The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Um, S. H.
Right arrow Articles by Behboudi, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Um, S. H.
Right arrow Articles by Behboudi, S.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Liver Cancer
The Journal of Immunology, 2004, 173: 1772-1778.
Copyright © 2004 by The American Association of Immunologists

{alpha}-Fetoprotein Impairs APC Function and Induces Their Apoptosis1

Soon Ho Um*, Catherine Mulhall*, Akeel Alisa*,{dagger}, Annette Robyn Ives*, John Karani{dagger}, Roger Williams*,{dagger}, Antonio Bertoletti* and Shahriar Behboudi2,*

* Institute of Hepatology, University College London, and {dagger} Liver Unit, Cromwell Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{alpha}-Fetoprotein (AFP) is a tumor-associated Ag, and its serum level is elevated in patients with hepatocellular carcinoma (HCC). In vitro, AFP induces functional impairment of dendritic cells (DCs). This was demonstrated by the down-regulation of CD40 and CD86 molecules and the impairment of allostimulatory function. Also, AFP was found to induce significant apoptosis of DCs, and AFP-treated DCs produced low levels of IL-12 and TNF-{alpha}, a cytokine pattern that could hamper an efficient antitumor immune response. Ex vivo, APCs of patients with HCC and high levels of AFP produced lower levels of TNF-{alpha} than that of healthy individuals. In conclusion, these results illustrate that AFP induces dysfunction and apoptosis of APCs, thereby offering a mechanism by which HCC escapes immunological control.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Structurally related to human albumin, {alpha}-fetoprotein (AFP)3 is a well-characterized oncofetal Ag. It is normally expressed during embryogenesis and is present in only trace amounts in normal adults (1). However, the expression of the AFP gene is reactivated in patients with testicular and hepatocellular carcinoma (HCC) with high levels of AFP being found in the sera and tumor tissues. The determination of serum AFP aids in the diagnosis and the management of patients with HCC. A serum AFP level of >200 ng/ml is shown to have a specificity of 100% for HCC (2). In a study of 68 Asian-American patients with HCC, serum AFP ranged from 0 to 636,000 ng/ml with the average being 5,200 ng/ml (3). AFP, like serum albumin, shows relatively strong binding affinities for a variety of ligands. Various other specific physiological roles for AFP are being proposed such as its possible role in the regulation of immune cells (1). A series of investigations has provided evidence that AFP causes selective down-regulation of MHC class II on monocytes (4, 5) and the suppression of T (6, 7, 8, 9) and B lymphocyte (10) responses. It has also been shown that AFP-mediated immunoregulation is an activity intrinsic to the molecule itself and cannot be attributed to either putative noncovalently bound moieties or to posttranslational modifications such as glycosylation and sialylation (11). Stimulation of leukotriene synthesis by AFP in macrophages has been suggested as a possible mechanism for its immunoregulatory effects (12). In addition, microscopic autoradiography has exhibited binding of AFP almost exclusively on human peripheral monocytes but not on lymphocytes (13), suggesting that the regulatory effects of AFP may be via APCs.

Dendritic cells (DCs) are the most potent APCs of the immune system and are crucial in the initiation of the immune response against pathogens and tumors. DCs exist in two differing states of maturation: immature and mature. Immature DCs express low levels of surface molecules such as CD80, CD86, CD40, and MHC class II, and have low T cell-stimulatory capacity. Several stimulatory agents, such as proinflammatory cytokines and viral or bacterial products, can trigger DC activation and thus maturation. This is defined by the up-regulation of costimulatory molecules, an increase in the levels of TNF-{alpha} and IL-12 production, and improved capacity to stimulate T cells (14, 15). The activated DCs can rapidly activate other innate immune cells such as NK (16) and NKT cells (17). Most knowledge about the biology of DCs has emerged from the ability to generate DCs in vitro from either CD34+ hemopoietic progenitors or peripheral blood monocytes.

