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The Journal of Immunology, 2007, 179, 7137 -7146
Copyright © 2007 by The American Association of Immunologists, Inc.
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Extracellular Branched-Chain Amino Acids, Especially Valine, Regulate Maturation and Function of Monocyte-Derived Dendritic Cells1

Eiji Kakazu*, Noriatsu Kanno{dagger}, Yoshiyuki Ueno2,* and Tooru Shimosegawa*

* Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan; and {dagger} Division of Gastroenterology, Iwai General Hospital, Iwate, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The functions of dendritic cells (DCs) are impaired in patients with liver cirrhosis. It is well-known that cirrhotic patients show decreased levels of plasma branched-chain amino acids (BCAA). Although amino acids are associated with maintaining the cell structure and function in many organs, limited data are available regarding the role of amino acids including BCAA in the immune system. We aimed to investigate the roles of BCAA in the function of human monocyte-derived DCs (MoDC). CD14-positive monocytes (CD14 +) were isolated from PBMC from healthy volunteers and hepatitis C virus (HCV) cirrhotic patients. In medium deprived of BCAA or valine, monocytes were able to differentiate into immature, but not into mature, DCs and showed weak expression of CD83. The deprivation of leucine or isoleucine did not affect this process. The MoDC allostimulatory capacity was significantly decreased in medium deprived of BCAA or valine (p = 0.017, p = 0.012, Bonferroni’s analysis, respectively). Annexin VFITC/propidium iodide staining showed that the DC yield and viability were not significantly different under any medium. Immunoblotting demonstrated that depletion of valine or leucine decreased phospho-S6 kinase expression. Valine increased dose-dependently the allostimulatory capacity and IL-12 production of MoDC from both healthy volunteers and HCV cirrhotic patients. An elevated extracellular concentration of valine could improve the DC function in cirrhotic patients. These data provide a rationale for nutrition therapy that could be beneficial to patients with cirrhosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hepatitis C virus (HCV)3 induces chronic liver disease in hosts, which can eventually progress to liver cirrhosis and chronic liver failure. Combination therapy with peginterferon and ribavirin has been shown to result in a sustained virological response in ~50% of patients (1, 2), but others continue to be viremic and progress to advanced fibrosis and liver cirrhosis. The number of advanced liver cirrhotic patients has been increasing, but combination therapy is poorly tolerated under cirrhosis and the response rate is low (3). Bacterial infection, such as spontaneous bacterial peritonitis and pneumonia, is one of the most frequent causes of death in immune-compromised cirrhotic patients. In such cirrhotic patients, a decrease in the levels of plasma branched-chain amino acids (BCAAs) is one of the characteristic features (4, 5). BCAA comprise the three essential amino acids L-leucine, L-isoleucine, and L-valine. BCAA granules (a mixture of L-leucine, L-valine, and L-isoleucine) have been used to effectively reverse the hypoalbuminemia and hepatic encephalopathy in patients with decompensated liver cirrhosis (6, 7), but little is known about the impact of changes in the BCAA levels on the immune system (8). In previous cohort studies, the BCAA-supplemented groups demonstrated elevations in the absolute lymphocyte count (9, 10). In previous in vitro studies, the omission of a single BCAA from the medium of cultured lymphocytes resulted in the complete abolition of protein synthesis or proliferation (11, 12, 13). These findings simply reflect the fact that BCAA are essential cell components.

Recently, it has become clear that amino acids are not only important as substrates for various metabolic pathways, but also activate a nutrient-sensitive signaling pathway in synergy with insulin (14). The mammalian target of the rapamycin (mTOR) signaling pathway is one of the most representative pathways, and this pathway has been shown to act as a major effecter of cell growth and proliferation via the regulation of protein synthesis. The pathway is activated by BCAA, especially by leucine (15, 16, 17). mTOR was identified and cloned (18, 19, 20) shortly after the discovery of the two yeast genes, TOR1 and TOR2, in the budding yeast Saccharomyces cerevisiae during a screening for resistance to the immunosuppressant drug rapamycin. Rapamycin, introduced to prevent allograft rejection, has been extensively studied for its effect on T lymphocytes, and is primarily known for its antiproliferative effect (21). Some studies have described that dendritic cell (DC) functions (viability, Ag uptake, cytokine production, and allostimulatory capacity) are impaired by rapamycin (22, 23, 24).

