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(Circulation Research. 1997;80:638-644.)
© 1997 American Heart Association, Inc.

Articles

Lysophosphatidylcholine Increases Expression of Heparin-Binding Epidermal Growth Factor–Like Growth Factor in Human T Lymphocytes

Eiichiro Nishi, Noriaki Kume, Hiroshi Ochi, Hideaki Moriwaki, Yoshio Wakatsuki, Shigeki Higashiyama, Naoyuki Taniguchi, , Toru Kita

From the Department of Geriatric Medicine (E.N., N.K., H.O., H.M., Y.W., T.K.), Graduate School of Medicine, Kyoto University, Kyoto, Japan, and the Department of Biochemistry (S.H., N.T.), Osaka University Medical School, Osaka, Japan.

Correspondence to Noriaki Kume, MD, PhD, Instructor, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. E-mail nkume{at}kuhp.kyoto-u.ac.jp

	Abstract

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Abstract Atherosclerotic lesions contain substantial numbers of activated T lymphocytes in addition to monocytes/macrophages. T cell–derived cytokines and growth factors may play a role in atherogenesis; however, stimuli responsible for T-cell activation in atherogenesis have not been fully elucidated. In this study, we provide evidence that lysophosphatidylcholine (lyso-PC), a polar phospholipid component increased in atherogenic lipoproteins and atherosclerotic lesions, can upregulate gene expression and secretion of heparin-binding epidermal growth factor–like growth factor (HB-EGF) in cultured T lymphocytes isolated from human peripheral blood. Effects of lyso-PC on T lymphocytes appear to be selective and specific, since lyso-PC also increases interleukin (IL)-2 receptor expression but does not affect mRNA levels for IL-2 or IL-4. Lyso-PC–induced upregulation of HB-EGF and IL-2 receptor mRNA in peripheral T cells is mostly dependent on exogenous IL-2 in conditioned medium. The effect of lyso-PC on HB-EGF induction was more potent in CD4⁺ cells than in CD8⁺ cells, although lyso-PC increases IL-2 receptor expression dramatically in both CD4⁺ cells and CD8⁺ cells. Lyso-PC similarly increased HB-EGF expression in Jurkat cells, a cell line for human CD4⁺ T lymphocytes. These results in vitro suggest that lyso-PC may be an important stimulus for T cells in atherogenesis in vivo to upregulate HB-EGF and that T cell–derived smooth muscle growth factors may modulate atherosclerotic progression.

Key Words: lysophosphatidylcholine • heparin-binding epidermal growth factor–like growth factor • T lymphocyte

	Introduction

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Atherosclerosis is a complex disease characterized by lipid deposition, cell proliferation, fibrosis, and inflammatory responses. One of the characteristic features of early atherogenesis is a localized accumulation of foam cells, most of which are derived from circulating blood monocytes. These fatty streak lesions subsequently develop into more complex lesions accompanied by migration and proliferation of medial SMCs.¹ Studies by Hansson and coworkers^{2 3} have demonstrated that substantial numbers of T lymphocytes, as well as monocytes/macrophages, are present in arterial intima at various stages of atherogenesis. The majority of T lymphocytes in atherosclerotic lesions have been suggested to be memory cells and in a state of chronic immunological activation, including their expression of major histocompatibility complex class II antigen and very late antigen-1.⁴ Furthermore, plaque-infiltrated T cells were demonstrated to express IL-2 receptor and interferon gamma in vivo⁵ ; however, mechanisms responsible for activation of T cells in atherosclerotic lesions have not been fully elucidated. Recent studies by Blotnick et al⁶ have revealed that T lymphocytes in atherosclerotic lesions produce smooth muscle growth factors, including HB-EGF, indicating that T cell–derived smooth muscle mitogens, in concert with other cytokines, may modulate fibroproliferative processes in the vessel wall.

Several lines of evidence, on the other hand, have indicated that oxidatively modified LDL plays a key role in atherogenesis.^{1 7 8 9 10} Lyso-PC, a major phospholipid component of oxidized LDL and ß-migrating very low-density lipoprotein,^{7 8 11 12 13} has been shown to activate endothelial cells and monocytes/macrophages to transcriptionally induce proatherogenic genes, including mononuclear leukocyte adhesion molecules and smooth muscle growth factors.^{14 15 16 17} Lyso-PC also appears to impair endothelium-dependent vasorelaxation and stimulate macrophage proliferation.^{13 18} In the present study, therefore, we have tested the hypothesis that lyso-PC may also modulate T-cell function to upregulate the expression of HB-EGF and IL-2 receptors.

	Materials and Methods

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Cells
Human peripheral blood mononuclear cells were harvested from healthy donors by Ficoll density gradient centrifugation. T lymphocytes were separated by direct positive panning on anti-CD3 monoclonal antibody–coated flasks (MicroCELLector, Applied Immune Sciences) according to the manufacturer's instructions. Briefly, mononuclear cells were incubated at room temperature in PBS containing 0.5% human -globulin to block Fc receptors for 15 minutes and were then introduced into anti-CD3 monoclonal antibody–coated flasks. After incubation at room temperature for 1 hour, nonadherent cells were removed, and RPMI 1640 with 20% FBS supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and IL-2 (50 U/mL) was added. Cells detached from flasks during an additional incubation for 24 hours were collected and maintained in the same medium. CD4⁺ and CD8⁺ lymphocyte subpopulations were obtained from peripheral blood mononuclear cells by similar positive-panning methods using anti-CD4 or anti-CD8 monoclonal antibody–coated flasks (Applied Immune Sciences). Human T-cell–line Jurkat was cultured in RPMI 1640 with 10% FBS (Irvine Scientific) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin. EP170.7 cells, which are 32D cells stably expressing the human EGF receptor, were maintained in RPMI 1640 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 10% FBS, and 5% WEHI-3 cell–conditioned medium, which contains high levels of IL-3.¹⁹ All experiments using Jurkat cells were performed in RPMI 1640 containing 5% FBS, and 50 U/mL of IL-2 was added for all experiments with peripheral T lymphocytes.

Reagents
Rabbit anti-human HB-EGF antibody H6 was raised against a synthetic peptide corresponding to amino acids 54 to 73 of the HB-EGF precursor.²⁰ Lyso-PC (palmitoyl, C16:0) was purchased from Avanti Polar Lipids. PMA, actinomycin D, and A23187 were obtained from Sigma Chemical Co. Recombinant human IL-2 was kindly provided by Takeda Chemical Industry (Osaka, Japan). FITC-conjugated anti–IL-2 receptor (ACT-1), nonimmune mouse IgG1, goat anti-rabbit IgG, anti-CD3 (UCHT1), anti-CD4 (MT310), and phycoerythrin-conjugated anti-CD8 (DK25) were purchased from DAKO.

Northern Blot Analysis
Total RNA, isolated from peripheral T lymphocytes and Jurkat cells by the acid–guanidinium phenol–chloroform method,²¹ was electrophoresed through 1% agarose gels containing formaldehyde and was transferred to nitrocellulose membranes (Schleicher & Schuell, Inc). Northern blots were hybridized with human HB-EGF cDNA probes labeled with [-³²P]dCTP (DuPont-New England Nuclear) using random hexanucleotide primers (DNA labeling kit, Pharmacia). Some blots were also hybridized with human IL-2, IL-4, and IL-2 receptor cDNA, and all blots were rehybridized with radiolabeled human ß-actin cDNA to control the amounts of RNA loaded. A 1.5-kb EcoRI–Kpn I fragment of human HB-EGF,²² a 0.6-kb Pst I fragment of human IL-2,²³ a 0.9-kb Xho I fragment of human IL-4 cDNA,²⁴ and a 1.4-kb HindIII fragment of human IL-2R²⁵ were used. Densitometric scanning was performed to quantify the amounts of mRNA using Image laser densitometer (Pharmacia). Human IL-2 and IL-2 receptor cDNA were obtained from Japanese Cancer Research Resources Bank, and human IL-4 cDNA was obtained from American Type Culture Collection.

Bioassay of Soluble HB-EGF
Bioassay of soluble HB-EGF was carried out using EP170.7 cells, which proliferate depending on IL-3 or EGF receptor ligand.²⁶ EP170.7 cells were resuspended with RPMI 1640 with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin without IL-3 and plated in 96-well microtiter wells (2x10⁴ cells/100 µL per well). After EP170.7 cells were incubated with conditioned medium (150 µL) from T lymphocytes or Jurkat cells for 36 hours, [methyl-³H]thymidine (1 µCi per well, DuPont-New England Nuclear) was added, and the amounts of [³H]thymidine incorporated into DNA were measured for the following 8 hours. To inhibit the mitogenic activity of HB-EGF, 5 µg/mL of CRM197, which is a nontoxic mutant form of diphtheria toxin that binds HB-EGF and thereby neutralizes mitogenic activity of HB-EGF, was included in culture medium of EP170.7 cells.²⁶

Flow Cytometry
To detect cell-associated forms of HB-EGF and IL-2 receptor (-chain of the IL-2 receptor complex, CD25), flow cytometric analysis was performed. For HB-EGF, cells were incubated with H6 IgG or nonimmune rabbit IgG, followed by incubation with FITC-conjugated goat anti-rabbit IgG. As for the IL-2 receptor, cells were incubated with ACT-1 or FITC-conjugated mouse IgG1. After washing twice with ice-cold PBS, cells were subjected to single color analysis using EPICS XL (Coulter). To confirm the purity of T cells and their subpopulations, flow cytometric analysis was also carried out using fluorescence-labeled antibodies directed to CD3, CD4, and CD8.

Statistical Analysis
The statistical significance of the differences among means of groups was determined using paired Student's t test.

	Results

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Lyso-PC Selectively Upregulates Gene Expression of HB-EGF in T Lymphocytes
To examine the effect of lyso-PC on HB-EGF gene expression in human peripheral blood T lymphocytes and the related cell line, Jurkat cells, Northern blot analysis was performed. Human peripheral T lymphocytes, isolated by direct positive-panning method using anti-CD3 antibody, showed >96% of the purity as measured by flow cytometry (data not shown). The addition of 50 µmol/L lyso-PC to 5% FBS–containing medium significantly increased the amount of HB-EGF mRNA after a 6-hour incubation in peripheral T lymphocytes (4.7-fold increase by densitometry) (Fig 1A) and after a 24-hour incubation in Jurkat cells (6.2-fold increase) (Fig 1B). These effects of lyso-PC on HB-EGF mRNA upregulation was dose dependent up to 75 µmol/L in both cells. In contrast, lyso-PC treatment did not affect mRNA levels for IL-2 or IL-4 in peripheral T cells (Fig 1A). Treatment with PMA (50 nmol/L) plus A23187 (300 ng/mL) for 6 hours, a well-known activator of T cells, dramatically increased IL-2 and IL-4 mRNA levels (Fig 1A) and exhibited nearly no effect on expression of HB-EGF. We did not detect any cytotoxicity, as evidenced by trypan blue dye exclusion test and flow cytometric determination with propidium iodide, at lyso-PC concentrations at least up to 75 µmol/L.

View larger version (75K): [in this window] [in a new window]   Figure 1. Dose-response relation of the lyso-PC effect on HB-EGF mRNA expression in T lymphocytes and Jurkat cells. A, Cultured human peripheral blood T cells were treated with the indicated concentrations of lyso-PC or PMA (50 nmol/L) in combination with A23187 (300 ng/mL) in RPMI 1640 supplemented with 5% FBS and 50 U/mL of IL-2 for 6 hours. B, Jurkat cells were treated with the indicated concentrations of lyso-PC in RPMI 1640 with 5% FBS for 24 hours. Total cellular RNA was isolated, and Northern blot analyses were performed as described in "Materials and Methods." Blots were rehybridized with IL-2, IL-4, and ß-actin cDNA. Ethidium bromide staining of the gel is also illustrated. Each lane contained 10 µg of total RNA. One of three similar results is shown.

Time-course experiments revealed that increased amounts of HB-EGF mRNA were detectable as early as 3 and 4 hours after lyso-PC treatment in peripheral T cells and Jurkat cells, respectively (Fig 2). A maximal effect of lyso-PC occurred at 6 hours in peripheral T lymphocytes (3.6-fold increase compared with sham treatment) (Fig 2A), whereas it peaked later (16 to 24 hours) in Jurkat cells (5.1-fold increase compared with sham treatment at 16 hours) (Fig 2B). The effect of lyso-PC nearly disappeared at 24 hours in peripheral T cells, whereas it was maintained for at least 48 hours in Jurkat cells (data not shown). Sham treatment showed modest time-dependent increases in HB-EGF mRNA levels in both human T lymphocytes and Jurkat cells, which were probably due to serum components or IL-2 in the assay medium (Fig 2).

View larger version (66K): [in this window] [in a new window]   Figure 2. Time course of HB-EGF gene upregulation by lyso-PC in T lymphocytes and Jurkat cells. A, Human peripheral blood T cells were incubated with or without lyso-PC (50 µmol/L) in RPMI 1640 containing 5% FBS and 50 U/mL of IL-2 for 3, 6, 10, and 24 hours. B, Jurkat cells were treated with or without lyso-PC (50 µmol/L) in RPMI 1640 with 5% FBS for 4, 8, 16, and 24 hours. Northern blot analysis was carried out as described in "Materials and Methods." Each lane contained 10 µg of total RNA. A representative figure from four independent experiments is shown.

Lyso-PC Stabilizes mRNA for HB-EGF
To test the hypothesis that lyso-PC might act to stabilize HB-EGF mRNA, we compared mRNA half-lives in both sham-treated and lyso-PC–treated cells. After peripheral T cells were incubated with or without lyso-PC for 6 hours, actinomycin D (5 µg/mL) was added to block new RNA synthesis, and mRNA levels for HB-EGF were chased up to an additional 3 hours. As seen in Fig 3, mRNA half-lives of HB-EGF were significantly increased in lyso-PC–treated cells (<45 minutes in sham-treated cells, 3 hours in lyso-PC–treated cells), indicating that lyso-PC stabilized mRNA for HB-EGF.

View larger version (27K): [in this window] [in a new window]   Figure 3. Stability of mRNA for HB-EGF in peripheral T cells. After pretreatment with or without lyso-PC (50 µmol/L) for 6 hours in RPMI 1640 with 5% FBS, T cells were incubated with actinomycin D (5 µg/mL) in the presence or absence of lyso-PC (50 µmol/L) for the indicated times. Total cellular RNA was isolated, and Northern blot analyses were performed as described in "Materials and Methods." Each lane contained 10 µg of total RNA. A representative figure from three independent experiments is shown in panel A. Intensities of bands relative to that of lyso-PC–treated cells before the addition of actinomycin D are shown in panel B. Intensities of bands in lyso-PC–treated and sham-treated cells relative to those of lyso-PC–treated and sham-treated cells before the addition of actinomycin D, respectively, are shown in panel C. Data are expressed as mean±SEM from three independent experiments.

Increased Production of HB-EGF in Lyso-PC–Treated Human Peripheral T Lymphocytes
To measure the amounts of HB-EGF secreted into conditioned medium, we performed a bioassay to evaluate mitogenic activity for EP170.7 cells, which proliferate in response to IL-3 or EGF receptor ligands. In the absence of IL-3, EP170.7 cells were incubated with conditioned medium from lyso-PC–treated or sham-treated T cells, and uptake of [³H]thymidine into DNA was measured. As shown in Fig 4, incubation of EP170.7 cells with conditioned medium from lyso-PC–treated cells resulted in significantly higher levels of [³H]thymidine uptake than that with conditioned medium from sham-treated T cells. CRM197, a nontoxic mutant of diphtheria toxin that has been shown to inhibit mitogenic activities of HB-EGF,²⁶ completely inhibited lyso-PC–induced mitogenic activity for EP170.7 cells. Fresh culture medium containing an equal amount of lyso-PC did not stimulate growth of EP170.7 cells, thus excluding the possibility that lyso-PC may directly act as a mitogen. Moreover, conditioned medium from lyso-PC–treated cells did not affect the growth of 32D cells that had not been transfected with the EGF receptor (data not shown). Since HB-EGF is synthesized as a membrane-spanning precursor (proHB-EGF) that can be cleaved to yield a secreted form of HB-EGF, we also measured the amount of proHB-EGF expressed on the surface of T cells and Jurkat cells by flow cytometry using a polyclonal antibody (H6) directed to the extracellular domain of HB-EGF. In Jurkat cells, 12.7% of lyso-PC–treated cells expressed significant amounts of HB-EGF, whereas 3.4% of the sham-treated cells were positive for HB-EGF. In contrast, we did not detect any increases in cell-associated proHB-EGF in lyso-PC–treated peripheral T lymphocytes (2.4% positive for lyso-PC–treated and 2.0% for sham-treated T cells, respectively; Fig 5). Taken together, lyso-PC increased the amounts of HB-EGF, which was mostly secreted into culture medium in human peripheral T lymphocytes, although increased levels of cell-associated proHB-EGF were detectable in Jurkat cells.

View larger version (15K): [in this window] [in a new window]   Figure 4. Effects of lyso-PC on amounts of HB-EGF secreted into conditioned media. After stimulating human peripheral T lymphocytes with or without lyso-PC (50 µmol/L) for 6 and 24 hours in RPMI 1640 with 5% FBS and IL-2 (50 U/mL), conditioned media were taken and incubated with EP170.7 cells with or without 5 µg/mL of CRM197 for 36 hours. EP170.7 cells were subsequently incubated with [3H]thymidine for an additional 8 hours, and radioactivities incorporated into DNA were measured. Data are expressed as relative values compared with the value for fresh medium containing lyso-PC (mean±SEM, n=8 from four separate experiments for sham-treated or lyso-PC–treated samples, n=4 from two separate experiments for lyso-PC–treated samples containing CRM197). *P<.01. **P<.001.

View larger version (21K): [in this window] [in a new window]   Figure 5. Effects of lyso-PC on expression of cell-associated proHB-EGF. Jurkat cells were treated with or without lyso-PC (50 µmol/L) for 24 hours in RPMI 1640 with 5% FBS. Cultured human peripheral T lymphocytes were similarly treated with or without lyso-PC for 6 hours in RPMI 1640 with 5% FBS and 50 U/mL IL-2. Cells were subsequently incubated with anti–HB-EGF antibody (H6) or nonimmune rabbit IgG (control), which was followed by incubation with FITC-conjugated goat anti-rabbit IgG and flow cytometry, as described in "Materials and Methods." D indicates H6 negative cells. I and E indicate H6 positive cells. Representative histograms are shown (top panel). Proportions of cells expressing cell-associated forms of HB-EGF are shown (lower panel). Values (mean±SEM) are from four and seven independent experiments for Jurkat cells and human T lymphocytes, respectively. *P<.05.

Lyso-PC Increases Cell-Surface Expression of IL-2 Receptors
Effects of lyso-PC on the cell-surface expression of the IL-2 receptor (-chain of the IL-2 receptor complex, CD25) was evaluated by flow cytometric analysis. Treatment with lyso-PC (50 µmol/L) for 6 hours, compared with sham treatment, dramatically increased the number of cells highly expressing IL-2 receptors. Upregulated expression of IL-2 receptors by lyso-PC remained for at least 24 hours (Fig 6). In sham-treated cells, as shown in the representative histograms, IL-2 receptor–positive cells were slightly increased after 6 and 9 hours and reduced to the basal level after 24 hours, suggesting that serum components or IL-2 present in the assay media may modestly stimulate IL-2 receptor expression. Since >40% of T cells express IL-2 receptor in response to lyso-PC, T-cell response to lyso-PC appears to be polyclonal and immunologically nonspecific.

View larger version (18K): [in this window] [in a new window]   Figure 6. Effects of lyso-PC on the cell-surface expression of IL-2 receptors. Cultured peripheral T cells were treated with or without lyso-PC (50 µmol/L) for the indicated period and were subjected to flow cytometry using anti–IL-2 receptor antibody. A, Representative histograms are shown. D indicates CD 25-negative cells, E indicates CD 25-positive cells. B, Mean percentages of the number of IL-2 receptors expressing cells in total T-cell population are indicated in bar graphs. Data are expressed as mean±SEM from five independent experiments. *P<.01, **P<.001.

Upregulation of HB-EGF and IL-2 Receptor Gene Expression by Lyso-PC in Peripheral T Lymphocytes Is Dependent on IL-2
To examine whether IL-2 is necessary for HB-EGF and IL-2 receptor gene upregulation elicited by lyso-PC in T cells, we excluded IL-2 from the assay medium and examined the effect of lyso-PC. After they were cultured in IL-2–containing medium for 7 days, human T lymphocytes were stimulated with lyso-PC in culture medium that did not contain IL-2. Under these conditions, lyso-PC showed negligible effects on gene expression of HB-EGF and IL-2 receptors (Fig 7).

View larger version (58K): [in this window] [in a new window]   Figure 7. Dependence of lyso-PC–induced HB-EGF and IL-2 receptor gene upregulation on exogenous IL-2 in human T lymphocytes. Cultured peripheral blood T cells were treated with lyso-PC (50 µmol/L) in RPMI 1640/5% FBS with or without IL-2 (50 U/mL) for 6 hours. Northern blot analysis was performed as described in "Materials and Methods." Each lane contained 10 µg of total RNA. One of four similar experiments is shown.

CD4⁺ and CD8⁺ T Lymphocytes Differentially Respond to Lyso-PC
CD4⁺ and CD8⁺ T-lymphocyte subpopulations were separately isolated by positive-panning method and cultured for 7 days. Purity of cultured CD4⁺ and CD8⁺ T lymphocytes used in our experiments was >95% and >98%, respectively, as determined by flow cytometry (data not shown). To test whether lyso-PC acts differentially on CD4⁺ and CD8⁺ cells, these subpopulations were separately treated with or without lyso-PC, and Northern blot analysis was performed. As shown in Fig 8, lyso-PC upregulated mRNA levels for HB-EGF dramatically in CD4⁺ cells but had negligible effects in CD8⁺ cells. In contrast, IL-2 receptor mRNA levels were elevated in both CD4⁺ and CD8⁺ cells in response to lyso-PC compared with sham treatment.

View larger version (91K): [in this window] [in a new window]   Figure 8. Different effects of lyso-PC on HB-EGF and IL-2 receptor mRNA expression between CD4+ and CD8+ T lymphocytes. Cultured human CD4+ or CD8+ T lymphocytes were treated with (+) or without (-) lyso-PC (50 µmol/L) in RPMI 1640/5% FBS and 50 U/mL IL-2 for 6 hours. RNA samples from lymphocytes before lyso-PC treatment (lane 0) also served as a control. Total cellular RNA was isolated, and Northern blot analysis was performed as described in "Materials and Methods." Blots were rehybridized with IL-2 receptor and ß-actin cDNA. Each lane contained 10 µg of total RNA. A representative figure from three independent experiments is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Accumulation of T lymphocytes has been demonstrated in atherosclerotic lesions, and T-cell–derived factors appear to modulate atherosclerotic progression.^{1 2 3 4 5 6} The present in vitro study demonstrates that lyso-PC can selectively upregulate the expression of HB-EGF and IL-2 receptors in T lymphocytes and suggests that this polar phospholipid increase in inflammatory and atherosclerotic lesions^{27 28} may be an important stimulus for T cells in atherogenesis in vivo.

Recent studies have demonstrated that human T lymphocytes isolated from peripheral blood can synthesize HB-EGF.⁶ Expression of HB-EGF has been shown in macrophages and SMCs in atherosclerotic lesions,²⁰ and T lymphocytes isolated from atherosclerotic plaques have been shown to produce HB-EGF.²⁹ Lyso-PC, therefore, may be a relevant stimulus to induce the expression of HB-EGF in atherosclerotic lesions, in addition to other pathophysiological stimuli, such as tumor necrosis factor,³⁰ thrombin,³¹ and platelet-activating factor.³² Lyso-PC exhibited more potent effects on HB-EGF expression in the CD4⁺ subset of T lymphocytes than in CD8⁺ cells (Fig 8). This appears to be consistent with the previous report that HB-EGF was produced mainly by CD4⁺ cells in vitro.⁶ Recent studies by Zhou et al³³ have shown that CD4⁺ T cells are more abundant in all phases of atherosclerotic lesions of apolipoprotein E knockout mice, an animal model of hypercholesterolemic atherosclerosis.³³ In human atherosclerosis, CD4⁺ T cells are reported to be dominant in relatively advanced stages of atherogenesis.³ Lyso-PC, a major phospholipid component of oxidized LDL, may act mainly on CD4⁺ T cells in atherosclerotic lesions and thereby induce a potent SMC growth factor.

Peripheral T cells in the present study were isolated by positive panning, using anti-CD3 antibody, and were subsequently cultured with IL-2. Without IL-2 in culture medium, lyso-PC did not upregulate the expression of HB-EGF or IL-2 receptors (Fig 7). These results indicate that lyso-PC may act on T lymphocytes, which have been activated, to some extent, by IL-2. Since T cells in the present study were isolated by the positive-panning method using either anti-CD3, -CD4, or -CD8 antibody, ligation to these antibodies might modulate T-cell functions, including the response to lyso-PC and requirement of IL-2. Jurkat cells, a cell line for human T cells, in contrast, may be already functionally activated; therefore, lyso-PC alone was sufficient to upregulate HB-EGF expression. In any case, our present results suggest that activated T cells in atherosclerotic lesions, but not naive T cells, may further be stimulated by lyso-PC and thereby produce HB-EGF and IL-2 receptors.

Studies by Stemme et al³⁴ have shown that CD4⁺ T cells in human atherosclerotic lesions contain subpopulations that proliferate in response to oxidized LDL, suggesting involvement of immune responses to oxidized LDL as an antigen. Our observation that lyso-PC stimulates HB-EGF expression in both peripheral T cells and Jurkat cells suggests that this response is polyclonal. Furthermore, lyso-PC can stimulate IL-2 receptor expression in a majority of both CD4⁺ and CD8⁺ T cells in culture (Figs 6 and 8), indicating that T-cell response to lyso-PC appears to be polyclonal and immunologically nonspecific. Conceivably, upregulated expression of IL-2 receptor by lyso-PC may further enhance the stimulatory effects of IL-2 on T cells and might result in augmented production of HB-EGF in response to lyso-PC.

HB-EGF is a potent mitogen for SMCs and fibroblasts that was originally identified as a macrophage-derived secreted protein that binds to the EGF receptor.²² HB-EGF is synthesized as a membrane-spanning precursor (proHB-EGF) that is tethered to the cell surface via a single transmembrane domain.³⁵ ProHB-EGF has been shown to act as a receptor for diphtheria toxin,³⁶ in addition to its action to stimulate the growth of adjacent cells, which express EGF receptors in a juxtacrine manner.¹⁹ ProHB-EGF can be cleaved and converted to a mature soluble form.³⁷ In cultured human peripheral T lymphocytes, lyso-PC increased mRNA levels for HB-EGF, which resulted in accumulation of secreted forms of HB-EGF in culture medium (Fig 4), although cell-surface expression of proHB-EGF was not significantly altered (Fig 5). This appears to indicate that in T lymphocytes, most of proHB-EGF was readily cleaved and secreted as mature HB-EGF. In lyso-PC–treated Jurkat cells, in contrast, increased expression of cell-surface proHB-EGF was detectable, suggesting that significant amounts of proHB-EGF remain on the cell surface in Jurkat cells (Fig 5).

Induced expression of HB-EGF by lyso-PC also has been demonstrated in cultured human endothelial cells¹⁵ and monocytes.¹⁷ These studies have shown that lyso-PC did not affect HB-EGF mRNA half-life^{15 17} but rather stimulated gene transcription in endothelial cells.¹⁵ Studies by Zembowicz and colleagues^{38 39} have also shown that lyso-PC can transcriptionally induce expression of endothelial constitutive nitric oxide and cyclooxygenase type II. In T lymphocytes, lyso-PC significantly stabilized mRNA for HB-EGF (Fig 3), which may be, at least in part, a mechanism responsible for increased levels of HB-EGF mRNA and protein in lyso-PC–treated T cells. These differences might partly result from the relatively shorter half-life of HB-EGF mRNA in T lymphocytes than in endothelial cells. Further studies are necessary to elucidate molecular mechanisms involved in lyso-PC–induced gene expression in T lymphocytes.

In summary, the present in vitro studies suggest that lyso-PC may be an important stimulus in atherogenesis to modulate T-cell functions. Elucidation of cellular and molecular mechanisms of lyso-PC actions on T lymphocytes and roles of T cell–derived SMC growth factors might provide new insights in atherogenesis.

	Selected Abbreviations and Acronyms

 

EGF = epidermal growth factor HB-EGF = heparin-binding EGF-like growth factor IL-2, IL-3, and IL-4 = interleukin-2, -3, and -4 LDL = low-density lipoprotein lyso-PC = lysophosphatidylcholine PMA = phorbol 12-myristate 13-acetate SMC = smooth muscle cell

	Acknowledgments

 
This study was supported by research grants 05404039, 06671022, 07557073, and 07NP0901 from the Ministry of Education, Science, and Culture of Japan, grants 5A-2 and 6B-1 for cardiovascular diseases from the Japanese Ministry of Health and Welfare, a research grant for the health sciences from the Japanese Ministry of Health and Welfare, and a research grant from the Japanese Foundation of Metabolism and Diseases. We would like to thank Dr Eisuke Mekada (Institute of Life Science, Kurume University) for his generous gift of CRM197.

Received August 5, 1996; accepted February 3, 1997.

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