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© 2006 American Society for Nutrition J. Nutr. 136:2384-2390, September 2006

Nutritional Immunology

Soy Isoflavones Attenuate Human Monocyte Adhesion to Endothelial Cell–Specific CD54 by Inhibiting Monocyte CD11a1

Shanmugam Nagarajan2,3,*, Bradford W. Stewart2 and Thomas M. Badger2,4

2 Arkansas Children's Nutrition Center, 3 Department of Microbiology and Immunology, and 4 Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR

* To whom correspondence should be addressed. E-mail: nagarajanshanmugam{at}uams.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Soy-based diets have been shown to protect against the development of atherosclerosis; however, the underlying mechanism(s) remain unknown. Interaction between activated monocytes and inflamed endothelial cells is an early event in atherogenesis. Therefore, we examined whether treatment of monocytes with soy phytochemicals could inhibit their adhesion to the endothelial cell–specific protein, CD54, a key factor in monocyte adhesion. Female Sprague-Dawley rats were fed AIN-93G diets containing soy protein isolate or casein. Sera from soy-fed rats inhibited CD54-dependent monocyte adhesion, whereas sera from casein-fed rats did not. To determine whether isoflavones in the sera of soy-fed rats were involved in this inhibition, monocytes were preincubated with soy isoflavones. Isoflavone treatment inhibited monocyte adhesion to CD54 protein, as well as to endothelial cells expressing CD54. Monocyte expression of CD11a, the cognate receptor for CD54, was unaffected by isoflavones. However, binding of the activation epitope–specific antibody mAb24, which binds specifically to the active form of CD11a, was significantly lower in soy isoflavone–treated monocytes than in media-treated cells. These findings suggest that inhibition of CD54-dependent monocyte adhesion by soy isoflavones is mediated in part by affinity regulation of CD11a. Inhibition of monocyte adhesion to endothelial cells by isoflavones resulted in reduced expression of the inflammatory cytokines IL-6 and IL-8. Collectively, these data suggest that the athero-protective effect of soy diets may be mediated by blocking monocyte-endothelial cell interaction.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Studies in humans have demonstrated that soy protein intake reduces serum cholesterol concentration, which is the major risk factor for atherosclerosis (1–3). Serum cholesterol levels and atherosclerotic lesions were also lower in monkeys fed soy protein–based diets than in those fed casein-based diets (4,5). In vitro studies using soy phytochemicals have suggested that the reduction in cholesterol levels may be attributed to the positive regulation of genes involved in cholesterol homeostasis, such as sterol regulatory element binding protein-2 (6). However, using the apolipoprotein E knockout (apoE–/–)5 mouse, preliminary findings from our study (7) and earlier reports (8,9) showed that atherosclerotic lesion sizes were reduced in animals fed the soy-based diet, despite unchanged serum lipid levels. These studies suggest that dietary soy can reduce atherosclerotic lesion development by mechanisms other than lowering serum cholesterol levels. However, the mechanism(s) and component(s) of soy responsible for these effects remain unknown.

Recent studies have demonstrated that atherosclerosis is a chronic inflammatory disease (10,11). Cell-cell interaction between circulating monocytes and endothelial cells is the primary event in the initiation of atherosclerosis (10,11). This interaction is mediated by endothelial cell–specific adhesion molecules intercellular adhesion molecule-1 (ICAM-1 or CD54) and vascular cell adhesion molecule-1 (VCAM-1 or CD106) binding to integrins (CD11a or CD49d) expressed on monocytes. Studies using gene knockout mice have demonstrated the causal relation between adhesion of monocytes to endothelial cells during the inflammatory process in atherosclerosis (12,13). Importantly, despite having hypercholesterolemic conditions, double knockout mice lacking the expression of apoE (apoE–/–) and cell adhesion molecules, such as CD54 or CD106, have been shown to have reduced numbers of atherosclerotic lesions; this highlights the critical role of cell adhesion molecules in the initiation and progression of atherogenesis (12,13).