DCs are professional APC that initiate and mediate immune responses against pathogens and tumors. Typically, immature DCs capture and process Ags to peptides which are then presented in the context of MHC class II or class I molecules. They migrate to lymphoid tissues and present antigenic peptides to naive T cells. The mature DCs, which characteristically express CD83 (25), can rapidly activate other innate immune cells including NK and NKT cells through the production of immunomodulatory cytokines such as ILs IL-10 and IL-12. Human DCs can be generated in vitro from peripheral blood CD14-positive monocytes, termed monocyte-derived DCs (MoDC) (26). The ability of monocytes to differentiate into DCs was originally demonstrated by Sallusto and Lanzavecchia (27), who reported the generation of DCs from human peripheral monocytes after in vitro culture with GM-CSF plus IL-4. Despite in vitro experimental evidence on the potential of monocytes to differentiate into DCs, whether this process occurs under physiological conditions is still controversial. In patients with chronic hepatitis C, the function of MoDC has been studied by various groups. These studies described that MoDC of patients had lower allostimulatory capacity and IL-12 production than MoDC of healthy subjects (28, 29, 30). It is considered that HCV proteins impair the hosts’ DC functions (31).

In this study, we demonstrated that BCAAs, especially valine, influenced the function of MoDC. Increasing the extracellular concentration of valine could improve the DC function in cirrhotic patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients and healthy volunteers

Liver cirrhotic patients with chronic HCV infection were diagnosed for persistent-positive HCV Ab and the presence of HCV-RNA in the serum. All patients had clinical and laboratory findings compatible with cirrhosis (Table I). Also, six nonviral cirrhotic patients and eight healthy volunteers were recruited to obtain MoDC. Written informed consent was obtained from each individual and the study protocol was approved by the Ethics Committee of Tohoku University School of Medicine (2003-326).


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  		Table I. Characteristics of study participantsa

 
Monocyte isolation and DC generation

PBMC were separated from the peripheral blood of healthy volunteers and patients by centrifugation on a density gradient (Ficoll-Paque Plus; Amersham Biosciences). The CD14-positive monocytes (CD14+) were isolated from PBMC using magnetic microbeads (Miltenyi Biotec). CD14+ were cultured at a density of 3.0 x 106 cells/well in 24-well flat-bottom plates (Falcon) for 5 days in the following culture medium: amino acid-free medium (D-MEM deprived of all amino acids) supplemented with 2 g/L BSA (Sigma-Aldrich), 1000 U/ml GM-CSF (PeproTech), 500 U/ml (human) IL-4 (PeproTech), 3.5 g/L glucose (Sigma-Aldrich), 6 g/L HEPES (Sigma-Aldrich), 1% insuline-transferin-selenium-X (Invitrogen Life Technologies), and variously conditioned amino acids. The culture medium containing all 20 kinds of amino acids was defined as the complete culture medium (CCM). The culture medium in which all amino acids—BCAA, valine, leucine, or isoleucine—were removed was defined as zero, {Delta}BCAA, {Delta}Val, {Delta}Leu, or {Delta}Ile, respectively (Table II). At day 5, 20 ng/ml TNF-{alpha} (R&D Systems) and 500 ng/ml LPS (Escherichia coli 026:B6; Sigma-Aldrich) were added and the culture was continued for an additional 24 h. Also, CD40L (1 µg/ml) or poly I:C (30 µg/ml) was added at day 5 to evaluate the CD83 expression. The CD14+ monocytes expressed high levels of CD14, HLA-DR, and CD86, and negligible levels of CD83. Immature DCs expressed lower levels of CD14 and CD86, and higher levels of HLA-DR and CD40, but they did not express CD83. Mature DC showed the up-regulation of costimulatory molecules (CD40, CD80, and CD86). These cells were also characterized by the induction of CD83 expression on their cell surface.


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  		Table II. Concentrations of amino acids in mediuma

 
MoDC surface marker analysis and viability assay

On day 5 or 6 of culture, MoDC were harvested and labeled with FITC- or PE-labeled mAbs (anti-human CD14, CD40, CD80, CD83, CD86 HLA-DR, or the relevant isotype controls: BD Pharmingen), according to the manufacturer’s directions. Briefly, cells (1 x 106) were incubated with 20 µl of Ab in a total volume of 100 µl (PBS) for 30 min at 4°C in the dark. Using a FACSCalibur (BD Immunocytometry Systems) flow cytometer, the cells were gated according to their size (forward light scatter (FSC)), granularity (side light scatter (SSC)), and surface marker expression and analyzed using the CellQuest (BD Immunocytometry Systems) program. On day 6, the viability of MoDC was determined using Annexin VFITC, with dead cells identified by propidium iodide (PI) staining (Annexin VFITC Apoptosis Detection kit; BioVision), according to the manufacturer’s directions.

