in the study.
The presence of soluble OX40 was confirmed by Western blotting, and its
molecular mass was determined as 
42 kDa (Fig. 1
) using the
molecular marker (Bio-Rad, Hercules, CA). The induction of
c-jun and c-fos mRNA expression was examined by
Northern blotting in human gp34-transfected mouse epithelial cell line
MMCE/gp34 or in empty vector-transfected MMCE/mock. The expression of
gp34 molecule in each transfectant was confirmed by FACS analysis
(Fig. 2A). To avoid the cellular state to which the early
gene mRNAs were strongly induced, through cell growth or proliferation
signals, and to minimize the background levels of these mRNAs, these
cells were cultured in RPMI 1640 with 5% FCS for 4 days without
changing medium, and the cells having reached in confluence and in
resting state were used for the subsequent assay. Soluble OX40 or mock
supernatants were added to the cell culture, and total RNA was isolated
after the treatment with either soluble OX40 or mock supernatants for
40 min. Subsequently, 20 µg of total RNA was subjected to Northern
blot analysis to detect c-jun and c-fos mRNA
(Fig. 2B). Increased c-jun and c-fos
mRNA expression was detected in MMCE/gp34 cells treated with soluble
OX40 supernatants (lane 2), but not with mock supernatants
(lane 3), as compared with those levels in steady
state (lane 1), and the induction of these early gene
mRNAs in MMCE/gp34 cells was blocked clearly by preincubation with
anti-gp34 Ab (ik-1; 50 µg/ml) for 30 min (lane 5), but
not with control Ab (MOPC-21; 50 µg/ml) (lane 4), before
the treatment with soluble OX40 supernatants. As expected, in MMCE/mock
cells, treatment with either soluble OX40 or mock supernatants or
pretreatment of Abs before the treatment with soluble OX40 supernatants
did not induce the expression of these early gene mRNAs (lanes
6–10).
Cytoplasmic portion of gp34 including the amino acid sequence 16–21 (RPRFER) was required for the increase in c-jun and c-fos mRNA transcription through gp34 stimulation
To determine the cytoplasmic portion of gp34 that is required for
the induction of c-jun and c-fos mRNA expression,
a series of MMCE/gp34 cytoplasmic deletion mutants were generated (Fig. 3A). The transfectant of each gp34 deletion mutant was
assayed by flow cytometry, and it was confirmed that the expression
level of cell surface gp34 was similar among the three transfectants
(Fig. 3B). These transfected cells were cultured in the same
condition as in examining c-jun and c-fos mRNA
induction of MMCE/gp34 cells. Total RNAs were prepared from the cells
stimulated with soluble OX40 supernatants, mock supernatants, or medium
alone. Twenty micrograms of total RNA was subjected to Northern blot
analysis. The levels of c-jun and c-fos mRNA
increased after treatment with soluble OX40 supernatants, but not with
mock supernatants, as compared with that in steady state, in
MMCE/gp34-del 3 (aa 7–183) cells (Fig. 3C, lanes
7–9) and MMCE/gp34-del 2 (aa 16–183) cells (lanes
4–6). In MMCE/gp34-del 1 (aa 22–183) cells (lanes
1–3), whose mutation contains only 2 amino acids in gp34
cytoplasmic portion, c-jun and c-fos mRNAs were
not induced to be expressed. These results indicated that the
cytoplasmic portion of gp34, including the amino acid sequence 16–21
(RPRFER), was required for the induction of c-jun and
c-fos mRNA through gp34 stimulation.
The level of c-jun mRNA increased in HUVECs through gp34 stimulation
The results of the above experiments employing gp34-transfected
cells prompted us to determine whether the similar intracellular events
are triggered in HUVECs that express gp34. In the assay, HUVECs were
seeded in M199 with 7.5% FCS, 45 µg/ml porcine intestinal heparin,
and 15 µg/ml endothelial cell growth supplement and left for 4 days
without changing medium to avoid early gene induction from growth
signaling. Total RNA was isolated from each culture of HUVECs
stimulated with soluble OX40 supernatants, mock supernatants, or medium
alone, and 10 µg of each total RNA was subjected to Northern blot
analysis. Increase in c-jun mRNA expression was observed in
HUVECs treated with soluble OX40 supernatants, but not with mock
supernatants (Fig. 4A). The
preincubation of HUVECs with anti-gp34 Ab (ik-1; 50 µg/ml) for 30
min, before the treatment with soluble OX40 supernatants, blocked the
induction of c-jun mRNA expression (Fig. 4B).