Endothelial cells treated with the soy isoflavone genistein inhibited monocyte adhesion, suggesting that the athero-protective effect of soy isoflavones could be mediated by regulating endothelial cell functions (14). Similarly, isoflavones such as genistein and daidzein, which are present in soy-based diets, have been shown to inhibit agonist-induced platelet aggregation (15). These reports suggest that modulation of interaction between inflammatory and endothelial cells may be a plausible mechanism for the beneficial effects of soy-based diets. However, to date there are no data in the literature on the effect of soy isoflavones on monocytes. Because interaction between the endothelial cell–specific protein CD54 and integrin CD11a expressed on monocytes play a key role in atherosclerosis, we hypothesized that the athero-protective effect of soy-based diets could be mediated by regulating the expression and functions of CD11a expressed on monocytes. Specifically, this study investigated whether soy isoflavones inhibit CD11a-mediated monocyte adhesion to endothelial cells.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
Cell culture

Human umbilical vein endothelial cells (HUVECs), purchased from Cambrex, were cultured in 0.2% gelatin-coated 25-cm2 flasks in endothelial cell medium EGM-2, supplemented with 2% fetal bovine serum (FBS) and growth factors and antibiotics present in SingleQuot kit (Cambrex). Human monocytic (U937) and T lymphocytic (Jurkat) cell lines were obtained from the American Type Culture Collection and cultured in DMEM-F12 medium supplemented with 10% fetal clone III (Hyclone) as described earlier (16).

PBMC isolation

Human peripheral blood mononuclear cells (PBMC) were isolated from blood samples by 6% dextran T500 sedimentation followed by lymphocyte separation medium 1.077 (Mediatech) density gradient centrifugation (17). PBMC were resuspended in EGM-2 medium containing 0.5% FBS. Protocols for blood collection from normal volunteers were approved by the Institutional Review Board at the University of Arkansas for Medical Sciences.

Antibodies and recombinant proteins

Murine hybridoma secreting monoclonal antibody (mAb) against human CD11a (TS1/22) was cultured in RPMI 1640 supplemented with 10% FBS (Mediatech). Mouse anti-human CD36 (FA6.152) and anti-CD106 mAb (1G11) were purchased from Beckman-Coulter, and anti-CD54 (LB-2) and CD11b (ICRF44) were purchased from BD-Biosciences. The anti-LFA-1 mAb (mAb24), which is specific for an activation epitope of CD11a, has been described previously (18) and was a kind gift from Dr. Nancy Hogg (Cancer Research UK, London Research Institute, London, UK). Unconjugated and conjugated secondary antibodies and streptavidin-HRP used in this study were purchased from Jackson Immunochemicals. Recombinant soluble human CD54-Ig was purchased from R&D systems.

Isoflavones

To obtain sera containing soy phytochemicals, female Sprague-Dawley rats (Harlan) were weaned to semipurified isocaloric diets (Harlan-Teklad), made according to the AIN-93G formulation (19), except that the sole protein sources were either casein or soy protein isolate as described previously (20). At postnatal d 50, rats were killed by decapitation, trunk blood was collected, and serum was stored at –20°C. Randomly pooled sera from 4 rats fed casein (Cas-sera) or soy protein isolate (Soy-sera) diets were used. The animal experiment described in this report was approved by the University of Arkansas for Medical Sciences Animal Care and Use Committee. Soy isoflavones, genistein, daidzein, and equol (LC labs) were mixed at indicated concentrations in appropriate buffers just before use. Because immune cells in vivo are exposed to a combination of isoflavones, we studied a mixture of soy isoflavones. All 3 soy isoflavones were mixed at equimolar concentrations before use.

OxLDL preparation

Oxidized-LDL (OxLDL) was either purchased from BTI or prepared by incubating LDL (200 mg/L in PBS) with 5 µmol/L freshly prepared CuSO4 at 37°C as previously described (16). The extent of oxidation was assessed by measuring TBARS, and the increase in electrophorectic mobility of oxLDL compared with nLDL was determined using TITAN agarose gel electrophoresis (Helena Labs) (16). OxLDL was extensively dialyzed against PBS and then against serum-free and phenol red-free RPMI prior to its use in the cell activation experiments. Protein estimation was performed using the BCA protein assay kit (Pierce) with bovine serum albumin (BSA) as the standard.

Monocyte activation

To determine the effect of isoflavones on monocyte adhesion, U937 cells (1 x 109/L in RPMI/0.3% BSA) were preincubated with soy isoflavones for 1 h at 37°C, followed by incubation with PMA (0.1 mg/L) or oxLDL (20 mg/L, unless indicated) for indicated times at 37°C in a CO2 incubator. Cell viability, determined by tyrpan blue exclusion, were >98% after treatment. After treatment, cells were used to determine the CD11a, CD11b, and CD36 expression by flow cytometric or in cell adhesion assays.