MLR and cytokine analysis

CD14+ monocytes isolated by MACS were cultured at a density of 1.0 x 105 cells/well in 96-well round-bottom plates (Falcon) containing 200 µl of various concentrations of valine or leucine in medium supplemented with 1000 U/ml GM-CSF, 500 U/ml IL-4 for the generation of immature DCs. On day 5, immature DCs were induced to mature using 500 ng/ml LPS and 20 ng/ml TNF-{alpha} for 24 h. On day 6, the allostimulatory capacity of 5.0 x 104 irradiated DCs (3000 rad) was tested in a one-way MLR with normal, allogeneic T CD4+ lymphocytes (isolated from PBMC using magnetic beads: 1 x 105cells/well) in duplicate or triplicate. Coculture cells were maintained for 4 days at 37°C in a 5% CO2 humidified atmosphere. The proliferation rate of the cells was measured using an MTS assay (CellTiter 96 aqueous one-solution cell proliferation assay; Promega). Forty microliters of CellTiter 96 aqueous one-solution were added to each well. After 2 h of incubation, the UV absorbance of the solution was measured at a wavelength of 490 nm. All MTS assays were done in triplicate. Supernatants were collected on day 6 and immediately IL-12 (p40 plus p70) and IL-10 were determined by specific cytokine ELISA kits (Bender MedSystems) according to the manufacturer’s instructions.

Immunoblotting

On day 6, MoDC were harvested and lysed using CelLyticTM-M Mammalian Cell Lysis/Extraction Reagent (Sigma-Aldrich). The lysed cells were centrifuged for 10 min at 12,000–20,000 x g to pellet the cellular debris. Thereafter, these protein concentrations were determined by a Modified Lowry Protein Assay kit (Pierce). The total 50 µg of protein were loaded onto SDS-PAGE gel and electrotransferred to a polyvinylidene fluoride (Immun-Blot PVDF membrane; Bio-Rad). After washing, the membranes were incubated in 25 ml of blocking buffer for 1 h at room temperature. Immunostaining was performed with rabbit polyclonal primary Ab (mTOR: no. 2972, p70 S6K: no. 9202, phopho-p70 S6K: no. 9205; Cell Signaling Technology), followed by incubation with a secondary Ab conjugated to HRP (Sigma-Aldrich). Immunoreactive proteins were revealed with an ECL reagent (ECL advance; Amersham Biosciences). To confirm the equal protein loading in all samples, the blot was stripped for 30 min in Ab stripping solution (Re-Blot Plus Western Blot Recycling kit; Chemicon International), washed extensively, and relabeled with anti β-actin Ab and secondary HRP Ab (Sigma-Aldrich). Densitometric analysis was performed using Scion Image for Windows.

Aminogram

The concentrations of plasma amino acids from 27 HCV cirrhotic patients were measured by HPLC. Also, these patients were classified according to the Child-Pugh classification.

Statistical analysis

The data were analyzed with ANOVA, and multiple comparisons were performed with Dunnett’s post-hoc procedure. When two groups were analyzed, the differences between groups were analyzed by the Wilcoxon t test. When, more than two groups were analyzed, the differences were analyzed by Bonferroni’s analysis. All data are expressed as mean SEM. In all analyses, a p value of <0.05 was considered statistically significant. All statistical analyses were performed with standard statistical software (SPSS 13.0 for Windows).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Depletion of extracellular BCAA did not influence the expression of costimulatory molecules on MoDC, but decreased the expression of CD83

