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Cardiovascular Research 2001 52(2):328-336; doi:10.1016/S0008-6363(01)00376-5
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Copyright © 2001, European Society of Cardiology

Effects of homocysteine on murine splenic B lymphocyte proliferation and its signal transduction mechanism

Qin Zhanga, Xiaokun Zenga, Jingxuan Guoa and Xian Wanga,b,*

aInstitute of Vascular Medicine, Third Hospital, Beijing 100083, People's Republic of China
bDepartment of Physiology, Health Science Center, Peking University, No. 38, Xue Yuan Road, Beijing 100083, People's Republic of China

* Corresponding author. Tel.: +86-10-6209-1443; fax: +86-10-6201-7700 xwang{at}mail.bjmu.edu.cn

Received 9 April 2001; accepted 5 June 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Elevated plasma homocysteine (Hcy) levels have been defined as an increased risk of atherosclerosis. However, the mechanisms that Hcy induces the development of atherosclerosis are not fully understood. Therefore, effect of Hcy on B lymphocyte proliferation and its cellular mechanism were examined in normal and hyperhomocysteinemia ApoE-knockout mice. Methods: Mouse B lymphocytes were incubated with Hcy, related compounds and/or antioxidants and/or inhibitors of PKC, p38 MAPK, NF-{kappa}B in the presence or absence of lipopolysaccharide. DNA synthesis, production of reactive oxygen species was measured. Results: Hcy (0.1–3.0 mM) and other compounds with thiol (–SH), such as cysteine and glutathione significantly increased resting and lipopolysaccharide-induced B lymphocyte proliferation. ApoE-knockout mice with hypercysteinemia (plasma Hcy levels were 20.3±2.9 vs. 2.6±0.6 µM in control, P<0.05) had a significant promotion of B cell proliferation in response to lipopolysaccharide. Hcy also increased intracellular reactive oxygen species production. Radical scavengers reduced Hcy-induced B lymphocyte proliferation. The promotion of Hcy was significantly inhibited by inhibitors of PKC (calphostin C and RO-31-8220), p38 MAPK (SB 202190 and PD 169316) and NF-{kappa}B (pyrrolidine dithiocarbamate). Conclusions: The reactive oxygen species generated by thiol (–SH) auto-oxidation of Hcy are essential, and PKC, p38 MAPK and NF-{kappa}B are involved in the Hcy-induced B lymphocyte proliferation. Hyperhomocysteinemia may increase B lymphocyte susceptibility to inflammatory progression of atherosclerotic lesions.

KEYWORDS Atherosclerosis; Free radicals; Immunology; Infection/inflammation; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Homocysteine (Hcy) is a sulfur-containing amino acid formed during the metabolism of methionine. Hyperhomocysteinemia was found in 20–30% of patients with premature atherosclerosis involving carotid, coronary and peripheral artery [1]. Elevated plasma Hcy levels have been defined as an independent risk factor for coronary heart disease [2]. Yet, the mechanism by which homocysteinemia induces atherosclerosis is unclear.

Recent studies demonstrate that Hcy enhances endothelial dysfunction [3] and promotes the proliferation of vascular smooth muscle cells [4]. Atherosclerosis fulfills many of the criteria of a chronic inflammatory process, and substantial evidence points toward the involvement of the humoral and cellular immune system in atherogenesis [5]. In the advanced atherosclerotic plaque, T lymphocytes make up nearly 20% of the cell population, 10% of which are in an activated state [6]. Atherosclerotic lesions also contain B lymphocytes [7]. Immunoglobulins and C5b–9 terminal complexes, which are resulting from the activation of the complement system, have been observed within atherosclerotic lesions, but not in non-atherosclerotic artery [8]. In our previous study, we have showed that Hcy could potentiate Con A-induced proliferation in mouse splenic T lymphocytes. However, effects of Hcy on B lymphocyte proliferation have not been determined in normal and ApoE-knockout mouse with elevated Hcy level.