Adhesion assays

    Monocyte adhesion to CD54 or oxLDL. CD54-dependent monocyte adhesion assays were performed in a 96-well ELISA plate (Dynex) and captured with CD54-Ig using fluorescent-labeled cells as described earlier (16). Fluorescent intensity was measured before and after inversion using a BioTek Synergy plate reader with a 485-nm excitation/530-nm emission filter. Media-treated cells without isoflavones were used as controls. A standard curve was generated using a known number of labeled cells to calculate the number of cells that adhered to the plates. The percentage of cell adhesion was calculated as follows: (mean fluorescence after inversion/mean fluorescence before inversion) x 100. The percentage of inhibition was calculated as follows: [(mean fluorescence of control cells – mean fluorescence of isoflavone-treated cells)/mean fluorescence of control cells] x 100. Background fluorescence was subtracted from each sample reading. Monocyte adhesion to oxLDL was performed as described above, except that oxLDL-coated plates (50 µL of 5 mg/L in borate buffer/10 mmol/L EDTA) were used. Additional controls included BSA-coated wells. The specific CD54-dependent U937 cell adhesion was obtained after subtracting the adhesion to the BSA-coated wells.

    Monocyte adhesion to endothelial cells. HUVECs were seeded (105/well) onto 0.2% gelatin-coated 96-well plates (Costar) 24 h prior to the experiment and incubated with TNF-{alpha} (0.01 mg/L) for 18 h (21) prior to the adhesion assay. Cell surface expression of CD54 and CD106 on HUVECs was determined by a modified cell ELISA method described previously, using anti-CD54 or anti-CD106 mAbs at 2 mg/L (22). Fluorescent-labeled U937 cells were activated with PMA in the absence or presence of soy isoflavones. To determine CD54-dependent adhesion, adhesion of fluorescent-labeled U937 cells to the HUVECs in the absence or presence of blocking mAb against CD54 or CD106 was performed as described above. In all adhesion assays, triplicate wells were run per condition, and results were expressed as mean ± SD of 3 independent experiments.

Cytokine assays

HUVECs (1 x104 cells/well) were plated on 0.2% gelatin-coated 96-well plates, cultured for 48 h, and fixed in buffered formalin/0.5% BSA for 1 h at room temperature. After washing to remove formalin, HUVECs were cocultured for 18 h with PBMC (1 x 105 cells/well) and treated with or without soy isoflavones at indicated concentrations in the presence of PMA (0.1 mg/L). Supernatants collected from HUVECs-PBMC coculture were analyzed for the secretion of pro-inflammatory cytokines, using a human inflammation panel Cytokine Bead Array kit from BD Biosciences, according to manufacturer's instruction, in a FACSCalibur flow cytometer (BD-Biosciences). Identically treated HUVECs or PBMC alone were used as controls.

Flow cytometric analysis

Cell surface expression of CD11a, CD11b and CD36 were analyzed in a FACSCalibur flow cytometer (Becton Dickinson) using corresponding mAbs (5 mg/L), followed by saturating concentrations of FITC-conjugated F(ab')2-goat anti-mouse IgG+IgM (16). The activation-dependent anti-CD11a mAb24 binding to monocytes were determined as described earlier (23). U937 cells (1 x 109/L in complete DMEM-F12 medium) were treated with the isoflavones (10 or 30 µmol/L; equimolar concentration of genistein, daidzein and equol) for 16 h. Cells treated identically in media without isoflavones were used as a control. Cells were stained with anti-CD11a mAb (TS1/22 or mAb24) at 2 mg/L, followed by saturating concentrations of donkey anti-mouse IgG-PE. Fluorescence intensity was determined in a FACSCalibur flow cytometer. Fluorescent intensity of cells treated similarly without isoflavones was taken as 100% for TS1/22 or mAb24 antibody binding.