c-fos mRNA was detectable in neither soluble OX40
supernatant- nor mock supernatant-treated HUVECs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To explore intracellular signaling events through gp34, we chose to examine whether immediate early genes are induced in response to gp34 stimulation. Transcription of c-jun and c-fos genes is induced very rapidly in response to a diverse spectrum of extracellular stimuli. Accordingly, as an initial approach, we prepared a model system in which human gp34 transfectant MMCE/gp34 was stimulated with the recombinant soluble form of OX40. We found that levels of c-jun and c-fos mRNA increased dramatically through gp34 stimulation in MMCE/gp34 cells upon recombinant soluble OX40 binding, which was blocked by the pretreatment with anti-gp34 Ab. Further studies with a series of gp34 deletion mutants demonstrated that the cytoplasmic portion of gp34, including the amino acid sequence 16–21 (RPRFER), was required for the induction of c-jun and c-fos mRNA expression via gp34 stimulation. As reported previously, it is characteristic that cytoplasmic domains of members of the TNF family are conserved across species, but not among family members, suggesting that the cytoplasmic domains of these ligands may have some physiologic relevance (37). Since the amino acid sequence 16–19 (RPRF) of each OX40 ligand is conserved in human (5), rat (38), mouse (4) and rabbit (GenBank accession number, AF037067), it is likely that this motif is conserved for its importance in transmitting intracellular signaling.
Next, we performed the experiment of immediate early gene mRNA induction in HUVECs through gp34. Northern blot analysis revealed that c-jun mRNA expression was strongly induced in HUVECs through gp34 stimulation upon OX40 binding, which was blocked by pretreatment with anti-gp34 Ab. In our hands, c-fos mRNA transcript was undetectable throughout the experiments in HUVECs. Detection of c-fos mRNA in HUVECs has been reported to be difficult and requires treatment with cycloheximide, an agent known to superinduce expression of this protooncogene, primarily via stabilization of transcripts (39, 40, 41). It is to be determined whether c-fos mRNA is not induced or induced at such low levels that we could not detect them. Our findings of c-jun mRNA induction in HUVECs via gp34 strongly indicated that HUVECs also receive signals mediated through OX40/gp34 pathway when they interact with OX40+ cells.
One obvious question that arises is what kinds of biological effects are elicited through gp34. There are several findings suggesting that OX40/gp34 system is involved in inflammation. In our preliminary studies, freshly isolated HUVECs and cryosection of umbilical cord specimen showed negative staining against anti-gp34 Ab with FACS analysis or immunohistochemical staining, respectively (data not shown). However, cultured HUVECs and blood vessels in some inflammatory skin diseases were shown to express gp34 (12), suggesting that the microvascular environment or some inflammatory mediators induce its expression, which may modulate immune response.
The roles of OX40 have been discussed in autoimmune disease
(42, 43, 44), acute graft-vs-host disease (45),
and immune responses against tumor cells (46). In
addition, OX40/gp34 interaction has been shown to be involved in
cytokine production. CD4+
OX40+ T cells are instructed to express IL-4
(47), and gp34+ dendritic cells are
primed to produce TNF-, IL-12, IL-1ß, and IL-6 (8).
Further studies are needed to elucidate the gp34-mediated biological
effects in vascular endothelial cells, which may play a key role in
inflammatory infiltration or tumor invasion.
In conclusion, our study demonstrated that OX40/gp34 signaling pathway is bidirectional and that reverse signaling pathways are distinctly activated through a member of the TNF family, which gives us a clue in further analysis of intracellular signaling events through other members of the TNF family.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Toshiyuki Hori, Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507, Japan. E-mail address:
