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The Journal of Immunology, 2001, 166: 130-136.
Copyright © 2001 by The American Association of Immunologists

Outside-to-Inside Signal Through the Membrane TNF-{alpha} Induces E-Selectin (CD62E) Expression on Activated Human CD4+ T Cells1

Shin-ichi Harashima*, Takahiko Horiuchi2,*, Nobuaki Hatta{ddagger}, Chika Morita*, Masanori Higuchi*, Takuya Sawabe*, Hiroshi Tsukamoto*, Tomoko Tahira{dagger}, Kenshi Hayashi{dagger}, Shigeru Fujita{ddagger} and Yoshiyuki Niho*

* First Department of Internal Medicine, Faculty of Medicine, and {dagger} Institute of Genetic Information, Kyushu University, Fukuoka, Japan; and {ddagger} First Department of Internal Medicine, School of Medicine, Ehime University, Ehime, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The membrane TNF-{alpha} is known to serve as a precursor of the soluble form of TNF-{alpha}. Although it has been reported the biological functions of the membrane TNF-{alpha} as a ligand, the outside-to-inside (reverse) signal transmitted through membrane TNF-{alpha} is poorly understood. Here we report a novel function mediated by outside-to-inside signal via membrane TNF-{alpha} into the cells expressing membrane TNF-{alpha}. Activation by anti-TNF-{alpha} Ab against membrane TNF-{alpha} on human T cell leukemia virus (HTLV) I-infected T cell line, MT-2, or PHA-activated normal human CD4+ 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-{alpha} (R78T/S79T) resistant to proteolytic cleavage was introduced into Jurkat or HeLa cells, E-selectin was induced by the treatment with anti-TNF-{alpha} Ab with the similar kinetics. Membrane TNF-{alpha}-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-{alpha}-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-{alpha}, but also presented evidence that E-selectin was inducible in cell types different from endothelial cells. It is strongly suggested that membrane TNF-{alpha} is a novel proinflammatory cell surface molecule that transmits bipolar signals in local inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-{alpha} is a multifunctional cytokine produced by activated macrophages as well as by several other cell types, such as lymphocytes and neutrophils (1, 2, 3). The precursor form of TNF-{alpha}, called membrane TNF-{alpha}, 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-{alpha} by TNF-{alpha}-converting enzyme and acts as a soluble form of 17-kDa polypeptide (4, 5). Most of the TNF-{alpha}-mediated biological effects, such as cell proliferation, apoptosis, and cytokine production, are attributed to the soluble form of TNF-{alpha}, whereas the function of membrane TNF-{alpha} is much less understood. The biological effects of TNF-{alpha} 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-{alpha}-mediated biological effects, the function of TNF-RII is not well understood.

The biological function of membrane TNF-{alpha} was first reported as cytotoxic activity (6, 7, 8), which was followed by the identification of several immunological functions. The membrane TNF-{alpha} 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-{alpha} (14). All of these biological effects by membrane TNF-{alpha} are mediated through a cell-to-cell contact fashion. The signal transmitted from membrane TNF-{alpha} to the target cells is likely to be mediated through both TNF receptors; however, it is more potent on TNF-RII than soluble TNF-{alpha} (15).

In contrast to the above functions of membrane TNF-{alpha} as a ligand, the reverse (outside-to-inside) signal via membrane TNF-{alpha} into the cells expressing membrane TNF-{alpha} 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-{alpha}-transfected HeLa cells (17), the cytoplasmic domain of membrane TNF-{alpha} should play some critical role in the cellular functions. Recently, our group first demonstrated the signaling through the membrane TNF-{alpha} into HTLV-I-infected human T cells that express this molecule on their surface. Upon activation of membrane TNF-{alpha} with anti-TNF-{alpha} Ab, IL-2 and IFN-{gamma} were induced with concurrent elevation of intracellular calcium concentration (10, 11). This finding of calcium mobilization by membrane TNF-{alpha} was confirmed subsequently by others (18). Membrane TNF-{alpha} has also been shown to modulate anti-CD3-triggered T cell cytokine expression in mice (19). Thus it is suggested that membrane TNF-{alpha} 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-{alpha} itself into T cells.