First, to investigate whether the depletion of extracellular BCAA influenced the generation of MoDC, we cultured the monocytes for 6 days under CCM and {Delta}BCAA. At day 5, the stimulants were added. We evaluated the expression of CD14, CD40, CD80, CD83, CD86, and HLA-DR on the surface of MoDC grown under either CCM or {Delta}BCAA (Fig. 1A) by flow cytometry. There was no difference in the percentage of MoDC expressing CD14, CD40, CD80, CD86, and HLA-DR between the two mediums. Negligible levels of CD14 and higher levels of HLA-DR, CD40, CD80, and CD86 indicated that the cells could differentiate into MoDC in both mediums. However, the CD83 expression was decreased under {Delta}BCAA as confirmed by the single-color staining. Similarly, when stimulated with either CD40L or poly I:C at day 5, the CD83 expression was impaired in {Delta}BCAA (data not shown). To investigate which amino acid in BCAA especially influenced the MoDC phenotype, we determined the MoDC phenotype (CD14, CD83, or CD86) in {Delta}Val, {Delta}Leu, and {Delta}Ile. In CCM, {Delta}Leu and {Delta}Ile, the MoDC phenotype was similar. However, in {Delta}Val, the CD83 expression of MoDC was significantly impaired compared with that in CCM. The CD86 expression was not significantly different in any medium (Table III). On microscopic appearance, depletion of BCAA also affected the morphological appearance and behavior of the cells in culture. Monocytes cultured under either CCM, {Delta}Leu, or {Delta}Ile were adherent with little tendency to form aggregations. On day 6, cells formed large, firmly adherent clusters (Fig. 1B), which were typical of mature DCs in vitro. In contrast, monocytes cultured under {Delta}BCAA and {Delta}Val formed much smaller clusters. Using a traditional FSC/SSC gate of the FACS plots, we evaluated the mean FSC and SSC values of the MoDC population in each medium. MoDC generated under {Delta}BCAA or {Delta}Val expressed lower FSC and SSC values than MoDC generated under CCM.


Figure 1
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  		FIGURE 1. MoDC cultured in BCAA-deprived medium express low levels of CD83. We cultured monocytes under CCM or {Delta}BCAA medium in 24-well tissue culture plates for 5 days and exposed them to LPS and TNF-{alpha} for an additional 24 h. A, Cells were harvested on day 6 of culture, stained with different mAbs, and analyzed using flow cytometry. Cells were stained with FITC-labeled anti-CD14, -CD40, -CD80, -CD83, -CD86, and -HLA-DR. For the histogram figure, filled traces represent isotype-matched control Ab staining; open traces indicate a marker-specific Ab; percentages indicate positive cells. Results are representative of five experiments from five different donors. B, Influence of BCAA on microscopic appearance of monocytes undergoing DC differentiation under serum-free conditions. Day 6, cells in firmly adherent clusters in CCM, {Delta}Leu, and {Delta}Ile, which are typical of DCs that mature in vitro. In contrast, cells in {Delta}BCAA and {Delta}Val formed much smaller clusters. Using a traditional FSC/SSC gate of the FACS plots, the figure expresses the mean FSC and SSC values of the MoDC population.

 

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  		Table III. Depletion of BCAA, especially valine, decreased MoDC CD83 expressiona

 
Depletion of extracellular BCAA or valine influenced the maturation but not the differentiation of MoDC

To further investigate at which point in time the amino acids influenced the MoDC phenotype, we cultured monocytes under CCM, {Delta}BCAA, {Delta}Val, or {Delta}Leu, and determined the phenotype of the MoDC before (day 5) and after (day 6) adding LPS and TNF-{alpha} by two-color staining (Fig. 2). Most monocytes (>95%), which were freshly isolated from PBMC, expressed the CD14-positive and CD83-negative phenotype. Before adding the stimulants (day 5), the MoDC phenotype was CD14 negative and CD83 negative in all medium compositions. These data indicated that the monocytes could differentiate into immature DCs in almost any medium. Interestingly, after adding the stimulus (day 6), under {Delta}BCAA and {Delta}valine, the percentage of CD14–/CD83+ mature DCs was lower than that under CCM or {Delta}Leu. These data indicated that depriving BCAA, especially valine, influenced the maturation of MoDC. After we cultured the monocytes under {Delta}Val for 5 days, we added 400 nM/ml valine with stimulus to the medium and cultured the cells for an additional 24 h. Then, the percentage of mature DCs was higher than that of {Delta}Val. We cultured the monocytes under CCM for 5 days, and with an additional 24 h under {Delta}Val, the percentage of mature DCs was decreased compared with that of CCM (data not shown).


Figure 2
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  		FIGURE 2. BCAA, especially valine, are necessary for MoDC maturation but not for differentiation. Monocytes were cultured under CCM, {Delta}BCAA, {Delta}Val, {Delta}Leu medium as described in Fig. 1. Cells were harvested on days 5 and 6 of culture, stained with different mAbs, and analyzed using flow cytometry. Cells were stained with FITC-labeled anti-CD14 and PE-labeled anti-CD83. For the dot-plot figure, percentages indicate the proportion of cells adopting the DC immunophenotype (CD14–/CD83+). Results are representative of four experiments from four different donors.