Statistical analysis

All tests were run in triplicate for each experimental condition and each experiment was repeated 2 or 3 times, as indicated in the figure legend. Data are expressed as means ± SD. Differences among means were tested for statistical significance by 1-way or 2-way ANOVA and Tukey's test was used for post-hoc comparisons. All analyses were carried out with SigmaStat 9 program (Jandel Corporation). Differences with P ≤ 0.05 were considered significant.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    OxLDL induces monocyte adhesion to endothelial cell adhesion molecule, CD54. Unactivated U937 cells (in medium) minimally adhered to CD54-Ig-coated plates, whereas activating monocytes with PMA increased monocyte adhesion (P < 0.001) to CD54-coated plates (Fig. 1A). Under similar conditions, no adhesion was seen in the control CD16A-Ig-coated wells, indicating monocyte adhesion to CD54-Ig is specific. U937 cells treated with oxLDL also showed a dose-dependent increase in monocyte adhesion to CD54-Ig coated plates (Fig. 1B). OxLDL at 10 mg/L showed 5% of monocytes adhere to CD54-coated plates, which was increased to 50% at 40 mg/L oxLDL (Fig. 1B). However, cells treated with nLDL or medium alone showed minimal adhesion (data not shown). OxLDL treatment of U937 cells showed a time-dependent increase in cell adhesion to CD54 (Fig. 1C). PMA- or oxLDL-induced enhanced CD54-dependent monocyte adhesion could be a result of the increase in the expression of CD11a or CD11b (cognate receptor for CD54) in U937 cells. However, flow cytometric analyses of PMA- or oxLDL-treated cells showed no significant changes in the CD11a or CD11b (data not shown).


Figure 1
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  		Figure 1  Prior activation of monocytes is necessary for CD54-dependent U937 cell adhesion. PMA-induced adhesion of U937 cells to CD54 (A). Fluorescent-labeled U937 cells were incubated with media or PMA. CD16A-Ig captured wells were used as a negative control. OxLDL pretreatment induces monocyte adhesion to CD54 (B). U937 cells were incubated with oxLDL at different concentrations. Cells incubated in medium (without oxLDL) or nLDL were used as negative controls. Kinetics of oxLDL-induced U937 cell adhesion to CD54 (C). U937 cells were treated with oxLDL at different time periods. Values are means ± SD, n = 9 (3 independent experiments performed in triplicate). Comparisons of means were made within media or PMA. Means without a common letter differ, P ≤ 0.05.

 
    Sera from soy diet fed rats inhibit CD54-dependent monocyte adhesion. We then investigated whether sera from soy protein–isolate fed rats could inhibit monocyte activation and subsequent adhesion. Sera from rats fed soy protein isolate (soy-sera) inhibited oxLDL-induced CD54-dependent monocyte adhesion, whereas sera from casein-fed rats (cas-sera) did not inhibit oxLDL-induced CD54-dependent monocyte adhesion (Table 1). Further, inhibition of oxLDL-induced CD54-dependent U937 cell adhesion was 2- to 6-fold greater in cells treated with soy-sera (another pool of sera from 4 rats fed soy-based diets) than in cells treated with cas-sera (Fig. 2).


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  		TABLE 1 Sera from soy diet–fed rats inhibit monocyte adhesion to CD541

 

Figure 2
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  		Figure 2  Sera from rats fed a soy-based diet inhibit CD54-dependent monocyte adhesion. U937 cells treated with oxLDL in the presence of different concentrations of soy-sera or cas-Sera were used for CD54-dependent adhesion assays. Values are means ± SD, n = 6 (2 independent experiments performed in triplicate). Means without a common letter differ, P ≤ 0.05.

 
    Soy isoflavones inhibit monocyte adhesion to CD54. We then determined whether the inhibition of monocyte adhesion is mediated by soy isoflavones, the primary phytochemicals present in sera after soy-based diet consumption. Preincubation of U937 cells with soy isoflavones resulted in a dose-dependent inhibition of U937 cell adhesion to CD54 (Fig. 3). There were 10, 12, and 18% inhibition of PMA-activated U937 cell adhesion to CD54 at 0.1, 1, and 3 µmol/L soy isoflavones, respectively (Fig. 3). Of the cells, >50% did not adhere to CD54-coated plates at 10 and 30 µmol/L concentrations of isoflavones (Fig. 3). About 10% inhibition of oxLDL-treated monocyte adhesion was seen on a short-term exposure (45 min) to soy isoflavones. Long-term (16 h) exposure of monocytes with isoflavones showed >80% inhibition of oxLDL-induced monocyte adhesion to CD54 (data not shown). Flow cytometric analysis of U937 cells treated with oxLDL in the presence of soy isoflavones showed no significant change in the expression of CD36, a receptor that binds to oxLDL (data not shown). Analyses for lectin-like oxidized-LDL binding protein-1 (LOX-1), another oxLDL binding protein, showed that LOX-1 was undetectable in untreated and oxLDL-treated cells in the presence or absence of soy-isoflavones (data not shown). Isoflavone treatment of monocytes also did not affect oxLDL binding to CD36 expressed on monocytes (data not shown).