In this study, we show that the treatment of monocyte-derived DCs with AFP induces DCs dysfunction as detected by the down-regulation of surface molecules and inhibition of their T cell-stimulatory capacity. In addition, AFP treatment reduces the ability of monocyte-derived DCs to produce TNF-{alpha} and IL-12 and induces apoptosis of DCs. Furthermore, we compare the ability of APCs from patients with HCC to produce TNF-{alpha} with that of control individuals. The data clearly show that HCC with high levels of serum AFP have reduced TNF-{alpha} production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

The patients involved in this study were all reviewed at the Liver Unit of the Cromwell Hospital (London, U.K.). The ethical committee’s approval was granted, and informed consent was obtained. In total, there were 16 patients with an AFP value ranging from 8 to 1,141,205 ng/ml. The average serum AFP level in the HCC group was 80,409 ng/ml. There were seven patients with hepatitis C cirrhosis, two with cryptogenic cirrhosis, three with hepatitis B cirrhosis, two with hepatitis B and C cirrhosis, one with alcoholic liver cirrhosis, and one with alcoholic liver and hepatitis C-related cirrhosis. Five of them were Child’s grade B and 11 were Child’s grade A. Laboratory tests including aspartate aminotransferase, alanine aminotransferase, total bilirubin, platelet count, prothrombin time, international normalized ratio, creatinine, hepatitis B surface Ag, anti-hepatitis C virus Ab, and hepatitis C virus RNA, were determined using standard, commercially available assays. All blood samples from patients with HCC were withdrawn before giving any therapy. The severity of cirrhosis was assessed by Child-Pugh score.

AFP measurement

Levels of serum AFP were measured using microparticle enzyme immunoassay (MEIA) kit obtained from Abbott Laboratories (Abbott Park, IL) and performed according to the manufacturer’s instruction. In brief, anti-AFP microparticles were incubated with the blood specimen, and an aliquot of the reaction mixture was transferred to the matrix cell. The matrix cell was washed, removing unbound materials, and the anti-AFP conjugate was dispensed onto the matrix cell. The substrate was added to the matrix cell, and the fluorescent product is measured by the MEIA optical assembly.

Cell culture

RPMI 1640 medium, penicillin and streptomycin, and 10% heat-inactivated FCS were purchased from Invitrogen Life Technologies (Carlsbad, CA). Purified human cord blood AFP (purity, >95%; SDS-PAGE) and purified human albumin (purity, >97%; SDS-PAGE) were obtained from Calbiochem (San Diego, CA) and Sigma-Aldrich (St. Louis, MO), respectively. Recombinant human GM-CSF and IL-4 were purchased from PeproTech (Rocky Hill, NJ).

Monocyte purification and generation of monocyte-derived DCs

Mononuclear cells were isolated from peripheral blood by centrifugation on Ficoll-Hypaque (Amersham Pharmacia, Uppsala, Sweden). Mononuclear cells from healthy individuals were incubated in 96-well plates in RPMI 1640 for 30 min, and nonadherent cells were removed by gentle wash. DCs were generated as described previously (18). Briefly, adherent cells were cultured in DC medium (RPMI 1640 supplemented with 10% FCS) containing GM-CSF (500 IU/ml) and IL-4 (250 IU/ml) and in the presence or absence of AFP or human serum albumin (HSA). On days 3 and 5, the cells were fed with the DC medium and the above cytokines. Where indicated, AFP or HSA was added on day 5 to study the effect of AFP on different stages of DC differentiation. DC maturation was induced by the addition of LPS (500 ng/ml; Sigma-Aldrich) to the culture on day 7 for 24 h.

Analysis of DC surface markers and apoptosis

DCs were stained with FITC- or PE-labeled mAbs (anti-human CD1a, CD11c, CD14, CD19, CD20, CD40, CD80, CD83, CD86, HLA-DR, or relevant isotype controls; BD Pharmingen, San Diego, CA) according to the manufacturer’s instructions. Cells were gated according to their size (forward light scatter) and granularity (side light scatter) using a FACScan flow cytometer (BD Immunocytometry Systems, San Diego, CA). The DC surface marker expression was analyzed using the CellQuest program (BD Immunocytometry Systems). DC apoptosis was detected using Annexin VFITC, with dead cells identified by propidium iodide (PI) staining (BD Pharmingen).

Mixed leukocyte reaction

To avoid DC maturation, DCs were harvested following gamma irradiation. Gamma-irradiated allostimulatory DCs were incubated in round-bottom microtiter plates with 105 allogeneic T cells. Triplicate cultures were maintained for 5 days at 37°C in a 5% CO2 humidified atmosphere. T cell proliferation was measured by pulsing cells with 1 µCi of methylthymidine (Amersham Pharmacia) for 18 h.

Intracellular cytokine assay

PBMCs or DCs were cultured in a medium containing LPS (500 ng/ml) and brefeldin A for 3 h. The cells were stained with anti-HLA-DR Ab, permeabilized, fixed, and stained with anti-human IL-12, IL-10, TNF-{alpha}, or isotype control Abs. The expression of intracellular cytokines was analyzed using flow cytometry.