These lines of evidence prompted us to extend our search for novel proinflammatory functions mediated through membrane TNF-{alpha} into T cells expressing this molecule. Here we report that activation of membrane TNF-{alpha} 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-{alpha} stimulation (21). Moreover, expression of E-selectin was also demonstrated by the treatment with anti-TNF-{alpha} Ab of the membrane TNF-{alpha}-transfected Jurkat and HeLa cells. Thus we confirmed the reverse signaling from membrane TNF-{alpha} 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 µg/ml of rabbit polyclonal anti-human TNF-{alpha} Ab (Genzyme, Cambridge, MA), anti-human TNF-{alpha} 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 x 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 x 105/ml) or MT-2 cells (2 x 105/ml), both of which expressed membrane TNF-{alpha} on the cell surface, were incubated in a 25-ml flask immobilized with anti-TNF-{alpha} Ab for 12–24 h in the presence or absence of various inhibitors, 100 ng/ml of herbimycin A (Wako Chemical), 40 µg/ml of genistein (Wako Chemical), 100 µM of N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7; Wako Chemical), 100 µM of manumycin, 100 ng/ml of wortmannin (Wako Chemical), 100 µM of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7; Wako Chemical), 100 µg/ml of cycloheximide (Wako Chemical), and 2 mM of EGTA (Wako Chemical). Jurkat cells (2 x 105/ml) were incubated in a 25-ml flask immobilized with anti-CD40L or FasL for 12–24 h. Rat anti-human TNF-{alpha} receptor (p75) mAb (Genzyme) and anti-human TNF-{alpha} receptor (p55) Ab (Bender MedSystems, Vienna, Austria) were used for blocking the function of TNF-{alpha}. These blocking Abs were added to the culture media concurrently with the stimulation of membrane TNF-{alpha} by immobilized anti-TNF-{alpha} Ab. The presence of soluble TNF-{alpha} in the supernatant was quantified by a commercial ELISA kit (Endogen, Woburn, MA). Recombinant human TNF-{alpha} (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 x 105 per sample) were stained on ice. Cell surface molecules including TNF-{alpha} and adhesion molecules were detected by FACScan (Becton Dickinson) using the following FITC-conjugated (/FITC) and PE-conjugated (/PE) Abs. For TNF-{alpha}, anti-human TNF-{alpha}/FITC mAb (R&D Systems, Minneapolis, MN) was used. For the detection of adhesion molecules, anti-human E-selectin (CD62E)/FITC mAb, anti-human 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 VCAM-1/FITC mAb (Immunotech, Marseilles, France) were used. Anti-human CD30 ligand (CD30L)/FITC mAb (Santa Cruz Biotechnology), anti-human CD40L/FITC (MBL, Nagoya, Japan), and rabbit polyclonal anti-human 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-{alpha}. 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-{alpha}, soluble TNF-{alpha} that might be bound to the cell surface was removed by washing with acidified buffer. PHA-activated CD4+ T cells (1 x 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-{alpha} on the cells was studied by FACS analysis.