Figure 3
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  		Figure 3  Soy isoflavones inhibit CD54-dependent U937 cell adhesion. Cells were incubated with variable concentrations of soy isoflavones (equimolar mixture of genistein, daidzein, and equol). Cell adhesion to CD54-Ig-coated plates in the presence of PMA was determined. Cells incubated without isoflavones were treated as a control. Values are means ± SD, n = 9 (3 independent experiments performed in triplicate). Means without a common letter differ, P ≤ 0.05.

 
    Soy isoflavones also inhibit monocyte adhesion to endothelial cells. HUVECs were used to extrapolate these findings to the soy isoflavones' role in inhibition of cell-cell interaction between monocytes and endothelial cells. HUVECs showed low-level expression of CD54 and CD106, 2 key endothelial cell adhesion molecules (data not shown). However, after overnight treatment with TNF-{alpha}, HUVECs showed ~6- and 2-fold increases in CD54 and CD106 expression, respectively (data not shown). The effect of isoflavones on CD54-dependent U937 adhesion to endothelial cells was then determined. U937 cells treated with as low as 1 µmol/L isoflavones showed 33% inhibition of CD54-dependent adhesion to vascular endothelial cells (Table 2). Inhibition of CD54-dependent U937 adhesion to endothelial cells increased with greater concentrations of soy isoflavones (Table 2).


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  		TABLE 2 Soy isoflavones inhibit PMA-induced U937 cell adhesion to endothelial cells1

 
    Soy isoflavones inhibit monocyte CD11a function. We sought to address whether inhibition of monocyte adhesion to CD54 by isoflavones is mediated via blocking the affinity regulation of CD11a (23–25). Affinity regulation of CD11a was determined by activation-dependent anti-CD11a mAb24 binding (18). Soy-isoflavone treatment of U937 cells did not affect activation-independent anti-CD11a mAb, TS1/22 binding (data not shown). However, when U937 cells were pretreated with isoflavones, the activation-dependent mAb24 binding was significantly inhibited by 10% and 30% at 10 and 30 µmol/L isoflavone concentrations, respectively, in comparison to media-treated cells (Table 3). Soy isoflavones also inhibited mAb24 binding to Jurkat (a human T cell line) cells (Table 3) without affecting TS1/22 binding (data not shown).


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  		TABLE 3 Soy isoflavones inhibit function of CD11a expressed on monocytic and lymphocytic cells1

 
    Secretion of IL-6 and IL-8 by PBMC is inhibited by soy isoflavones. Endothelial cells or PBMC alone showed very minimal secretion of IL-6 (Fig. 4A) and IL-8 (Fig. 4B). However, interaction of human PBMC with endothelial cells resulted in elevated IL-6 and IL-8 secretion (Fig. 4). Addition of soy isoflavones to PBMC and endothelial cell coculture resulted in a dose-dependent inhibition of IL-6 and IL-8 secretion (Fig. 4). Soy isoflavones at 1 µmol/L showed 25% inhibition, and >90% inhibition was seen at 30 µmol/L soy isoflavones (Fig. 4).


Figure 4
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  		Figure 4  PBMC-endothelial cell interaction–dependent secretion of IL-6 and IL-8 is inhibited by soy isoflavones. HUVECs were cocultured with PBMC treated with or without (EC+PBMC) soy isoflavones at different concentrations. Supernatants collected from HUVECs-PBMC coculture (EC+PBMC) were analyzed for the secretion IL-6 (A) and IL-8 (B), using human cytokine bead array. HUVECs (EC) or PBMC alone were used as controls. Values are means ± SD, n = 6 (2 independent experiments performed in triplicate). Means without a common letter differ, P ≤ 0.05.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The athero-protective effects of soy-based diets have been attributed to its effect in reducing serum cholesterol levels in humans (1–3). Similar findings have also been reported in monkeys fed soy-based diets compared with those fed control diets (4,5). However, recent studies in the atherosclerosis-susceptible apoE knockout mouse showed that atherosclerotic lesions are reduced, despite unchanged serum lipid levels (7–9). These data suggest that additional mechanism(s) may contribute to the athero-protective effects of soy-based diets. As interactions between endothelial and inflammatory cells play a pivotal role in the initiation and progression of atherogenesis (12,13,26), modulation of these interactions could be a basis for the beneficial effects of soy-based diets. In this article, we show that preexposure to soy isoflavones inhibits monocyte adhesion to endothelial cells through the endothelial cell adhesion molecule CD54. This inhibition by isoflavones also leads to lesser inflammatory cytokine production by monocytes. Furthermore, the mechanism by which soy isoflavones block monocyte adhesion is through inhibition of CD11a affinity to CD54.

The main indicator of initiation of atherogenesis is the adhesion of activated-monocytes to inflamed-vascular endothelium (10,11). Soy feeding has been shown to increase the soy phytochemicals in serum (27,28), suggesting a possibility that the athero-protective effect of soy isoflavones could be mediated by regulating the functions of 2 key cellular components of atherosclerosis, the endothelial and inflammatory cells. Indeed, our findings show that preexposure of monocytes to soy-sera or soy isoflavones inhibited their adhesion to recombinant CD54 protein and CD54+ endothelial cells. During the inflammatory process, interaction between circulating leukocytes and endothelium has been shown to result in secretion of pro-inflammatory cytokines (29,30). Coculture of PBMC with endothelial cells resulted in increased secretion of pro-inflammatory cytokines, such as IL-6 and IL-8, and addition of soy isoflavones to the monocyte/endothelial cell coculture inhibited the secretion of these pro-inflammatory cytokines. These findings suggest that soy isoflavones not only affect the inflammatory cell adhesion to endothelial cell, but they also inhibit endothelial cell-monocyte interaction-dependent, pro-inflammatory cytokine responses. Although the effect of isoflavones on endothelial cells has been reported (14,15), the effect of soy isoflavones on the monocytes and its adhesive functions has not been studied. This is, to our knowledge, the first report to directly assess the role of isoflavones on monocytes.

Soy isoflavones circulate in several molecular forms, including glucuronide and sulfatide conjugates, free aglycones, and protein-bound aglycones (27,31,32). Because the aglycones have been shown to influence cellular metabolism, such as cholesterol homeostasis (6), it has been widely assumed that the aglycones are biologically active molecules. We chose to study a mixture of isoflavone aglycones (genistein, daidzein, and equol) because these are the major soy isoflavones in the circulation and the target tissues of rats fed soy-based diets. Of particular interest is equol, because equol is among the most potent isoflavones (in terms of estrogen receptor binding) and is present in very high concentrations in rats (27) and monkeys fed soy-based diets (28,33). It is interesting to note that much of the previous work in defining the role of soy-based diets on prevention of atherosclerosis have been performed in monkey model (4,5).

We show that although >50% inhibition of monocyte adhesion to endothelial cell adhesion molecule CD54 and endothelial cells was seen at high isoflavone concentrations (30 µmol/L), significant inhibition of monocyte was observed at a physiological concentration of 1 µmol/L isoflavones (a combination of 0.33 µmol/L each of genistein, daidzein, and equol). We recognize that the concentrations of soy isoflavones used in this study and other in vitro studies may be higher (6,34) than the levels present in target tissues (0.02–3 nmol/g). However, these levels are within the plasma concentrations of infants fed soy formula or animals fed soy-containing diets reported earlier (27,28,31,32). Pharmacokinetics studies have also reported that plasma concentration of soy isoflavones, particularly genistein and daidzein, can reach a total of 1–2 µmol/L after consumption of soy meal. Furthermore, in most of the in vitro studies, the cells are exposed only transiently (for a short time) in contrast to the constant exposure of target tissues in vivo with low concentrations of soy isoflavones. Total soy isoflavones in circulation would also include glucuronide and sulfatide conjugates (27,31,32). We have not evaluated the soy isoflavones conjugates, as we had limited access to sufficient quantities of the glucuronide or sulfatide conjugates. Instead, we have tested sera from rats fed soy-based diets (soy-sera) in the adhesion assays. Interestingly, sera from soy-based diet–fed rats blocked monocyte adhesion to CD54, whereas minimal effect was observed with sera from rats fed casein-based diets. The concentration of total soy isoflavones in soy-sera was ~1.5 µmol/L; of which genistein, daidzein, and equol are a~0.4, 0.3, and 0.64 µmol/L, respectively (27,28). However, the level of inhibition by soy-sera is higher than that observed with 1 µmol/L isoflavones (0.33 µmol/L of each isoflavones), suggesting that sera effect may not be due to isoflavones. Alternatively, soy isoflavone aglycones, conjugated forms of these isoflavones, other unidentified soy phytochemicals, and/or peptides in sera from soy-based diet rats could have also contributed to the soy diet–based inhibition.