ELISA

Levels of biologically active IL-12 p70 and nonactive IL-12 p40 were measured using ELISA kit obtained from R&D Systems (Minneapolis, MN). ELISAs were performed in duplicate according to the manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AFP impairs DC function in vitro

In this study, we have examined the effects of AFP on the function of monocyte-derived DCs generated from healthy individuals. AFP was added at concentration similar to that of AFP level reported in the sera of patients with HCC. As assessed by flow cytometry, monocyte-derived DCs cultured for 7 days in the presence of GM-CSF and IL-4 developed into DCs characterized by the acquisition of CD1a and CD11c and loss of CD14 molecules. They expressed high levels of HLA-DR and CD1a molecules but did not express CD14, CD16, CD19, or CD20 (Fig. 1a). Addition of AFP (as low as 2500 ng/ml) on day 0 of culture induced phenotypical alteration of DCs (Fig. 1b). However, this was not demonstrated with the addition of HSA. CD86 expression was substantially down-regulated, and a slight reduction of CD40 median fluorescence was detected. The expressions of CD1a, HLA-DR, and CD83 did not alter (Fig. 1a). Similar results were obtained when DCs were cultured in a medium containing 10% human serum (data not shown). When AFP was added on day 5 or 6 instead of day 0, a less pronounced decrease in CD86 expression was observed (data not shown). Addition of LPS on day 7 resulted in up-regulation of CD40, CD83, and CD86 molecules 24 h later (Fig. 1c). However the expression levels of the surface molecules on the AFP-treated mature DCs were still significantly lower than that of the nontreated mature DCs (Fig. 1c).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 1. DCs treated with AFP express low levels of CD86 molecules. Monocytes were cultured with GM-CSF and IL-4, in the presence or absence of different concentrations (25,000 ng/ml in a and c; 12,500, 5,000, or 2,500 ng/ml in b) of AFP or 25,000 ng/ml HSA. Cells were harvested on day 7 of culture, stained with different mAbs, and analyzed using flow cytometry. Cells were stained with PE-labeled mAbs. A PE-labeled, isotype-matched control Ig was used (a–c). Cells were also stained with FITC-labeled anti-HLA-DR and PE-labeled anti-CD14, CD16, CD19, and CD20 (a). Numbers represent the percentage of cells expressing CD83, CD86, CD40, HLA-DR, and CD1a. Sideward light scatter (SSC) and forward light scatter (FSC) characteristics are shown (a and b). Results were similar in four different independent experiments. On day 7, cells were treated with LPS to induce DC maturation, and the expression of surface molecules was analyzed on day 8 (c). Results were similar in three different independent experiments.

 
AFP-treated DCs are poor stimulators of allogeneic T cell response in vitro

AFP-treated DCs, but not HSA-treated or nontreated DCs, were shown to have low allostimulatory capacity. Graded numbers of viable DCs were cocultured with allogeneic T cells for 5 days in an MLR assay. The allostimulatory function of AFP-treated immature DCs was significantly reduced in a dose-dependent manner (Fig. 2, a and b). HSA did not inhibit allostimulatory function of DCs. A significant inhibitory effect of AFP (25,000 ng/ml) was also detected when AFP was added on day 5 (data not shown).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. AFP reduces the DC allostimulatory function in a dose-dependent manner. Monocytes were cultured in the presence of GM-CSF and IL-4. AFP or HSA was added at different concentrations (25, 12.5, 5, or 2.5 µg/ml) (b) or 25 µg/ml (a and c) on day 0 of culture. Immature DCs (a and b) (day 7) or mature DCs (c) (day 8 after LPS stimulation for 24) were harvested, and graded numbers of viable cells were added to allogeneic T lymphocytes. After 5 days, T cell proliferation was assessed by the addition of thymidine for 18 h. The data shown are average counts per minute of three replicate determinations ± SD and are representative of three similar experiments.

 
AFP-treated immature DCs were stimulated with LPS for 24 h to induce DC maturation. The allostimulatory ability of AFP-treated mature DCs (10,000 cells/well) was analyzed. AFP-treated mature DCs had significantly lower T cell-stimulatory capacity than that of nontreated mature DCs (Fig. 2c).