RNA preparation

MT-2 cells were stimulated by immobilized rabbit anti-human TNF-{alpha} 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 µg of total RNA was reverse transcribed in the presence of Moloney murine leukemia virus (MoLV) reverse transcriptase, oligo dT, and 25 µM dNTP at 25°C for 60 min in a total reaction mixture of 25 µl. 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 {beta}-actin was described previously (22), and the expected size of the PCR product was 314 bp. PCR were performed using 1 µl of the cDNA product as template, 0.2 µM of each primer, 25 µM of dNTP, 2 µCi [{alpha}-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 µl. 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 µg 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 [{alpha}-32P]dATP using Multiprime DNA labeling systems (Amersham) and was used as probes. After hybridization, filters were washed with 0.2 x 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-{alpha} on Jurkat and HeLa cells

Wild-type membrane TNF-{alpha} cDNA was obtained by using RT-PCR of the total RNA from normal human PBMC. The primer sequences were 5'-ATGAGCACTGAAAGCATGATCCGGGACGTG-3' and 5'-TCACAGGGCAATGATCCCAAAGTAGACCTG-3' and the product size was 702 bp, encompassing the entire coding region of the membrane TNF-{alpha}. The uncleavable mutant form of membrane TNF-{alpha} 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-{alpha} was subcloned into pKF19. Both the codon AGA for Arg77 at position +2 (2 aa downstream from the TNF-{alpha}-converting enzyme cleavage site) and the codon TCA for Ser78 at position +3 were substituted to the codon ACA for Thr and the codon ACA for Thr, respectively, which has already been shown to result in the effective reduction in the cleavage of membrane TNF-{alpha} (17). The oligonucleotide sequence to introduce those mutations in the membrane TNF-{alpha} was 5'-CAGGCAGTCACAACATCTTCTC-3'. The oligonucleotide sequences of the wild-type and mutant forms of membrane TNF-{alpha} were confirmed by direct sequencing using Amplicycle sequencing kit (Perkin-Elmer) (26). Wild-type and uncleavable mutant forms of membrane TNF-{alpha} 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 x 105 cells/ml) and HeLa cells were stimulated with rabbit anti-human TNF-{alpha} polyclonal Ab (diluted to 1:1000) to see the up-regulation of E-selectin. The competition by an excess amount of soluble TNF-{alpha} was studied by adding 1 µg/ml of recombinant soluble TNF-{alpha} to the culture medium.

Expression of TNF-RII on HeLa cells

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.

Coculture of membrane TNF-{alpha}-expressing T cells with TNF-RII-expressing 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-{alpha} (5 x 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-{alpha}-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 x 105 cells/ml) or Jurkat cells transfected with uncleavable mutant form of membrane TNF-{alpha} (2 x 105 cells/ml) were incubated in a 25-ml flask immobilized by rabbit anti-human TNF-{alpha} polyclonal Ab for 1, 2, 5, 10, 20, and 40 min. Then 10 µl of the MT-2 cells were mixed with an equal amount of 2 x 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-{alpha}-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-{alpha} polyclonal Ab (Genzyme) followed by the visualization with HRP-conjugated anti-rabbit Ig Ab (Amersham).

Phosphorylation of serine residues of membrane TNF-{alpha} was studied in membrane TNF-{alpha}-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
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Membrane TNF-{alpha}-mediated expression of E-selectin (CD62E) on the HTLV-I-infected T cell line, MT-2

To study the effects of membrane TNF-{alpha} 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-{alpha} on the surface by FACS analysis as well as Western blot analysis (10). As shown in Fig. 1Goa, the expression of membrane TNF-{alpha} on the surface of MT-2 cells was confirmed by FACS analysis. After the stimulation of membrane TNF-{alpha} with immobilized rabbit anti-human TNF-{alpha} 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 analysis. Of the molecules studied, only E-selectin showed a pronounced increase in the expression. Fig. 1Gob shows the kinetics of E-selectin expression. Although, in the absence of stimulation of membrane TNF-{alpha}, 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-{alpha}. The E-selectin expression on MT-2 cells was decreased to the undetectable level by 48 h after the stimulation of membrane TNF-{alpha}. In contrast, the stimulation of membrane TNF-{alpha} with immobilized anti-human TNF-{alpha} 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-{alpha} Ab treatment was due to neutralization of soluble TNF-{alpha}, we next studied the expression of E-selectin in the presence of the blocking Abs against TNF-RI (p55) and TNF-RII (p75) (Fig. 1Goc). The kinetics of E-selectin expression by the stimulation of membrane TNF-{alpha} by anti-TNF-{alpha} Ab was not altered in the presence of blocking Abs against TNF-RI and TNF-RII. Moreover, the addition of human recombinant soluble TNF-{alpha} 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-{alpha} in the culture medium (data not shown). Thus it is concluded that E-selectin expression by anti-TNF-{alpha} Ab is mediated by direct stimulation through membrane TNF-{alpha}, not by the indirect effect on soluble TNF-{alpha}.



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  		FIGURE 1. a, Expression of membrane TNF-{alpha} on the surface of the HTLV-I-infected T cell line, MT-2. Cells are stained with FITC-conjugated mouse anti-human TNF-{alpha} 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-{alpha} with immobilized rabbit polyclonal anti-TNF-{alpha} Ab for 0, 12, 24, and 48 h (filled peaks). Background staining is shown by black lines. c, Membrane TNF-{alpha}-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-{alpha} 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).

 
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-{alpha} with immobilized rabbit anti-TNF-{alpha} Ab. In contrast, ICAM-1 and VCAM-1 were constitutively expressed on MT-2 cells, which were slightly down-regulated by the treatment of immobilized anti-TNF-{alpha} Ab (data not shown).

Induction of E-selectin on activated normal human CD4+ T cells

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-{alpha} Ab showed that unstimulated CD4+ T cells from normal individuals did not express membrane TNF-{alpha}, whereas treatment of normal CD4+ T cells with 100 ng/ml of PHA for 12 h resulted in the expression of membrane TNF-{alpha} (Fig. 2Goa). The treatment with acidified buffer to dissociate the cell surface-soluble form of TNF-{alpha} did not alter the intensity of this staining by anti-TNF-{alpha} Ab, which confirmed that the membrane TNF-{alpha} 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. 2Gob). Then, the membrane TNF-{alpha} expressed on the activated human CD4+ T cells was stimulated with immobilized rabbit anti-human TNF-{alpha} 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-{alpha} is shown as a representative in Fig. 2Goc. This membrane TNF-{alpha}-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. 2God). We next analyzed the expression of E-selectin in normal human CD8+ T cells. Although membrane TNF-{alpha} 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 anti-TNF-{alpha} Ab (data not shown).



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  		FIGURE 2. E-selectin expression on normal human CD4+T cells stimulated with anti-TNF-{alpha} 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-{alpha} (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-{alpha} Ab for 12 h in the absence (c) or presence (d) of blocking Abs against TNF-RI and TNF-RII. 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-{alpha}

To investigate E-selectin expression in the cells that are stably and abundantly expressing membrane TNF-{alpha}, we transfected the wild-type or uncleavable mutant form of membrane TNF-{alpha} 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-{alpha} Ab. Although membrane TNF-{alpha} was not expressed on the surface of steady-state Jurkat or HeLa cells, transfection with the uncleavable mutant form of TNF-{alpha} resulted in the constitutive expression of membrane TNF-{alpha} when assessed by FACS analysis and Western blot analysis (Fig. 3Go, 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-{alpha} 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. 3Goc). 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-{alpha} (1 µg/ml) almost completely inhibited this E-selectin up-regulation by anti-TNF-{alpha} Ab on membrane TNF-{alpha}-expressing Jurkat cells, suggesting again the specificity of anti-TNF-{alpha} 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-{alpha}. Fig. 3God shows a representative result in the transfected HeLa cells at 4 h after the treatment with polyclonal anti-TNF-{alpha} Ab. The Jurkat or HeLa cells transfected with the wild-type membrane TNF-{alpha} expressed a considerable amount of membrane TNF-{alpha} on their surface. However, soluble TNF-{alpha} was more significantly secreted in the culture medium of the wild-type membrane TNF-{alpha} transfectants compared with that of uncleavable mutant transfectants as assessed by Western blot analysis. Upon activation with polyclonal anti-TNF-{alpha} Ab, E-selectin was similarly induced on the Jurkat or HeLa cells transfected with wild-type membrane TNF-{alpha} (data not shown).