CD11a is expressed as an inactive low-affinity form (24,25) in circulating (unactivated) monocytes and lymphocytes, so that under physiological conditions these cells do not firmly adhere to CD54+ vascular endothelial cells (Fig. 5). Activation of monocytes and T lymphocytes by agonists such as PMA results in the transformation of a low affinity form of CD11a to a high affinity form (24,25), which in turn results in firm adhesion of these cells to the endothelium (Fig. 5). Transformation of a low to high affinity form of CD11a can be detected using activation-specific anti-CD11a mAb24 (18,23,35). Our findings show that the anti-CD11a mAb24 does not bind to unactivated monocytes, but it can bind to CD11a only upon monocyte activation (Table 3); this is in agreement to earlier reports (18,23,35). Furthermore, isoflavone treatment significantly inhibited mAb24 (which recognized the high affinity form of CD11a) binding to monocytes. These findings suggest that soy isoflavones inhibit the activation-induced transformation of a low to a high affinity form of the CD11a molecule, and subsequent CD54 and mAb24 binding to monocytes (Fig. 5). Inhibition of CD11a-dependent monocyte adhesion to endothelial cells by soy isoflavones could be mediated by 2 possible mechanisms. Tyrosine kinase inhibitors have been shown to inhibit CD11a binding to CD54 (36,37); hence genistein, an isoflavone with kinase inhibiting properties, could have contributed to the inhibition of monocyte adhesion. Furthermore, the phytoestrogenic activity of soy isoflavones (38) may be a contributing factor to the inhibition of monocyte adhesion to CD54. This possibility is supported by a recent article by Friedrich et al. (39), which reports that monocytes treated with 17ß-estradiol poorly adhere to endothelial cells. These probable mechanisms(s) warrant further investigation and work is in progress in our laboratory to address these 2 mechanisms.


Figure 5
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  		Figure 5  Model depicting the functional regulation of CD11a during activation of inflammatory cells and the effect of soy isoflavones on CD11a function.

 
In conclusion, the in vitro studies presented here demonstrate that a blood-borne factor present in soy-sera and soy isoflavones inhibits the monocyte-endothelial interaction, which is the principal event in atherogenesis. Based on these findings, we speculate that similar mechanism(s) may have a role in soy-fed rats in vivo in preventing the initiation and progression of atherosclerosis.


   ACKNOWLEDGMENTS
 
We thank Dr. Nancy Hogg, Cancer Research UK, London Research Institute, London, UK, for anti-CD11a mAb24. We also thank Drs. Martin Ronis, Rick Helm, Prajitha Thampi, and Uma Nagarajan for their critical review of the manuscript, Mr. James Wilkerson for technical assistance, and Mr. Mark Robinette for data analysis.


   FOOTNOTES
 
1 This work was supported by a grant from U.S. Department of Agriculture (CRIS 6251-51000-005-00D) and an Arkansas Children's Hospital Research Institute Lyon New Scientist Award from the Marion B. Lyon Revocable Trust (S.N.). Back

5 Abbreviations used: ApoE–/–, apolipoprotein E knockout; BSA, bovine serum albumin; cas-sera, sera from casein-fed rats; FBS, fetal bovine serum; HUVECs, human umbilical vein endothelial cells; LOX-1, lectin-like oxidized-LDL binding protein-1; mAb, monoclonal antibody; oxLDL, oxidized-LDL; PBMC, peripheral blood mononuclear cells; soy-sera, sera from soy diet–fed rats. Back

Manuscript received 8 May 2006. Initial review completed 2 June 2006. Revision accepted 26 June 2006.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Clarkson TB. Soy, soy phytoestrogens and cardiovascular disease. J Nutr. 2002;132:566S–9S.[Abstract/Free Full Text]

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