AFP induces apoptosis of DCs in vitro

The actual DC and PBMC recovery was determined after 7-day culture in the presence or absence of AFP. The cell recovery in the AFP-treated DCs, but not AFP-treated PBMCs, was significantly reduced. In the DC cultures, the number of cells recovered was 113,000 ± 10,000 for the AFP-treated group, and 236,000 ± 10,000 for the nontreated group. In PBMC cultures, the number of cells recovered was 143,000 ± 12,000 for the AFP-treated group, and 123,000 ± 4,000 for the nontreated group. The reduction of cell viability in the DC cultures after exposure to AFP may be due to induction of apoptosis. To investigate this possibility, cells were treated with AFP on day 0 of culture and analyzed on day 7 for the presence of apoptotic cells. AFP induced a significant increase in the number of apoptotic DCs as assessed by Annexin VFITC/PI staining (Fig. 3a). To test whether AFP exerts apoptotic effects on lymphocytes, PBMCs were treated with AFP for 7 days and annexin V binding was assessed on CD3+ cells. AFP did not induce apoptosis of CD3+ cells (Fig. 3a).



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3. AFP induces apoptosis of monocyte-derived DCs but not in CD3+ cells. Monocytes were cultured in the presence of GM-CSF and IL-4, and AFP or HSA (25,000 ng/ml) was added on day 0 of culture. PBMC were also cultured in the presence of AFP or HSA (25,000 ng/ml) for 7 days. A two- or three-color staining with annexin V and PI with (c) or without (a) anti-CD86 Ab was conducted on day 7 of DC culture. A three-color staining was performed on PBMC culture (annexin V, PI, and anti-CD3 Ab). Numbers represent the percentage of cells in each quadrant. The cells in PBMC culture were gated on CD3+ cells, and the expression of annexin V-positive, PI-negative cells (apoptotic cells) was analyzed (a). The cells in DC culture were gated on nonapoptotic cells (annexin V-negative, PI-negative), and the expression levels of CD86 molecules were analyzed (b). The counted cells for the AFP-treated group were 340,000 cells, and for nontreated and HSA-treated group was 10,000 cells. AFP-treated or nontreated nonapoptotic DCs, i.e., annexin V-negative, PI-negative cells, were added (20,000 cells/well) to allogeneic T lymphocytes. After 5 days, T cell proliferation was assessed by the addition of thymidine for 18 h (c). The data shown are average counts per minute of three replicate determinations ± SD. The data are representative of three similar experiments.

 
AFP inhibitory effects on DCs could be due to the induction of DC apoptosis or the suppression of DC function or both. We characterized the phenotype (Fig. 3b) and the function (c) of nonapoptotic DCs. The cells were gated on annexin V-negative, PI-negative cells (nonapoptotic), and the expression levels of CD86 molecule were analyzed. The nonapoptotic DCs exposed to AFP expressed slightly lower levels of CD86 than that on nonapoptotic, nontreated DCs (Fig. 3b). To test the functional ability of nonapoptotic DCs, we first determined the percentage of apoptotic cells in the cultures (annexin V-positive, PI-negative cells). The ability of nonapoptotic cells to stimulate T cell proliferation was determined in an MLR assay. The number of nonapoptotic cells in unsorted cells was estimated, and AFP-treated or nontreated nonapoptotic DCs, i.e., annexin V-negative, PI-negative cells, were added (20,000 cells/well) to allogeneic T lymphocytes. After 5 days, T cell proliferation was assessed by the addition of thymidine for 18 h. The nonapoptotic cells from AFP-treated group had a low ability to stimulate T cell proliferation (Fig. 3c).

AFP-treated DCs produce lower levels of TNF-{alpha} and IL-12 in vitro

DCs were stimulated with LPS for 3 h, and the production of intracellular TNF-{alpha}, IL-12, and IL-10 was analyzed. DCs that were differentiated in the presence of AFP had an impaired ability to produce IL-12 and TNF-{alpha}. The lowest level of AFP used in vitro with inhibitory effects on IL-12 production was 12,500 ng/ml. HSA treatment did not significantly reduce the ability of DCs to produce TNF-{alpha} and IL-12. When AFP was added on day 5 instead of day 0 of culture, a significant decrease in IL-12 production occurred. However, a greater decrease was seen when AFP was added on day 0 (Fig. 4). The serum AFP levels in patients with HCC (n = 16) are shown in Fig. 5. The lowest amount of AFP used in vitro to induce CD86 down-regulation on DC was 2,500 ng/ml and for inhibition of IL-12 production was 12,500 ng/ml. There was an undetectable level of IL-10-producing cells in all three experimental groups (data not shown).