 
Depletion of extracellular BCAA does not influence MoDC viability

To elucidate the possibility that impaired MoDC maturation under {Delta}BCAA or {Delta}Val was caused by decreased cell viability, we evaluated the cell recovery, yield of DCs, and viability on day 6 in each medium. The cell recovery and yield of DCs was not significantly different between any medium. The percentage of cells recovered was 54 ± 8.4 (±SD), 49 ± 12.1, 49 ± 11.3, and 50 ± 8.9 for the CCM, {Delta}BCAA, {Delta}Val, and {Delta}Leu, respectively. The DC yield percentages were 57 ± 9.2, 56 ± 10.4, 58 ± 8.4, and 57 ± 8.1 for CCM, {Delta}BCAA, {Delta}Val, and {Delta}Leu, respectively. The viability of MoDC on day 6 was determined by annexin V/PI staining. The percentages of necrotic cells, living cells, and early apoptotic cells were not different under any medium (Fig. 3).


Figure 3
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  		FIGURE 3. Depletion of extracellular BCAA did not influence the MoDC viability. Monocytes were cultured under CCM, {Delta}BCAA, {Delta}Val, and {Delta}Leu medium as described in Fig. 1. Cells were harvested on day 6 of culture. Annexin VFITC/PI staining was performed to determine the cell viability. In the quadrant statistics, PI-negative and annexin V-negative indicated live cells. PI positive (upper) indicated necrotic cells. PI negative and annexin V positive (lower right) indicated early apoptotic cells. Data shown are representative of four independent experiments with cells from different donors.

 
BCAA, especially valine, modulated MoDC allostimulatory capacity and cytokine production

Based on the result that MoDC maturation was suppressed by the lack of extracellular BCAA, especially valine, we hypothesized that the concentration of extracellular BCAA could influence the function of MoDC. To investigate this hypothesis, we cultured monocytes for 6 days under CCM, Zero, {Delta}Val, {Delta}Leu, {Delta}Ile, and {Delta}BCAA. The MoDC (5.0 x 104) were cocultured with normal allogeneic CD4+ lymphocytes under CCM and evaluated for their allostimulative capacity by the MLR. Expectedly, the allostimulatory capacity of MoDC cultured under {Delta}BCAA and {Delta}Val was significantly impaired (p = 0.017, p = 0.012, Bonferroni’s analysis, respectively), although there was no significant difference among {Delta}Leu, {Delta}Ile, and CCM (Fig. 4A). There was no statistically difference between {Delta}Val and {Delta}Leu (p = 1.000, Bonferroni’s analysis). Furthermore, to examine whether the addition of valine enhanced the function of MoDC, we cultured monocytes under various mediums that contained 0–800 nM/ml valine or leucine, and evaluated the allostimulatory capacity of the MoDC. The addition of valine increased the allostimulatory capacity of MoDC in a dose-dependent manner. However, the concentration of leucine did not influence the pharmacological effect (Fig. 4B). Cytokines play key roles in determining the strength and the phenotypes of the T cell response. Thus, on day 6, we measured the cytokine production from MoDC. The addition of valine increased the IL-12 production of MoDC in a dose-dependent fashion. Interestingly, the IL-10 production was not influenced by the concentration of valine (Fig. 4C).


Figure 4
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  		FIGURE 4. BCAA, especially valine, modulated MoDC allostimulatory capacity. A, We cultured monocytes under each medium for 5 days (detailed amino acid composition is shown in Table II) in 96-well tissue-culture plates and irradiated MoDC after exposing them to LPS and TNF-{alpha} for additional 24 h. The MoDC yielded (5.0 x 104) were cocultured with normal, allogeneic CD4+ T lymphocytes (1 x 105cells/well) under CCM for 4 days and evaluated for their allostimulative capacity by the MLR. B, We cultured monocytes in the same way under various mediums that contained 0–800 nM/ml valine or leucine. Zero nanomoles per milliliter of valine or leucine medium are represented by {Delta}Val or {Delta}Leu, respectively. CCM contained 800 nM/ml valine and leucine medium. C, After 6 days, the supernatants were removed and assayed for the cytokine concentrations. Mean ± (A) SEM values from five different donors are shown. B and C, Mean ± SEM values from four different donors are shown. Statistical significance for all conditions was determined by one-way ANOVA and Bonferroni’s post-hoc procedure for A, one-way ANOVA and Dunnett’s post-hoc procedure for B and C. **, p < 0.01; *, p < 0.05 vs CCM (A and B) or vs {Delta}Val (C).