3 Current address: Systemix, Inc., Palo Alto, CA 94304.
4 Current address: Department of Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
5 Abbreviation used in this paper: HTLV-I, human T cell leukemia virus type I.
Received for publication March 16, 1999. Accepted for publication June 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B by TNF receptor 2 and CD40. Science 269:1424.B activation. Genes Dev. 10:963.B by the CD30 receptor is mediated by TRAF1 and TRAF2. Mol. Cell. Biol. 17:1535.[Abstract]
B activation. J. Biol. Chem. 272:2042.B and putative signal transducer for the lymphotoxin-ß receptor. J. Biol. Chem. 271:14661.B activation. J. Biol. Chem. 273:5808.B. Mol. Cell. Biol. 18:558.This article has been cited by other articles:
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  M. Sun and P. J. Fink A New Class of Reverse Signaling Costimulators Belongs to the TNF Family J. Immunol., October 1, 2007; 179(7): 4307 - 4312. [Abstract] [Full Text] [PDF]
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M. Sun, K. T. Ames, I. Suzuki, and P. J. Fink The Cytoplasmic Domain of Fas Ligand Costimulates TCR Signals J. Immunol., August 1, 2006; 177(3): 1481 - 1491. [Abstract] [Full Text] [PDF]
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P. Soroosh, S. Ine, K. Sugamura, and N. Ishii OX40-OX40 Ligand Interaction through T Cell-T Cell Contact Contributes to CD4 T Cell Longevity J. Immunol., May 15, 2006; 176(10): 5975 - 5987. [Abstract] [Full Text] [PDF]
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 Y. Diaz-de-Durana, G. T. Mantchev, R. J. Bram, and A. Franco TACI-BLyS signaling via B-cell-dendritic cell cooperation is required for naive CD8+ T-cell priming in vivo Blood, January 15, 2006; 107(2): 594 - 601. [Abstract] [Full Text] [PDF]
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R. Morita, T. Uchiyama, and T. Hori Nitric Oxide Inhibits IFN-{alpha} Production of Human Plasmacytoid Dendritic Cells Partly via a Guanosine 3',5'-Cyclic Monophosphate-Dependent Pathway J. Immunol., July 15, 2005; 175(2): 806 - 812. [Abstract] [Full Text] [PDF]
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A. Zingoni, T. Sornasse, B. G. Cocks, Y. Tanaka, A. Santoni, and L. L. Lanier Cross-Talk between Activated Human NK Cells and CD4+ T Cells via OX40-OX40 Ligand Interactions J. Immunol., September 15, 2004; 173(6): 3716 - 3724. [Abstract] [Full Text] [PDF]
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R. Arens, M. A. Nolte, K. Tesselaar, B. Heemskerk, K. A. Reedquist, R. A. W. van Lier, and M. H. J. van Oers Signaling through CD70 Regulates B Cell Activation and IgG Production J. Immunol., September 15, 2004; 173(6): 3901 - 3908. [Abstract] [Full Text] [PDF]
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T. Ito, R. Amakawa, M. Inaba, T. Hori, M. Ota, K. Nakamura, M. Takebayashi, M. Miyaji, T. Yoshimura, K. Inaba, et al. Plasmacytoid Dendritic Cells Regulate Th Cell Responses through OX40 Ligand and Type I IFNs J. Immunol., April 1, 2004; 172(7): 4253 - 4259. [Abstract] [Full Text] [PDF]
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R. Zhang, C. J. Fichtenbaum, D. A. Hildeman, J. D. Lifson, and C. Chougnet CD40 Ligand Dysregulation in HIV Infection: HIV Glycoprotein 120 Inhibits Signaling Cascades Upstream of CD40 Ligand Transcription J. Immunol., February 15, 2004; 172(4): 2678 - 2686. [Abstract] [Full Text] [PDF]
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N. Martin-Orozco, Z. Chen, L. Poirot, E. Hyatt, A. Chen, O. Kanagawa, A. Sharpe, D. Mathis, and C. Benoist Paradoxical Dampening of Anti-Islet Self-Reactivity but Promotion of Diabetes by OX40 Ligand J. Immunol., December 15, 2003; 171(12): 6954 - 6960. [Abstract] [Full Text] [PDF]
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 Y. Takahashi, Y. Tanaka, A. Yamashita, Y. Koyanagi, M. Nakamura, and N. Yamamoto OX40 Stimulation by gp34/OX40 Ligand Enhances Productive Human Immunodeficiency Virus Type 1 Infection J. Virol., August 1, 2001; 75(15): 6748 - 6757. [Abstract] [Full Text]
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V. Malmstrom, D. Shipton, B. Singh, A. Al-Shamkhani, M. J. Puklavec, A. N. Barclay, and F. Powrie CD134L Expression on Dendritic Cells in the Mesenteric Lymph Nodes Drives Colitis in T Cell-Restored SCID Mice J. Immunol., June 1, 2001; 166(11): 6972 - 6981. [Abstract] [Full Text] [PDF]
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 J. ATEN, A. ROOS, N. CLAESSEN, E. J. M. SCHILDER-TOL, I. J. M. TEN BERGE, and J. J. WEENING Strong and Selective Glomerular Localization of CD134 Ligand and TNF Receptor-1 in Proliferative Lupus Nephritis J. Am. Soc. Nephrol., August 1, 2000; 11(8): 1426 - 1438. [Abstract] [Full Text]
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A. Cerutti, A. Schaffer, R. G. Goodwin, S. Shah, H. Zan, S. Ely, and P. Casali Engagement of CD153 (CD30 Ligand) by CD30+ T Cells Inhibits Class Switch DNA Recombination and Antibody Production in Human IgD+ IgM+ B Cells J. Immunol., July 15, 2000; 165(2): 786 - 794. [Abstract] [Full Text] [PDF]
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 A. Kunitomi, T. Hori, A. Imura, and T. Uchiyama Vascular endothelial cells provide T cells with costimulatory signals via the OX40/gp34 system J. Leukoc. Biol., July 1, 2000; 68(1): 111 - 118. [Abstract] [Full Text]
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