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 4. AFP treatment impairs IL-12 and TNF-{alpha} production from monocyte-derived DCs. Monocytes were cultured in the presence of GM-CSF, IL-4, and different concentrations of AFP (25,000 ng/ml in a and b; 12,500 or 5,000 ng/ml in c) or HSA (25,000 ng/ml) were added on day 0 (a and c) or 5 (b) of culture. Cells were harvested on day 7 of culture and stimulated in vitro with LPS. Cells were stained with PE-conjugated mAb for intracellular IL-12 or TNF-{alpha}, and analyzed by flow cytometry. A PE-labeled isotype-matched mAb was also used. Numbers represent the percentage of cells expressing the intracellular cytokines. The results were similar in three independent experiments.

 


View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 5. AFP induces functional impairment of DCs at concentrations close to those detected in the sera from patients with HCC. The level of serum AFP in patients with HCC was measured using MEIA. The dots represent results from patients. The lowest levels of AFP used in culture that had inhibitory effects (down-regulation of CD86 molecule and reduced IL-12 production) are shown.

 
The level of IL-12 (p70) and IL-12 (p40) in supernatant of AFP-treated DCs (1 x 105/well) was measured using ELISA after 48-h stimulation with LPS. AFP-treated DCs produced lower levels of IL-12 (p40) and bioactive IL-12 (p70) than that of nontreated DCs. No IL-12 (p40) or IL-12 (p70) was detected in the supernatant of AFP-treated DCs. The levels of IL-12 (p40) and IL-12 (p70), produced by nontreated DCs, were 2200 ± 230 and 508 ± 127 pg/ml, respectively.

APCs of patients with HCC produce low levels of TNF-{alpha} ex vivo

Flow cytometry was used to examine TNF-{alpha} secretion profiles of PBMCs in patients with HCC (n = 16) and healthy individuals (n = 7), in response to stimulation with LPS. The cells were gated on HLA-DR-positive or CD14-positive cells, and the expression of intracellular TNF-{alpha} was determined. In both healthy individuals and HCC patients, 70–98% of TNF-{alpha}-producing cells expressed high levels of HLA-DR molecule and 71–93% expressed CD14 molecule. The percentage of TNF-{alpha}-producing cells was lower in HCC patients with high levels of serum AFP than that of the healthy group (Fig. 6). To examine whether the reduction of TNF-{alpha} production could be attributed to the loss of HLA-DR-positive cells, the percentage and mean fluorescence intensity of cells expressing HLA-DR molecules were analyzed. No significant loss of HLA-DR-positive cells was observed (Fig. 6, b and c). When the ability of HLA-DR-positive cells to produce IL-10 was analyzed, there was no significant difference between patients with HCC and healthy individuals (data not shown).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 6. PBMCs of HCC patients with high levels of serum AFP produced lower TNF-{alpha} ex vivo. PBMCs were stimulated with LPS for 3 h, harvested, and stained with FITC-labeled anti-HLA-DR mAb for surface molecules and PE-labeled TNF-{alpha} for intracellular cytokine. Cells were analyzed using flow cytometry, and the percentages of HLA-DR+TNF-{alpha}+ cells were determined. The closed dots ({blacksquare}) represent results obtained from patients with HCC (n = 16), and open dots ({otimes}) represent results obtained from healthy controls (n = 7), with the y-axis representing percentage of HLA-DR+TNF-{alpha}+ cells and the x-axis representing the levels of serum AFP (a). The percentage (b) and mean fluorescent intensity (c) of cells expressing HLA-DR molecules in PBMC of patients with HCC and healthy individuals are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inadequate presentation of tumor Ags by the host professional APCs is one potential mechanism by which tumors evade the host’s immune response. Several clinical observations suggest that DC infiltration of solid tumors correlates with better prognosis (19). Conversely, impairment of the DCs is associated with increased immunosuppression and tumor progression (20, 21, 22, 23, 24). It has been shown that both circulating DCs ex vivo (12) and monocyte-derived DCs (25) are functionally impaired in patients with HCC. The impairment of DC function has also been reported in other tumors, a process mostly related to tumor-derived soluble factors, several of which have been identified (20, 21, 26). A large variety of murine and human tumor cell lines produce as-yet-unidentified soluble factors that cause increased apoptosis of DCs.

In this study, the effect of AFP on the functions and viability of DCs was analyzed in vitro. The data clearly show that DCs treated with AFP (as low as 2,500 ng/ml) become dysfunctional. These cells express low levels of costimulatory molecules and produce low levels of IL-12 and TNF-{alpha}, which correlates with their low allostimulatory capacity. Furthermore, the results suggest that the impairment of DCs is not only due to apoptosis. Further studies are required to determine the inhibitory effects of AFP on DC differentiation in vivo. We used a concentration of AFP (2,500–25,000 ng/ml) that is in a range similar to that detected in the sera of patients with HCC (ranging from 8 to 1,141,205 ng/ml).