 
Depletion of extracellular valine down-regulated the mTOR/S6K-signaling pathway of MoDC

The mTOR-signaling pathway, one of the most representative pathways, is known as a major effecter of cell growth and proliferation via the regulation of protein synthesis. A previous study showed that the removal of extracellular amino acids, especially leucine, inhibited the ability of mTOR to signal to p70 S6 kinase (15, 16, 17). We hypothesized that BCAA modulate the mTOR/S6K-signaling pathway of MoDC and influences the maturation markers. First, to investigate whether the mTOR/S6K inhibitor rapamycin could influence the MoDC phenotype, we cultured monocytes under CCM for 5 days. At day 5, LPS and TNF-{alpha} were added to the medium with or without rapamycin (1 µM). Under CCM with rapamycin, the percentage of CD14–/CD83+ mature DCs was lower than under CCM without rapamycin (Fig 5A). These data indicated that rapamycin could suppress the maturation of MoDCs similarly as when depriving them of BCAA or valine. Also, we recovered MoDCs cultured under CCM for 5 days, and matured these MoDCs by stimulant in either CCM, CCM added with rapamycin, {Delta}Val, {Delta}Leu, or {Delta}Ile for 24 h. On day 6, we determined the expression of mTOR, p70 S6K, and phospho-p70 S6K by immunoblotting. MoDC expressed similar levels of mTOR, p70 S6K, and β-actin among all mediums. MoDCs cultured in {Delta}Val and {Delta}Leu expressed significantly lower levels of phospho-p70 S6K than those cultured in CCM (Fig. 5B). Expression of phospho-p70 S6K expression by MoDC in {Delta}Val was recovered by adding 400 nM/ml valine to medium during stimulation.


Figure 5
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  		FIGURE 5. Depleting extracellular valine or leucine down-regulated the mTOR/S6K-signaling pathway of MoDC. A, We cultured monocytes under CCM for 5 days in 24-well tissue culture plates and exposed them to LPS and TNF-{alpha} for an additional 24 h in CCM, {Delta}Val, and CCM plus rapamycin (1 µM). Representative results from three subjects are shown. B, We cultured monocytes under CCM for 5 days and exposed them to LPS and TNF-{alpha} for 24 h in either CCM, CCM plus rapamycin (1 µM), {Delta}Val, {Delta}Leu, or {Delta}Ile. Also at day 6, we added 400 nM/ml valine to {Delta}Val. Cells were harvested on day 6 or 7 and lysed. Equal amounts of protein were loaded and the levels of mTOR, p70 S6K, and phosho-p70 S6K were determined by Western blot analysis. Densitometry shows the mean and SEM of the relative levels of phospho-p70 S6K to p70 S6K. Data shown are representative of four independent experiments with cells from different donors. **, p < 0.01; *, p < 0.05 vs CCM.

 
Elevating the extracellular valine concentration improved the allostimulatory capacity and IL-12 production dose-dependently in MoDC from HCV cirrhotic patients

The functions of DCs are impaired in patients with chronic hepatitis C (28, 29, 30). As in the in vivo study, we measured the concentrations of BCAAs in the peripheral blood of 27 HCV cirrhotic patients by HPLC. We confirmed that the plasma concentrations of BCAAs were decreased along with the Child-Pugh grade (Fig. 6). In Child-Pugh B or C patients, the concentrations of BCAA, especially valine, were significantly decreased compared with those of healthy subjects. As in the in vitro study, we evaluated the function of MoDC from HCV cirrhotic patients (Table I). First, we determined the phenotype of MoDC from the patients and controls (Fig. 7A). There was no difference regarding the percentage or mean fluorescence intensity (MFI) of MoDC expressing CD14, CD80, and CD86 between healthy volunteer and patients. However, the CD40, CD83, and HLA-DR expression by MoDC from the patients was significantly decreased compared with healthy volunteers (Table IV). Second, as shown in Fig. 4, we cultured monocytes under the medium that contained 0–800 nM/ml valine and evaluated the expression of CD83, cytokine production, and allostimulatory capacity by MoDC. The addition of valine dose-dependently increased the percentage of CD83+/CD14– mature MoDC in both healthy volunteers and patients (Fig. 7B). Regarding the cytokine production, the addition of valine increased the IL-12 production by MoDC in a dose-dependent manner, although the IL-10 production was not influenced by the concentration of valine in the culture medium (Fig. 7C). These tendencies were similar to those in healthy controls. Regarding the allostimulatory capacity, the values were maximum under 2.4 µM/ml valine, and those of cirrhotic patients were lower than those of healthy controls (Fig. 7D). In contrast, the allostimulatory capacities of MoDC from nonviral cirrhotic patients were at the intermediate level between healthy controls and HCV cirrhotic patients (Fig. 7D).