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  		FIGURE 3. FACS analysis and Western blot analysis of the expression of membrane TNF-{alpha} on Jurkat (a) or HeLa (b) cells after the transfection of the uncleavable form of membrane TNF-{alpha} (R78T/S79T) using expression vector pCXN2. Stable expression of membrane TNF-{alpha} 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-{alpha} (Mt). c, Untransfected cells. E-selectin expression 4 h after the stimulation of rabbit polyclonal anti-human TNF-{alpha} 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-{alpha}. 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).

 
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-{alpha} (membrane TNF-{alpha}-expressing T cells) (Fig. 4Goa) 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. 4Gob). The expression of E-selectin was induced as early as 6 h after the start of coculture on membrane TNF-{alpha}-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. 4Goc). This up-regulation of E-selectin was abolished in the presence of TNF-RII-specific neutralizing Ab. In addition, membrane TNF-{alpha}-expressing Jurkat cells did not show any induction of E-selectin by the coculture with untransfected HeLa cells (data not shown).



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  		FIGURE 4. Up-regulation of E-selectin on TNF-{alpha}-expressing Jurkat cells cocultured with TNF-RII-expressing HeLa cells. a, Membrane TNF-{alpha} expression on Jurkat cells. The uncleavable mutant form of membrane TNF-{alpha} was stably expressed on Jurkat cells as described in Fig. 3Goa. 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-{alpha}-expressing Jurkat cells when brought into cell-to-cell contact with TNF-RII-expressing HeLa cells. When membrane TNF-{alpha}-expressing Jurkat cells were cocultured with TNF-RII-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-{alpha}-expressing Jurkat cells after 24 h of coculture was shown as a representative. Background staining is shown by black lines (mouse IgG1/FITC).

 
Up-regulation of E-selectin message after the stimulation of membrane TNF-{alpha}

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-{alpha}, which was increased upon activation of the membrane TNF-{alpha} as early as 1 h after the stimulation of membrane TNF-{alpha} (Fig. 5Goa). The E-selectin message was stably increased until 12 h after the stimulation. The amount of control {beta}-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. 5Gob).



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  		FIGURE 5. Up-regulation of E-selectin message in MT-2 cells after the stimulation of membrane TNF-{alpha}. a, RT-PCR analysis. Total RNA purified from MT-2 cells after the incubation in the immobilized anti-TNF-{alpha} 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-{alpha} (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 {beta}-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 µg) 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-{alpha}. The size of the messages in MT-2 cells was identical with that of HUVEC.

 
Intracellular signal transduction mechanism mediated by membrane TNF-{alpha}

To understand the intracellular signals essential for the membrane TNF-{alpha}-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 µM of H-7 (protein kinase C inhibitor), 100 ng/ml of herbimycin A (tyrosine kinase inhibitor), 40 µg/ml of genistein (tyrosine kinase inhibitor), 100 ng/ml of wortmannin (myosine light chain kinase inhibitor), 100 µM of manumycin (farnesyl transferase inhibitor), 100 µM of W-7 (calmodulin inhibitor), and 2 mM of EGTA, the membrane TNF-{alpha}-mediated E-selectin expression was not affected (data not shown). We next studied the tyrosine phosphorylation induced by membrane TNF-{alpha} stimulation in MT-2 and Jurkat cells transfected with membrane TNF-{alpha}. Upon stimulation of membrane TNF-{alpha} with immobilized anti-TNF-{alpha} Ab, altered tyrosine phosphorylation patterns were not clearly observed. In addition, membrane TNF-{alpha}, but not soluble TNF-{alpha}, was constitutively phosphorylated at the serine residues, which confirmed the data reported by Pócsik (17) (data not shown). Treatment of membrane TNF-{alpha}-expressing Jurkat cells with 100 ng/ml of cycloheximide resulted in the significant, but not complete, inhibition of anti-TNF-{alpha}-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).

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. 6Goa). Stimulation with immobilized rabbit polyclonal anti-CD40L or anti-FasL did not result in the up-regulation of E-selectin in Jurkat cells (Fig. 6Gob).



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  		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 x 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We here presented the evidence that membrane TNF-{alpha} expressed on activated CD4+ T cells or stably expressed on Jurkat or HeLa cells, when treated with polyclonal anti-TNF-{alpha}, induced the expression of an adhesion molecule, E-selectin. E-selectin up-regulation was also observed in membrane TNF-{alpha}-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-{gamma} and IL-2 was induced in HTLV-I-infected human T cells by the stimulation of membrane TNF-{alpha} (10, 11). Moreover, ligation of soluble TNF receptor to membrane TNF-{alpha} 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-{alpha}.

The members of TNF-{alpha}/TNF-{alpha} receptor superfamily are rapidly increasing in recent years. The TNF-{alpha} (TNF-ligand) family comprises over 10 proteins, such as lymphotoxin-{alpha}, 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-{alpha} in the carboxyl-terminal receptor-binding region (~145-aa residues) and are believed to act as trimers (28, 29, 30). The N-terminal cytoplasmic domain is conserved across different species, but not between family members. Although lymphotoxin-{alpha} is entirely secreted and TNF-{alpha} 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 well-characterized function as ligands, evidence for the reverse signaling 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-{alpha}, strongly suggest that TNF-{alpha} 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-{alpha}, as stimulation of FasL or CD40L did not cause E-selectin up-regulation in Jurkat cells (Fig. 6Go).

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. E-selectin, 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-{alpha}, IL-1, as well as LPS up-regulate the expression of E-selectin (25), whereas TGF-{beta} acts as a suppressor (36). The time course of the E-selectin expression is different between CD4+ T cells induced by membrane TNF-{alpha} (Fig. 2Goc) and HUVEC induced by the soluble form of TNF-{alpha}, 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. 5Gob). It has been reported that TNF-{alpha}-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-{alpha} 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-{alpha}. However, it is possible that some Ser/Thr kinase might be important for its effect on E-selectin expression, as membrane TNF-{alpha} 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-{alpha}-associated proteins would contribute to further understanding of the biological functions of membrane TNF-{alpha}. The cytoplasmic domain of the membrane TNF-{alpha} should be essential for the binding of membrane TNF-{alpha}-associated proteins because substitution of a particular amino acid residue by site-directed mutagenesis resulted in the complete loss of E-selectin expression without affecting the expression of membrane TNF-{alpha} 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-to-inside signal is demonstrated in the case of L-selectin (42).

In conclusion, we presented the evidence that membrane TNF-{alpha} upon activation with anti-TNF-{alpha} 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-{alpha} in Jurkat or HeLa cells followed by the stimulation with polyclonal anti-TNF-{alpha} Ab also resulted in the expression of E-selectin. In addition, stimulation with TNF-RII-expressing HeLa cells also induced E-selectin expression in membrane TNF-{alpha}-expressing Jurkat cells in cell-to-cell contact manner. Taking account of our previous findings that membrane TNF-{alpha} mediates Th1 type cytokine production (10, 11), the function of membrane TNF-{alpha} 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-{alpha}, which will contribute to the cell-to-cell adhesion and interaction. Thus it is suggested that membrane TNF-{alpha} is a bipolar molecule that transmits signals positively, regulating local inflammation both as a ligand and as an acceptor.


   Footnotes
 
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. Back

2 Address correspondence and reprint requests to Dr. Takahiko Horiuchi, First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan. Back

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. Back

Received for publication December 1, 1999. Accepted for publication September 29, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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