Many patients with HCC have a serum AFP level of <12,500 ng/ml, and this may imply that the level of serum AFP in most patients is not high enough to exert immunoregulatory effects. However, one can hypothesize that higher concentrations of AFP can be found in the vicinity of the tumors where tissue-specific or infiltrating APCs may be exposed to high levels of AFP and therefore become dysfunctional. Our ex vivo results support this notion and show that the ability of monocytes, isolated from patients with elevated levels of serum AFP (>1,000 ng/ml), to produce TNF-{alpha} is impaired. This was not observed for monocytes isolated from PBMCs of patients with low serum AFP (<200 ng/ml). In this study, we decided to analyze monocytes rather than DCs as the majority of TNF-{alpha}-producing cells in peripheral blood are monocytes (10% of PBMCs) and DC subsets comprise only a very small percentage. In addition, to analyze DC subsets, we would have needed large amounts of blood from patients who had already severe pancytopenia. Further studies are required to determine the functional ability of DC subsets in patients with HCC. The impairment of TNF-{alpha} production by monocytes may have important clinical implications and prognostic values. However, one cannot assume that the dysfunction of the monocytes (i.e., impairment of TNF-{alpha} production) is solely attributable to high levels of AFP. Other regulatory soluble factors released by the tumors may contribute to the impairment of APCs. For example, an elevation of TGF-{beta} level has been reported in the serum of patients with HCC (27). TGF-{beta} is known to have the ability to suppress APCs (28). In addition to soluble factors, regulatory T cells can also suppress DC function (29), and several studies have shown that the frequency of the regulatory T cells is significantly increased in some patients with cancer (30, 31), including HCC (our unpublished data). However, the mechanisms by which regulatory T cells suppress APCs in patients with HCC remains poorly understood.

In conclusion, this study demonstrates for the first time that AFP severely impairs the function of DCs and induces their apoptosis. In addition, the ability of APCs, of patients with HCC and high levels of serum AFP, to produce proinflammatory cytokines is reduced. This provides new insights into understanding the mechanisms underlying the suppression of immune recognition of tumor in patients with HCC.


   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 CRDC project grant. We are also grateful to de Laszlo Foundation, Citrina Foundation, and Alex and Madelaine von Nolcken for their support. S.H.U. was a visiting scientist from Department of Internal Medicine at Korea University College of Medicine, and he was supported by Clinical Research Fund of Korean Association for the Study of the Liver and GlaxoSmithKline Korea. Back

2 Address correspondence and reprint requests to Dr. Shahriar Behboudi, Institute of Hepatology, University College London, 69-75 Chenies Mews, London WC1E 6HX, U.K. E-mail address: s.bedhoudi{at}ucl.ac.uk Back

3 Abbreviations used in this paper: AFP, {alpha}-fetoprotein; HCC, hepatocellular carcinoma; DC, dendritic cell; MEIA, microparticle enzyme immunoassay; HSA, human serum albumin; PI, propidium iodide. Back