Figure 6
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  		FIGURE 6. The plasma concentrations of BCAA in cirrhotic patients were decreased according to the Child-Pugh grade. Twenty-seven liver cirrhotic patients were classified by Child-Pugh classification. The levels of plasma BCAAs in these patients were measured using HPLC. The dots represent the value from each patient and the circles represent the averages. **, p < 0.01, *, p < 0.05 vs healthy control.

 

Figure 7
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  		FIGURE 7. Elevating the extracellular valine concentration dose-dependently increased the allostimulatory capacity of MoDC and IL-12 production in cirrhotic patients. A, Monocytes isolated from the peripheral blood of HCV liver cirrhotic patients were cultured under CCM and analyzed the phenotypes as described in Fig. 1. For the histogram figure, filled traces represent isotype-matched control Ab staining; open traces indicate a marker-specific Ab; percentages indicate positive cells. Results are representative of five experiments from five different patients. B, We cultured monocytes under various mediums that contained 0–800 nM/ml valine and determined the percentage of CD83-positive mature MoDC. C, Similarly as in Fig. 4C, cytokine productions were measured with ELISA. D, We cultured the monocytes under various mediums that contained 0–8.0 µM/ml valine. Zero nanomoles per milliliter of valine is indicated by {Delta}Val. 0.8 µM/ml valine medium is identical with CCM. Patient number (B) 10-13, (C) 1-4, (D) 5-12, 15-20 in Table I. Statistical significance for all conditions was determined by one-way ANOVA and Dunnett’s post-hoc procedure. *, p < 0.05 vs {Delta}Val.

 

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  		Table IV. Comparison of the phenotypic characteristics of MoDC derived from HCV cirrhotic patients and healthy volunteera

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we showed that BCAAs, especially valine, influenced the function of MoDCs. Cultures of human MoDCs are typically made in medium containing human or FCS supplements. Medium that contains serum varies in its concentration of amino acids according to each lot number, which can influence the phenotypes of the cells and their functional properties. Thus, the current study evaluated the concentrations of the amino acids strictly (details in Materials and Methods).

First, we found that depletion of extracellular BCAA did not influence the expression of costimulatory molecules (CD40, CD80, and CD86) on MoDC, but decreased the CD83 expression. Human CD83 is a 45-kDa glycoprotein which belongs to the Ig superfamily. CD83 is expressed on MoDCs after stimulation with inflammatory cytokines (26), and CD83 is considered as a maturation marker. Although the function of CD83 is still unknown, inhibition of CD83 expression by interfering with a specific RNA-exporting pathway leads to a dramatic reduction of the DC-mediated T cell stimulation (32). This study supports our hypothesis that the impairment of the allostimulatory capacity of MoDC cultured in medium deprived of valine was caused by the lower expression of CD83. We also found that during the generation of MoDC, depletion of extracellular BCAA, especially valine, did not influence the differentiation but impaired the maturation of MoDC. Moreover, this phenomenon was accompanied by a suppression of the mTOR/S6K-signaling pathways. Monti et al. (22) recently reported that rapamycin-treated DCs were less capable of up-regulating CD83 after exposure to CD40L. This observation partially supports our result, although the relation between mTOR/S6K signaling and CD83 expression should be evaluated in future studies. Although the depletion of leucine is believed to suppress the mTOR/S6K pathway in general, our study demonstrated that the depletion of valine also suppressed P-S6K. This conflicting observation could have resulted from differences in the cell sources evaluated.

We next showed that the addition of valine increased the IL-12 production by MoDC in a dose-dependent manner, and that the IL-10 production was not influenced by the concentration of valine in either healthy controls or patients. IL-12 is an IL that is naturally produced by macrophages, B-lymphoblastoid cells, and DCs in response to antigenic stimulation. It is involved in the differentiation of naive T cells into Th1 cells, which is important in the resistance to foreign pathogens. IL-10 is naturally produced by monocytes and type 2 Th cells. It is believed to have important suppressive functions on immune responses and also may be involved in the maintenance of tolerance. Our results raised the possibility that elevating the extracellular valine concentration could modulate Th1/Th2 differentiation in both healthy subjects and patients. To examine this possibility, it was necessary to coculture MoDC and naive CD4 T cells, and determine the phenotype of T cells and cytokine production. In addition, we found that depriving extracellular valine decreased the IL-12 production by MoDC with impaired mTOR/S6K signaling. In a previous study, active S6K1 suppressed the PI3K-Akt pathway by inactivating the insulin receptor substrate (33), whereas PI3K negatively regulated IL-12 synthesis by DCs (34). These results permit us to speculate that valine influences IL-12 production by MoDC through the PI3K/mTOR/S6K pathway.

In this study, we found that an increased concentration of valine could recover the impaired function of DCs in cirrhotic patients. However, the degree of this improvement was very modest, which lead to the speculation that persistent HCV infection itself could suppress the function of DCs in such patients. The MoDCs from hepatitis C patients have been previously reported by several studies (28, 29, 30), although their results regarding allostimulatory capacity or phenotype had been conflicting. Our results demonstrated the decreased allostimulatory capacity and decreased expression of CD40, CD83, and HLA-DR. In our series, the medium was serum-free medium and the patients’ backgrounds were all cirrhotics. This is similar to study by Auffermann-Gretzinger (28) in which serum-free medium was used and had a more dominant cirrhotic population. It seemed that the expression of CD83 is decreased in either serum-free medium or progressed liver disease including cirrhosis. However, the allostimulatory capacities of HCV-infected cirrhotic patients tended to be lower compared with those of non-B, non-C cirrhotics. This impaired DC function could be possibly due to HCV infection itself, although we have not proved this hypothesis in the current study. We also found that the increase of extracellular valine could increase the phenotype (CD83 expression), allostimulatory capacity and cytokine production in HCV cirrhotic patients. The allostimulatory functions of DCs were maximum at a considerably higher than physiological concentration in both normal subjects and patients. However, the concentrations of valine in either the liver, portal blood flow, or lymph nodes could be higher than that in the peripheral blood. This issue should be evaluated in future studies. Furthermore, the changes of this allostimulatory capacity were most apparent at ranges near the physiological concentration in peripheral blood.

Recently, Osugi et al. (35) showed several differences between MoDC and the myeloid DCs present in vivo. In contrast, monocytes could differentiate into DCs in vivo (36, 37, 38). Further evaluations using circulating DCs will be needed to clarify this issue.

As previously described in a review (8), BCAA are essential for the synthesis of proteins required for cellular proliferation. However, little information is available regarding the immunologic effect of variations in the concentrations of BCAA at ranges that might occur physiologically or pathophysiologically. In this study, we have demonstrated that 1) depriving extracellular BCAA for 6 days does not influence the viability of MoDC, 2) depriving extracellular isoleucine did not decrease the allostimulatory capacity of MoDC, 3) CD40, CD80, CD86, and HLA-DR molecules were equally expressed in both CCM and {Delta}BCAA medium, 4) IL-10 production was not influenced by the extracellular valine concentration, 5) mTOR signaling was associated with decreased DC function in valine or leucine depletion.

These data suggest that BCAA are important for cell function through a nutrient-sensitive signaling pathway rather than through acting as substrates for various metabolic pathways and cell structures. Also, it is preferable to measure the intracellular concentration of amino acids, although their uptake was reported to be either sodium dependent or independent (39). Finally, we need to clarify why the depletion of valine itself caused a more potent inhibition of the allostimulatory capacity compared with depletion of all three components of BCAA.

In clinical situations, the administration of BCAA was reported to increase the number of peripheral lymphocytes and improve opportunistic infections or immune functions (9, 10). In advanced cirrhosis, long-term nutritional supplementation with oral BCAA has been shown to be useful to prevent progressive hepatic failure and to improve surrogate markers and the perceived health status (7). Our data concerning immune functions provide the rationale for future nutrition therapy, which could be beneficial to patients with cirrhosis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Health and Labour Sciences Research Grants (from the Ministry of Health, Labour, and Welfare of Japan) for the Research on Measures for Intractable Diseases and Grant-in-Aid for Scientific Research C (17590609) from the Japan Society for the Promotion of Science (to Y.U.). Back

2 Address correspondence and reprint requests to Dr. Yoshiyuki Ueno, Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aobaku, Sendai, 980-8574 Japan. E-mail address: yueno{at}mail.tains.tohoku.ac.jp Back

3 Abbreviations used in this paper: HCV, hepatitis C virus; BCAA, branched-chain amino acid; mTOR, mammalian target of rapamycin; DC, dendritic cell; MoDC, monocyte-derived DC; CCM, complete culture medium; FSC, forward light scatter; SSC, side light scatter; PI, propidium iodide; MFI, mean fluorescence intensity. Back

Received for publication November 8, 2006. Accepted for publication August 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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