The thiol (–SH) group of Hcy is readily oxidized. During oxidation of the thiol group, superoxide anion radical (O2–), hydrogen peroxide (H2O2) and hydroxyl radical (OH·) are generated [9]. These oxygen-derived molecules are believed to account for the endothelial cytotoxicity and vascular smooth muscle cell proliferation of Hcy [3,10]. Recently, it was reported that the increase of reactive oxygen species (ROS) following various external stimuli at low concentration may function as cellular signaling intermediators and associates with cellular proliferation [11]. Therefore, we hypothesized that there might be relationships between Hcy stimulation, ROS and B lymphocyte proliferation.

ROS play a role as second messengers to regulate signal transduction pathways that ultimately control gene expression and post-translational modifications of proteins. A substantial body of evidence indicates that activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-{kappa}B) may be controlled by ROS [12]. Some other studies suggest that the activation of protein kinase C (PKC) and MAPK are involved in lipopolysaccharide (LPS)-induced B lymphocyte proliferation [13]. In addition, the NF-{kappa}B activation induced by LPS in B lymphocytes serves as a critical regulator of the expression of genes involved in inflammatory immune [14]. Therefore, we hypothesized that PKC, MAPK and NF-{kappa}B transcription factor were involved in Hcy-induced B lymphocyte proliferation.

In the present study, our results indicate that the Hcy-induced B lymphocyte proliferation is mediated by oxygen radicals such as O2–, OH· and H2O2 generated by the thiol (–SH) auto-oxidation. Furthermore, our data indicate that PKC, p38 MAPK and NF-{kappa}B transcription factor are involved in the proliferative response of murine splenic B lymphocyte following the stimulation with Hcy. The hyperhomocysteinemia may lead to inflammatory progression of atherosclerotic lesions in concert with other stimuli.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
The treatment of the laboratory animals and experimental protocols of the present study adhered to the guidelines of Health Science Center of Peking University and were approved by the Institutional Authority for Laboratory Animal Care. Experiments were carried out in healthy, male Balb/C mice (6–8 weeks old, 18–22 g) and male ApoE-knockout mice (3–4 weeks old) obtained from the animal laboratory of Health Science Center of Peking University. They were housed in wire-mesh cages at 22°C ambient temperature and maintained on standard mouse chow, which contained 4.76% fat by weight, and water ad libitum with a 12-h light/dark cycle for 1–2 weeks prior to all experiments. The ApoE-knockout mice were fed with a diet of 400 mg/kg/day methionine for 6 weeks by which the hyperhomocysteinemia was performed as previously reported [15].

2.2 Preparation of B lymphocytes
Lymphocytes were obtained from spleens of male Balb/C mice and ApoE-knockout mice [16]. B cells were purified by negative selection as previously described [17]. Briefly, single cell suspension of spleen was centrifuged. After lysis of red blood cells, single cell suspensions were incubated for 2 h at 37°C to allow adherence of macrophages. Nonadherent cells were collected and incubated in Petri dishes coated with mouse anti-IgG for 1 h at room temperature. Cell suspensions (T lymphocytes) were removed by RPMI-1640 medium at 37°C. The antibody-conjugated cells (B lymphocyte) were collected by iced RPMI-1640 medium. Purity of the B cells was shown to be 94% as determined by the flow cytometry (Becton Dickinson). Cell viability was evaluated by trypan blue exclusion. Only cell preparations with a 95% viability or greater were used.

2.3 B Cell proliferation assay
Cell proliferation was determined by [3H]thymidine incorporation, an index of DNA synthesis. Cells were plated in triplicate samples in flat bottom, 96-well culture plates at a density of 2x105 cells/well in RPMI-1640 medium containing 5% fetal calf serum. Cells were treated for 72 h with Hcy (0.1–10 mM), or related compounds including homoserine, homocystine, cysteine, glutathione and methionine, and/or with antioxidants including catalase, superoxide dismutase (SOD), and 1% DMSO, and/or the inhibitors of PKC, MAPK and NF-{kappa}B for 30 min before exposed to Hcy, in the presence or absence of LPS 0.5 µg/ml. [3H]thymidine (0.2 µCi/well) was added during the last 6 h, and the cells were then harvested onto glass fiber filters. After drying at 80°C for 2 h, the radioactivity was determined by liquid scintillation counting (Beckman, USA) and presented as counts per minute.

2.4 Measurement of plasma Hcy levels by EIA
Immediately following removal of blood of the ApoE-knockout mice with or without a diet of methionine, the samples were placed in EDTA-containing test tubes. Following centrifugation, the plasma fractions were transferred to other polypropylene tubes and stored at –20°C for not more than 2 months before analysis for Hcy levels by Hcy EIA reagent kit (Bio-Rad, CA, USA).

2.5 Measurement of intracellular ROS generation
Hcy (0.1–10 mM) was added to the cells together with the dye 2',7'-dichlorofluorescein diacetate (DCFH-DA, 5 µM) in the presence or absence of LPS (0.5 µg/ml) for 25 min. Determination of intracellular oxidant production was based on the oxidation of DCFH-DA by intracellular ROS, resulting in the formation of the fluorescent compound 2',7'-dichlorofluorescein (DCF) [18]. DCF fluorescence was monitored with a confocal laser scanning microscope (Leica, Germany).

2.6 Measurement of LDH release
B cells were treated with Hcy (0.1–10 mM) for 72 h. Cell suspensions were collected for determination of LDH. LDH activity was then measured by spectrophotometric enzyme activity method. LDH activity was expressed as units per liter medium.

2.7 Chemicals
D,L-Hcy, D,L-cysteine, D,L-homoserine, D,L-homocystine, glutathione (reduced form), and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma Co. (St. Louis, MO). 2'-7'-dichlorofluorescein diacetate was obtained from Molecular Probes (Eugene, OR). LPS (lipopolysaccharide B from S. enteridis) was purchased from Difco Laboratories (Detroit, MI). PD 169316, PD 98059, SB 202190, calphostin C, RO-31-8220 were purchased from Calbiochem Co. (La Jolla, CA). RPMI-1640 was purchased from Gibco (Grand island, NY). Catalase, SOD and other chemicals were purchased from the Chinese Chemical Co. (Beijing, China).

2.8 Statistical analysis
The results were expressed as the mean±S.E.M. The number of experimental animals used for each group is presented as the n value in the figure legends. Data were analyzed using one-way ANOVA and further analyzed using the Student–Newman–Keuls’ test for multiple comparisons between treatment groups. A P value of less than 0.05 was considered a significant difference between treatment group means.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effects of Hcy on B lymphocyte proliferation
To test whether Hcy affected B lymphocyte proliferation, splenic cells were cultured with Hcy (0.1–10 mM) in the presence or absence of LPS (0.5 µg/ml) for 72 h. As shown in Fig. 1A, Hcy (0.1–10 mM) caused a significant enhancement of resting B lymphocyte proliferation with 3H-TdR incorporation increased to 2238.9±573.2% at Hcy 3 mM. In addition, Hcy (0.3–3.0 mM) also potentiated LPS-induced B lymphocyte proliferation, as compared with LPS alone; the 3H-TdR incorporation was increased to maximum at Hcy 1 mM (Fig. 1B). However, when the concentration of Hcy was in excess of 3.0 mM, both directing and potentiating effects of Hcy declined.


Figure 1
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  		Fig. 1 Effects of Hcy on B lymphocyte proliferation. B cells were incubated with Hcy (0.1–10 mM) in the absence (A) or presence (B) of LPS 0.5 µg/ml for 72 h. Cell proliferation was assayed by 3H-TdR incorporation. Data are expressed as mean±S.E.M. of the percentage of 3H-TdR incorporation of control or LPS alone. * P<0.05, compared with control (A) or LPS alone (B); n=5 for each group.

 
3.2 B lymphocyte proliferation in ApoE-knockout mice with hyperhomocysteinemia
To further investigate the effect of Hcy on B lymphocyte proliferation in vivo, hyperhomocysteinemia was induced after 6 weeks with a diet of methionine 400 mg/kg/day (plasma Hcy levels were 20.3±2.9 vs. 2.6±0.6 µM in control; P<0.05, n=4). Spleen B lymphocytes were incubated in the presence or absence of LPS (0.5 or 1.0 µg/ml) for 72 h. As shown in Fig. 2, the proliferation of B cells in response to LPS obtained from ApoE-knockout mice with hyperhomocysteinemia was greatly increased as compared with control (ApoE-knockout mice without methionine treatment).


Figure 2
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  		Fig. 2 B lymphocyte proliferative response in ApoE-knockout mice with hyperhomocysteinemia. ApoE-knockout mice were treated with methionine 400 mg/kg/day for 6 weeks. B lymphocytes isolated from spleen were cultured for 72 h with or without LPS (0.5 or 1.0 µg/ml). Data were expressed as mean±S.E.M. of CPM. * P<0.05, compared with control group (no methionine treatment); n=5 for each group.

 
3.3 Involvement of thiol (–SH) in the action of Hcy
To determine whether the thiol (–SH) was involved in the promotion of Hcy-induced B lymphocyte proliferation, several related compounds of Hcy including D,L-cysteine, D,L-homoserine, D,L-homocystine, D,L-methionine and glutathione were added to the cells in the presence or absence of LPS (0.5 µg/ml) for 72 h. As shown in Fig. 3A, the compounds, such as D,L-Hcy, D,L-cysteine and glutathione could increase the resting B lymphocyte proliferation to 525.7±199.2, 167.5±20.8 and 237.1±34.9%, respectively. They also could potentiate LPS-induced B lymphocyte proliferation to 778.2±127.2, 201.3±39.9 and 181.5±32.7%, respectively (Fig. 3B). In comparison with cysteine and glutathione, Hcy was three to four times more potent. However, other compounds without thiol (–SH) had no effects on B lymphocyte proliferation.


Figure 3
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  		Fig. 3 Effects of related compounds with Hcy on B lymphocyte proliferation. Each compound was added in the absence (A) or presence (B) of LPS 0.5 µg/ml for 72 h. Cell proliferation was assayed by 3H-TdR incorporation. Data are expressed as mean±S.E.M. and represented the percentage of 3H-TdR incorporation of control or LPS alone. * P<0.05, compared with control (A) or LPS alone (B); n=5 for each group.

 
3.4 Effect of Hcy on intracellular ROS production
To investigate the possibility that Hcy could induce intracellular ROS production, we measured the ROS level using the redox sensitive fluorescent dye DCFH-DA. This is a process that has been demonstrated to be dependent on intracellular production of ROS [19]. Our data showed that the peak fluorescence intensity occurred within 25 min of adding the Hcy (data not shown). Therefore, B cells were cultured with Hcy (0.3–10 mM) for 25 min in the presence or absence of LPS 0.5 µg/ml. As shown in Fig. 4, Hcy at 0.3–3.0 mM could significantly enhance resting ROS production from 11.1±1.2 to 38.1±2.2 units at Hcy 1.0 mM. In addition, Hcy (0.3 and 1.0 mM) could potentiate LPS-induced ROS production from 18.2±1.5 to 24.7±1.8 and 34.7±6.8, respectively. However, when the concentration of Hcy exceeded 3.0 mM, no significant increase of intracellular ROS was observed.


Figure 4
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  		Fig. 4 Effect of Hcy on DCF formation in B lymphocyte. Cells were incubated with Hcy in the absence (A) or presence (B) of LPS 0.5 µg/ml. DCFH-DA was added to monitor the intracellular ROS production. DCF fluorescence was monitored for 25 min. Results were means±S.E.M. of a.u. * P<0.05, compared with control (A) or LPS alone (B); n=4 for each group.

 
3.5 Effect of antioxidants on Hcy-induced B lymphocyte proliferation
To evaluate the role of oxygen radicals in Hcy-induced B lymphocyte proliferation, cells were pretreated for 30 min with catalase 250 µg/ml (a scavenger of H2O2) or 1% DMSO (a scavenger of OH·) or SOD 500 U/ml (a scavenger of O2–) and then stimulated by Hcy 1.0 mM with or without LPS 0.5 µg/ml for 72 h. As demonstrated in Fig. 5, these scavengers of oxygen radicals depressed Hcy-induced B lymphocyte proliferation from 606.5±191.3 to 217.3±35.1, 179.5±45.1 and 128.3±37.1%, respectively. Potentiation of LPS-induced B lymphocyte proliferation by Hcy was also inhibited by these antioxidants, from 5147.9±1454.8 to 1135.8±648.2, 1387.9±589.6 and 104.9±21.3%, respectively.


Figure 5
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  		Fig. 5 Inhibitory effect of antioxidants on Hcy-induced B lymphocyte proliferation. Cells were pretreated with antioxidants, catalase 250 µg/ml, 1% DMSO or SOD 500 U/ml for 30 min and then stimulated with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 72 h. Cell proliferation was assayed by 3H-TdR incorporation. Data were expressed as means±S.E.M. and represented the percentage of 3H-TdR incorporation of control. * P<0.05, compared with LPS alone; # P<0.05, compared with Hcy alone; + P<0.05, compared with LPS+Hcy; n=5 for each group.

 
3.6 Role of PKC in Hcy-induced B lymphocyte proliferation
To define whether the activation of PKC could contribute to Hcy-induced B lymphocyte proliferation, the splenic cells were pretreated for 30 min with the PKC inhibitors, calphostin C 500 nM and RO-31-8220 100 nM before stimulation with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 72 h. As shown in Fig. 6, both calphostin C and RO-31-8220 decreased LPS-induced B lymphocyte proliferation from 343.7±87.8 to 129.5±17.0 and 265.6±58.9%, respectively, and depressed Hcy-induced B lymphocyte proliferation from 293.6±37.8 to 108.1±16.5 and 201.8±39.9%, respectively. The proliferation induced by LPS plus Hcy was reduced by 91 and 83%, respectively.


Figure 6
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  		Fig. 6 Role of PKC in Hcy-induced B lymphocyte proliferation. Cells were pretreated with calphostin C (Cal, 500 nM) or RO-31-8220 (RO, 100 nM) for 30 min and then stimulated with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 72 h. Cell proliferation was assayed by 3H-TdR incorporation. Data were expressed as means±S.E.M. of the percentage of 3H-TdR incorporation of control. * P<0.05, compared with LPS alone; # P<0.05, compared with Hcy alone; + P<0.05, compared with LPS+Hcy; n=4 for each group.

 
3.7 Effects of PKC inhibitors on intracellular ROS production induced by Hcy
To further strengthen the correlation between the redox state and PKC activation, we pretreated the cells with calphostin C 500 nM or RO-31-8220 100 nM for 30 min. Intracellular ROS levels were measured following stimulation with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 25 min. Treatment with calphostin C or RO-31-8220 had no effect on intracellular ROS production induced by Hcy (data not shown).

3.8 Role of MAPK in Hcy-induced B lymphocyte proliferation
First, we determined whether p38 MAPK, one isoform of MAPK, was involved in the signaling mechanisms that regulate B cell proliferation upon Hcy activation. Cells were pretreated with two specific inhibitors of p38 MAPK, SB 202190 (5.0 µM) and PD 169316 (1.0 µM) for 30 min, following stimulation with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 72 h. As could been seen in Fig. 7A, these inhibitors not only reduced LPS-induced B lymphocyte proliferation from 236.3±68.8 to 134.5±35.7 and 130.5±34.1%, respectively, but also decreased Hcy alone induced B lymphocyte proliferation from 303.6±75.2 to 172±42.5 and 129.5±27.9%, respectively. The potentiation of Hcy on LPS-induced B lymphocyte proliferation was inhibited by 48 and 33%, respectively.


Figure 7
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  		Fig. 7 Roles of p38 MAPK (A) and ERK1/2 (B) in Hcy-induced B lymphocyte proliferation. Cells were pretreated with SB 202190 5.0 µM or PD 169316 1.0 µM or PD 98059 (2 or 20 µM) for 30 min, then stimulated with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 72 h. Cell proliferation was assayed by 3H-TdR incorporation. Data were expressed as means±S.E.M. of the percentage of 3H-TdR incorporation of control. * P<0.05, compared with LPS alone; # P<0.05, compared with Hcy alone; + P<0.05, compared with LPS+Hcy; n=5 for each group.

 
We next assessed whether ERK1/2, another isoform of MAPK, contributed to the potentiation of Hcy-induced B lymphocyte proliferation. Cells were pretreated with PD 98059 (2 or 20 µM), a specific inhibitor of ERK1/2, for 30 min before stimulation with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 72 h. Data showed that PD 98059 had no inhibitory effects on B lymphocyte proliferation induced by Hcy, LPS or LPS plus Hcy (Fig. 7B).

3.9 Role of NF-{kappa}B in Hcy-induced B lymphocyte proliferation
To determine whether NF-{kappa}B transcription factor was involved in Hcy-induced B lymphocyte proliferation, cells were pretreated for 30 min with the inhibitor of NF-{kappa}B, PDTC (0.1–3.0 µM) and then stimulated with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 72 h. As shown in Fig. 8, the addition of PDTC could concentration-dependently decrease LPS- or Hcy-induced B lymphocyte proliferation. Potentiation of LPS-induced B lymphocyte proliferation by Hcy was also reduced.


Figure 8
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  		Fig. 8 Role of NF-{kappa}B in Hcy-induced B lymphocyte proliferation. Cells were pretreated with PDTC (0.1–3.0 µM) for 30 min and then stimulated with Hcy 1.0 mM in the presence or absence of LPS 0.5 µg/ml for 72 h. Cell proliferation was assayed by 3H-TdR incorporation. Data were expressed as means±S.E.M. of the percentage of 3H-TdR incorporation of control. * P<0.05, compared with LPS alone; # P<0.05, compared with Hcy alone; + P<0.05, compared with LPS+Hcy; n=4 for each group.

 
3.10 Effect of Hcy on LDH release from B lymphocyte
To determine the effect of Hcy on LDH release, cells were incubated with Hcy at 0.3–10 mM for 72 h. Hcy (3.0–10 mM) significantly caused the increase in LDH release as compared with control (Fig. 9).


Figure 9
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  		Fig. 9 Effect of Hcy on the release of LDH production in B lymphocyte. Cells were treated with Hcy (0.3–10 mM) for 72 h. The suspensions were collected for determination of LDH levels by spectrophotometric enzyme activity method. The data were expressed as mean±S.E.M. * P<0.05, compared with control; n=5 for each group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Our results clearly demonstrate for the first time that Hcy not only promotes resting B lymphocyte proliferation, but also potentiates LPS-induced B lymphocyte proliferation. ApoE-knockout mice with hyperhomocysteinemia cause an enhancing susceptibility of B cell proliferation comparing to non-hyperhomocysteinemia group. Related compounds with thiol (–SH), such as cysteine, glutathione can also promote B lymphocyte proliferation. The effects of Hcy can be significantly reduced by scavengers of ROS and by the inhibitors of PKC, p38 MAPK and NF-{kappa}B transcription factor, suggesting that ROS such as O2–, OH· and H2O2 generated by thiol (–SH) autoxidation and PKC, p38 MAPK and NF-{kappa}B pathways are involved in Hcy-induced B lymphocyte proliferation. Hyperhomocysteinemia may be involved in the pathogenesis of atherosclerosis by modulating B cell activity.

The mechanisms that Hcy induces the development of atherosclerosis are not fully understood. Previous studies have focused on the injury of endothelial cells and proliferation of vascular smooth muscle cells by Hcy [3,4]. Increasing evidences suggest that humoral and cellular immune reactions are involved in all stages of atherogenesis [20]. B cells and significant amounts of Ig-secreting plasma cells are found in the developing atherosclerotic plaques [21]. CD22+ B cells and abundant IgM are contained in atherosclerotic plaques of hypercholesterolaemic ApoE-knockout mice [22]. Ig-secreting B cells, immunoglobulins and C5b–9 terminal complexes resulting from the activation of the complement system have all been reported to be detected in human atherotic lesions [23]. It is also reported that in the initial stages, inflammatory infiltrates composed of macrophages, T cells, dentritic cells, mast cells, but B cells and plasma cells only occasionally, in the intimal plaque. In chronic advanced stages of atherosclerosis, inflammatory infiltrates are often present in the adventitia adjacent to plaques that the majority of cells in these infiltrates are B cells rather than T cells [24,25] and that selection of specific B cell subsets takes place [26]. Thus, it increases with the severity of atherosclerotic plaque formation. However, effects of Hcy on B lymphocyte proliferation have never been defined. In the present study, we show that Hcy can not only activate resting B lymphocyte proliferation, but also act as a modulator to potentiate LPS-induced B lymphocyte proliferation. In addition, by assessing IgG levels, we recently observed that Hcy elevated resting and 0.5 µg/ml LPS-induced IgG production from B lymphocyte of these mice (unpublished observation). Interestingly, we demonstrate that ApoE-knockout mice with hyperhomocysteinemia significantly increase susceptibility of B cell proliferation in the presence of LPS. Therefore, we postulate that elevated Hcy in vivo might be involved in activated B lymphocyte in the immune response of atherosclerosis.

Thiol (–SH) groups participate in the regulation of lymphocyte proliferation [27]. In another study, we demonstrated that thiol (–SH) group was involved in Hcy-potentiated T lymphocyte proliferation induced by T cell mitogen (paper submitted for publication). In the present study, we showed that the related compounds with thiol (–SH) such as cysteine, glutathione not only activated resting B lymphocyte proliferation but also potentiated LPS-induced B lymphocyte proliferation (Table 1). Other compounds without thiol (–SH) had no such effects. This suggests that thiol (–SH) plays a key role in the action of Hcy. In addition, we observed that the effect of Hcy was the strongest as compared with cysteine and glutathione. This difference might be due to its chemical property. The ammonium group of cysteine (HSCH2CHNH2COOH) exerts a strong electron withdrawing effect on the thiol group. However, with Hcy (HSCH2CH2CHNH2COOH), due to its having one more methylene group than cysteine, the withdrawing effect of the ammonium group on the thiol group is weaker. Therefore, the thiol group of Hcy is more active and easier to be autoxidated than that of cysteine.


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  		Table 1 Effects of Hcy and related compounds on B lymphocyte proliferation

 
Growing evidence supports the concept that low concentrations of ROS play some physiological roles in activation and proliferation of lymphocytes [28]. To clarify the mechanism that ROS acts as a mediator in Hcy-induced B lymphocyte proliferation, we first assessed the production of intracellular ROS using DCFH-DA. We found that 0.3–1.0 mM Hcy, the concentration which enhanced B lymphocyte proliferation, could increase the intracellular oxidation of DCFH, which is produced equally by the intracellular superoxide anion (O2–), hydroxyl ion (OH·) or hydrogen peroxide (H2O2) [18]. However, when the concentration of Hcy exceeded 3.0 mmol/l, it could not further increase the level of intracellular ROS. This might be due to its toxic effect on the cells as determined by LDH release. In addition, the potentiating effect was prevented by pretreatment with different antioxidants that are well known as radical scavengers. For example, SOD catalyses the dismutation of superoxide anion radicals, catalase catalyses the reduction of hydrogen peroxide and 1% DMSO scavenges hydroxyl radicals (OH·). Treatment with antioxidants attenuates Hcy-induced B lymphocyte proliferation, suggesting that the production of intracellular ROS induced by thiol (–SH) autoxidation is involved in the effect of Hcy-induced B lymphocyte proliferation. The types of ROS include superoxide anion (O2–), hydroxyl ion (OH·) and hydrogen peroxide (H2O2).

PKC plays a critical role in signal transduction pathways leading to a variety of cellular functions, such as cell growth and differentiation. Our results clearly demonstrated that the two PKC selective inhibitors blocked Hcy-induced B lymphocyte proliferation, suggesting a necessary role for the PKC pathway during the Hcy induced B lymphocyte proliferation. However, the addition of PKC inhibitors had no effect on the intracellular ROS production, suggesting that ROS production does not require PKC activation.

At least three types of MAP kinase have been described in mammalian cells: extracellular regulated kinase (ERK), the c-terminal jun kinase (JNK), and the p38 MAP kinase. ERK corresponds to the classical mitogen-activated kinase, whose activity is induced by mitogenic stimuli. The two other types of kinases, JNK and p38 MAP were originally found to be activated by a variety of stimuli associated with cellular stress, inflammatory cytokines or LPS [29,30]. In the present study, the specific inhibitors of p38 MAPK activity (PD 169316 and SB 202190) blocked Hcy-induced responses, suggesting that the p38 MAPK is a critical pathway in the action of Hcy. However, the proliferation did not require ERK activation.

NF-{kappa}B, a redox-sensitive transcription factor, can rapidly activate the expression of genes involved in inflammatory immune and acute phase responses. Potent inducers of NF-{kappa}B include cytokines, LPS, oxygen free radicals, etc [31]. Upon oxidant stimulation, the inhibitory subunit (I{kappa}B) is released, allowing migration of the heterodimer to the nucleus where it presumably binds to DNA and increases transcription of several stimuli-responsive genes [32]. In the present study, we have investigated the effect of the NF-{kappa}B inhibitor, PDTC, on the proliferation induced by Hcy, LPS, and LPS plus Hcy. Our results indicated that Hcy and/or LPS-induced B cell activation, were highly sensitive to NF-{kappa}B inhibitor, suggesting the involvement of NF-{kappa}B pathway in Hcy activation. A recent study has demonstrated ROS effect on NF-{kappa}B translocation [33]. The increased ROS production induced by Hcy may be partially responsible for the NF-{kappa}B activation.

Hcy concentrations of 0.2–0.25 mM appear to be associated with atherosclerosis, and plasma concentration up to 0.5 mM has been found in patients who suffer from homocysteinuria [34]. In a local plaque, the concentration of Hcy is still unknown; it may be higher than that of plasma. The exact effect of Hcy on B lymphocyte function in atherosclerosis plaque in vivo is unknown. The roles of Hcy-activated B cells in animals and patients atherosclerosis are currently investigated in our laboratory.

In conclusion, this study has demonstrated that Hcy significantly potentiated resting and LPS-induced proliferation in splenic B lymphocyte. In ApoE-knockout mice, hyperhomocysteinemia in vivo caused a significant promotion of B lymphocyte proliferative response. The thiol (–SH) group of Hcy and its oxidative products were involved in the action of Hcy. Furthermore, our data indicated the signaling mechanism for the activation of B lymphocyte, involving protein kinase C, p38 MAPK and NF-{kappa}B transcription factor. Elucidation of the effect of Hcy on B lymphocyte proliferation might be helpful explaining the mechanisms of homocysteinemia-induced atherosclerosis.

Time for primary review 25 days.


   Acknowledgements
 
This research project was supported by Major National Basic Research Program of People’s Republic of China (No. G2000056908) and a grant from the National Natural Science Foundation of China awarded to XW. The authors thank Professor Robert D. Wurster, Department of Physiology, Loyola University Medical Center in Chicago, for his revision of this paper.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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