Received for publication October 7, 2003. Accepted for publication May 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Deutsch, H. F.. 1991. Chemistry and biology of {alpha}-fetoprotein. Adv. Cancer Res. 56:253.[Medline]
  2. Nguyen, M. H., R. T. Garcia, P. W. Simpson, T. L. Wright, E. B. Keeffe. 2002. Racial differences in effectiveness of {alpha}-fetoprotein for diagnosis of hepatocellular carcinoma in hepatitis C virus cirrhosis. Hepatology 36:410.[Medline]
  3. Tong, M. J., S. Govindarajan. 1988. Primary hepatocellular carcinoma following perinatal transmission of hepatitis B. West. J. Med. 148:205.[Medline]
  4. Laan-Putsep, K., H. Wigzell, P. Cotran, M. Gidlund. 1991. Human {alpha}-fetoprotein (AFP) causes a selective down regulation of monocyte MHC class II molecules without altering other induced or noninduced monocyte markers or functions in monocytoid cell lines. Cell. Immunol. 133:506.[Medline]
  5. Lu, C. Y., P. S. Changelian, E. R. Unanue. 1984. {alpha}-Fetoprotein inhibits macrophage expression of Ia antigens. J. Immunol. 132:1722.[Abstract]
  6. Murgita, R. A., L. C. Andersson, M. S. Sherman, H. Bennich, H. Wigzell. 1978. Effects of human {alpha}-foetoprotein on human B and T lymphocyte proliferation in vitro. Clin. Exp. Immunol. 33:347.[Medline]
  7. Murgita, R. A., E. A. Goidl, S. Kontianen, H. Wigzell. 1977. {alpha}-Fetoprotein induces suppressor T cells in vitro. Nature 267:257.[Medline]
  8. Peck, A. B., R. A. Murgita, H. Wigzell. 1978. Cellular and genetic restrictions in the immunoregulatory activity of {alpha}-fetoprotein. I. Selective inhibition of anti-Ia-associated proliferative reactions. J. Exp. Med. 147:667.[Abstract/Free Full Text]
  9. Peck, A. B., R. A. Murgita, H. Wigzell. 1982. Cellular and genetic restrictions in the immunoregulatory activity of {alpha}-fetoprotein. III. Role of the MLC-stimulating cell population in {alpha}-fetoprotein-induced suppression of T cell-mediated cytotoxicity. J. Immunol. 128:1134.[Abstract]
  10. Murgita, R. A., T. B. Tomasi, Jr. 1975. Suppression of the immune response by {alpha}-fetoprotein on the primary and secondary antibody response. J. Exp. Med. 141:269.[Abstract/Free Full Text]
  11. Semeniuk, D. J., R. Boismenu, J. Tam, W. Weissenhofer, R. A. Murgita. 1995. Evidence that immunosuppression is an intrinsic property of the {alpha}-fetoprotein molecule. Adv. Exp. Med. Biol. 383:255.[Medline]
  12. Aussel, C., M. Fehlmann. 1987. Effect of {alpha}-fetoprotein and indomethacin on arachidonic acid metabolism in P388D1 macrophages: role of leukotrienes. Prostaglandins Leukotrienes Med. 28:325.[Medline]
  13. Suzuki, Y., C. Q. Zeng, E. Alpert. 1992. Isolation and partial characterization of a specific {alpha}-fetoprotein receptor on human monocytes. J. Clin. Invest. 90:1530.
  14. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  15. Mellman, I., R. M. Steinman. 2001. Dendritic cells: specialized and regulated antigen processing machines. Cell 106:255.[Medline]
  16. Moretta, A.. 2002. Natural killer cells and dendritic cells: rendezvous in abused tissues. Nat. Rev. Immunol. 2:957.[Medline]
  17. Smyth, M. J., N. Y. Crowe, Y. Hayakawa, K. Takeda, H. Yagita, D. I. Godfrey. 2002. NKT cells—conductors of tumor immunity?. Curr. Opin. Immunol. 14:165.[Medline]
  18. Behboudi, S., D. Chao, P. Klenerman, J. Austyn. 2000. The effects of DNA containing CpG motif on dendritic cells. Immunology 99:361.[Medline]
  19. Vicari, A. P., C. Caux, G. Trinchieri. 2002. Tumour escape from immune surveillance through dendritic cell inactivation. Semin. Cancer Biol. 12:33.[Medline]
  20. Gabrilovich, D. I., I. F. Ciernik, D. P. Carbone. 1996. Dendritic cells in antitumor immune responses. I. Defective antigen presentation in tumor-bearing hosts. Cell. Immunol. 170:101.[Medline]
  21. Menetrier-Caux, C., G. Montmain, M. C. Dieu, C. Bain, M. C. Favrot, C. Caux, J. Y. Blay. 1998. Inhibition of the differentiation of dendritic cells from CD34+ progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor. Blood 92:4778.[Abstract/Free Full Text]
  22. Enk, A. H., H. Jonuleit, J. Saloga, J. Knop. 1997. Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma. Int. J. Cancer 73:309.[Medline]
  23. Bell, D., P. Chomarat, D. Broyles, G. Netto, G. M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K. A. Palucka, J. Banchereau. 1999. In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J. Exp. Med. 190:1417.[Abstract/Free Full Text]
  24. Kunitani, H., Y. Shimizu, H. Murata, K. Higuchi, A. Watanabe. 2002. Phenotypic analysis of circulating and intrahepatic dendritic cell subsets in patients with chronic liver diseases. J. Hepatol. 36:734.[Medline]
  25. Ninomiya, T., S. M. Akbar, T. Masumoto, N. Horiike, M. Onji. 1999. Dendritic cells with immature phenotype and defective function in the peripheral blood from patients with hepatocellular carcinoma. J. Hepatol. 31:323.[Medline]
  26. Peguet-Navarro, J., M. Sportouch, I. Popa, O. Berthier, D. Schmitt, J. Portoukalian. 2003. Gangliosides from human melanoma tumors impair dendritic cell differentiation from monocytes and induce their apoptosis. J. Immunol. 170:3488.[Abstract/Free Full Text]
  27. Sacco, R., D. Leuci, C. Tortorella, G. Fiore, F. Marinosci, O. Schiraldi, S. Antonaci. 2000. Transforming growth factor {beta}1 and soluble Fas serum levels in hepatocellular carcinoma. Cytokine 12:811.[Medline]
  28. Kobie, J. J., R. S. Wu, R. A. Kurt, S. Lou, M. K. Adelman, L. J. Whitesell, L. V. Ramanathapuram, C. L. Arteaga, E. T. Akporiaye. 2003. Transforming growth factor {beta} inhibits the antigen-presenting functions and antitumor activity of dendritic cell vaccines. Cancer Res. 63:1860.[Abstract/Free Full Text]
  29. Shevach, E. M.. 2002. CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2:389.[Medline]
  30. Woo, E. Y., C. S. Chu, T. J. Goletz, K. Schlienger, H. Yeh, G. Coukos, S. C. Rubin, L. R. Kaiser, C. H. June. 2001. Regulatory CD4+CD25+ T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer Res. 61:4766.[Abstract/Free Full Text]
  31. Wolf, A. M., D. Wolf, M. Steurer, G. Gastl, E. Gunsilius, B. Grubeck-Loebenstein. 2003. Increase of regulatory T cells in the peripheral blood of cancer patients. Clin. Cancer Res. 9:606.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
D.-M. Kuang, Q. Zhao, J. Xu, J.-P. Yun, C. Wu, and L. Zheng
Tumor-Educated Tolerogenic Dendritic Cells Induce CD3{epsilon} Down-Regulation and Apoptosis of T Cells through Oxygen-Dependent Pathways
J. Immunol., September 1, 2008; 181(5): 3089 - 3098.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Alisa, S. Boswell, A. A. Pathan, L. Ayaru, R. Williams, and S. Behboudi
Human CD4+ T Cells Recognize an Epitope within {alpha}-Fetoprotein Sequence and Develop into TGF-{beta}-Producing CD4+ T Cells
J. Immunol., April 1, 2008; 180(7): 5109 - 5117.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
D. S. Wilkinson, W.-W. Tsai, M. A. Schumacher, and M. C. Barton
Chromatin-Bound p53 Anchors Activated Smads and the mSin3A Corepressor To Confer Transforming Growth Factor {beta}-Mediated Transcription Repression
Mol. Cell. Biol., March 15, 2008; 28(6): 1988 - 1998.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
G. J. Mizejewski
Physiology of Alpha-Fetoprotein as a Biomarker for Perinatal Distress: Relevance to Adverse Pregnancy Outcome
Experimental Biology and Medicine, September 1, 2007; 232(8): 993 - 1004.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Ayaru, S. P. Pereira, A. Alisa, A. A. Pathan, R. Williams, B. Davidson, A. K. Burroughs, T. Meyer, and S. Behboudi
Unmasking of {alpha}-Fetoprotein-Specific CD4+ T Cell Responses in Hepatocellular Carcinoma Patients Undergoing Embolization
J. Immunol., February 1, 2007; 178(3): 1914 - 1922.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
L. H. Butterfield, A. Ribas, V. B. Dissette, Y. Lee, J. Q. Yang, P. De la Rocha, S. D. Duran, J. Hernandez, E. Seja, D. M. Potter, et al.
A Phase I/II Trial Testing Immunization of Hepatocellular Carcinoma Patients with Dendritic Cells Pulsed with Four {alpha}-Fetoprotein Peptides.
Clin. Cancer Res., May 1, 2006; 12(9): 2817 - 2825.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Cui, T. T. Nguyen, J. H. Taube, S. A. Stratton, M. H. Feuerman, and M. C. Barton
Family Members p53 and p73 Act Together in Chromatin Modification and Direct Repression of {alpha}-Fetoprotein Transcription
J. Biol. Chem., November 25, 2005; 280(47): 39152 - 39160.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
A. Alisa, A. Ives, A. A. Pathan, C. V. Navarrete, R. Williams, A. Bertoletti, and S. Behboudi
Analysis of CD4+ T-Cell Responses to a Novel {alpha}-Fetoprotein-Derived Epitope in Hepatocellular Carcinoma Patients
Clin. Cancer Res., September 15, 2005; 11(18): 6686 - 6694.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Um, S. H.
Right arrow Articles by Behboudi, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Um, S. H.
Right arrow Articles by Behboudi, S.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Liver Cancer

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS