Pepro Tech

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakamoto, Y.
Right arrow Articles by Kobayashi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakamoto, Y.
Right arrow Articles by Kobayashi, K.
(Journal of Leukocyte Biology. 2002;72:49-55.)
© 2002 by Society for Leukocyte Biology

Increased susceptibility to apoptosis and attenuated Bcl-2 expression in T lymphocytes and monocytes from patients with advanced chronic hepatitis C

Yasunari Nakamoto, Shuichi Kaneko and Kenichi Kobayashi

Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, Japan

Correspondence: Shuichi Kaneko, M.D., Ph.D., Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan. E-mail: skaneko{at}medf.m.kanazawa-u.ac.jp


arrow
ABSTRACT
 
Some viral infections are reported to influence the susceptibility of peripheral blood mononuclear cells (PBMC) to apoptosis, which is related to disease progression. The current study was designed to monitor apoptosis in separated PBMC subsets, CD4+ and CD8+ T lymphocytes, and CD14+ monocytes under apoptotic stimuli in patients with chronic hepatitis C. Apoptosis was induced by serum starvation and by incubation with anti-CD3 antibody and with phorbol 12-myristate 13-acetate. With the escalating severity of liver disease, susceptibility of all PBMC subsets to apoptosis increased under the apoptotic stimulus of serum starvation (P<0.05). Consequently, increased susceptibility to apoptosis was associated with diminished intracellular expression of the antiapoptotic protein Bcl-2 (P<0.05). The current observations demonstrate that the abnormality of PBMC subsets in undergoing apoptosis as a result of the down-regulation of Bcl-2 expression may contribute to viral persistence and progression of liver disease in chronic hepatitis C.

Key Words: peripheral blood mononuclear cells • hepatitis C virus • serum starvation


arrow
INTRODUCTION
 
Hepatitis C virus (HCV) causes a prolonged and persistent infection that progresses to liver cirrhosis (LC) and eventually induces an approximately 400-fold increase in the risk of developing hepatocellular carcinoma (HCC) [1 , 2 ]. The mechanisms responsible for viral persistence and disease progression in HCV infection are not well defined; however, at least two hypotheses have been proposed [3 ]. One hypothesis states that the virus has evolved a mechanism to evade the immune response, probably through the generation of viral variants during infection. These variants are capable of escaping neutralizing antibody or cytolytic T lymphocyte recognition. We, and others, have demonstrated evidence that heterogeneity in the hypervariable domain within the envelope E2 protein may be accompanied by failure to mount a humoral immune response against the virus [4 5 6 7 8 ]. This strategy is consistent with the high error rate of the viral polymerase present during viral RNA replication. Furthermore, this hypothesis may account for the high frequency of HCV quasispecies detected in HCV-infected patients [4 ].

An alternative hypothesis maintains that immune dysfunction of effector cells results in insufficient clearance of the virus. Consequently, persistent infection occurs. CD4+ and CD8+ T lymphocytes and monocytes play a critical role in the control of viral replication [9 ]. In patients presenting with chronic HCV infection, cell-mediated immune response by CD4+ and CD8+ T lymphocytes to the virus is not as strong as that observed in acutely infected patients in which a vigorous T lymphocyte response is displayed, demonstrating virus clearance and eradication of infected cells [10 11 12 ]. Monocytes/macrophages are known to be activated in inflamed liver and to secrete antiviral cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}) [13 ]. Conversely, dysfunction of these cells results in insufficient T cell response. Thus, the predominant cause of viral persistence in chronic HCV infection may be the development of an insufficient antiviral immune response. However, the immunological basis has yet to be determined.

It has been reported that some viral infections influence the susceptibility of peripheral blood mononuclear cells (PBMC) to apoptosis with dysfunction of the Fas ligand (FasL)/Fas and TNF-{alpha} death pathways and the Bcl-2 family, which are directly related to viral persistence and disease progression [14 15 16 17 18 19 20 21 ]. Thus, changes in the susceptibility of PBMC subsets to apoptosis may be a plausible mechanism describing the insufficient antiviral immune responses leading to persistent viral infection and disease progression.

The susceptibility of PBMC to apoptosis is not well defined in patients presenting with chronic HCV infection. We have previously observed apoptosis of unseparated PBMC obtained from patients exhibiting various degrees of chronic viral hepatitis in the absence of apoptotic stimuli in vitro. We found no differences in mean (%) PBMC mortality in these patients [22 ]. In the current study, we monitored apoptosis of separated PBMC subsets, CD4+ and CD8+ T lymphocytes, and CD14+ monocytes under apoptotic stimuli. The results suggest that the susceptibility of CD4+ and CD8+ T lymphocyte and CD14+ monocyte subsets to apoptosis escalates under the apoptotic stimulus of serum starvation. This observation is most likely a result of the down-regulation of Bcl-2 expression in patients with advanced chronic hepatitis (CH) C.


arrow
MATERIALS AND METHODS
 
Patients
Sixty-four patients attending Kanazawa University Hospital between May 1999 and June 2000 were enrolled in this study with their informed consent (Table 1 ). Forty-eight of these individuals tested positive for antibodies against HCV (anti-HCV). Fourteen of the 48 participants presented with histological diagnosis of CH, 14 subjects demonstrated LC, and 20 patients displayed cirrhosis with HCC (HCC). The remaining 16 patients without chronic liver disease and negative for anti-HCV (nonC) were also enrolled as controls. Clinical and laboratory characteristics of patients are summarized in Table 1 . When assessed by the Kruskal-Wallis and Mann-Whitney U tests or by Fisher’s exact test, no significant differences were observed between groups with respect to mean age, sex, white cell count, lymphocyte count, and serum levels of alanine transaminase and total bilirubin. Platelet and hepaplastin tests decreased significantly (P<0.01) with increasing severity of liver disease.


View this table:
[in this window]
[in a new window]
  		Table 1. Clinical Characteristics of Patients Studied

Cell isolation and culture
PBMC were isolated from heparinized venous blood via separation with Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) density gradient centrifugation. Subset separation of CD4+ and CD8+ T lymphocytes and CD14+ monocytes was performed by positive selection using magnetic cell sorting systems (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, the separated cell subsets were analyzed by double staining with fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibodies (mAb) specific for CD4 (RPA-T4), CD8 (HIT8a), or CD14 (M5E2; PharMingen, San Diego, CA) and phycoerythrin (PE)-conjugated mAb specific for CD3 (UCHT1) or CD11c (B-ly6; PharMingen). Quantitation was conducted via flow cytometric analysis using an EPICS® Elite (Coulter, Hialeah, FL). Subsets exhibiting purities of greater than 96% were incubated in complete RPMI culture medium (Gibco, Gaithersburg, MD) containing 10% heat-inactivated fetal calf serum (FCS; Gibco), 2 mM L-glutamine, 1 mM sodium pyruvate, streptomycin (50 µg/ml), penicillin (50 U/ml), and gentamicin (2 µg/ml) at 1 x 106 cells/ml at 37°C with 5% CO2 for 18 h.

Induction of apoptosis
Following an 18 h incubation, each PBMC subset was individually incubated at 2.5 x 105 cells/ml in 96-well culture plates (Corning, Corning, NY) at 37°C with 5% CO2. Apoptosis was induced by serum starvation or by incubation with 1 µg/ml anti-CD3 antibody (UCHT1, PharMingen) for T cell receptor stimulation or with 10 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.) as a mitogen. Subsets were incubated with 10 µg/ml anti-TNF-{alpha} (1825.121, R&D Systems, Minneapolis, MN)- or anti-FasL (NOK2, PharMingen)-neutralizing antibodies or control immunoglobulin G (IgG) to determine the molecular basis of apoptosis induction as a consequence of the apoptotic stimuli.

Quantitation of apoptosis
Apoptotic cells of each subset were detected by double staining with propidium iodide (PI) and FITC-labeled Annexin V according to the protocols supplied by the manufacturer (PharMingen) 0, 12, and 24 h after incubation. Quantitation was conducted via flow cytometric analysis using an EPICS® Elite (Coulter).

RNase protection assay for Bcl-2 family gene expression
Total RNA (5–10 µg) was extracted from freshly isolated and 9 h serum-starved 5 x 106 PBMC and was subjected to RNase protection analysis to monitor the expression of the bcl-2 family genes, bcl-W, bcl-X(L), bfl-1, bad, bik, bak, bax, and bcl-2. In addition, the housekeeping gene L32 was evaluated. Analysis was performed using the multiprobe hAPO-2c according to the protocols supplied by the manufacturer (PharMingen). Autoradiography was conducted and analyzed on a BAS 1000 image analyzer (Fuji Photo Film, Tokyo, Japan). Each band corresponding to a bcl-2 family gene was quantitated and expressed as a percentage of the L32 band.

Intracellular staining for Bcl-2
Single staining of cell surface molecules was performed by a 30-min incubation at 4°C with PE-conjugated mouse mAb specific for CD4 (RPA-T4), CD8 (HIT8a), and CD14 (M5E2) purchased from PharMingen and PE-conjugated control mouse IgG in phosphate-buffered saline containing 1% bovine serum albumin. For double staining of cell surface molecules and intracytoplasmic Bcl-2, PBMC were successively fixed/permeabilized with 1% paraformaldehyde for 15 min at room temperature followed by 70% methanol for 45 min at 4°C. Cells were washed and incubated with FITC-conjugated mouse anti-Bcl-2 mAb (124, Dako, Carpinteria, CA) for 30 min at 4°C as described [23 , 24 ]. After washing, cells were analyzed on an EPICS® Elite (Coulter).

Immunoblotting for Bcl-2
Protein extraction and immunoblotting were performed as described [25 ] with minor modifications. Samples (20 µg) from PBMC were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes. On the same gel, the indicated amounts of Jurkat cell (human T leukemia cell, American Type Culture Collection, Manassas, VA) lysates were loaded as standards. Following probing with anti-Bcl-2 mAb (Bcl-2 100, 1:200 dilution) from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-ß-actin (AC-74, 1:5000 dilution) from Sigma Chemical Co., the protein bands were detected by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK).

Statistical analysis
Results are expressed as means ± SD. Differences between groups were analyzed for statistical significance by the Kruskal-Wallis and Mann-Whitney U tests. Qualitative variables were compared by means of Fisher’s exact test. All tests were two-tailed, and a P value of <0.05 was considered statistically significant.


arrow
RESULTS
 
Induction of apoptosis in PBMC subsets
Freshly isolated PBMC subsets, CD4+, CD8+, and CD14+, exhibiting purities in excess of 96%, were exposed to apoptotic stimuli (Fig. 1 ). Detection of apoptosis was conducted by double staining with PI and FITC-labeled Annexin V and flow cytometric analysis. A small number of cells demonstrated characteristics of early apoptosis; i.e., cells were positive for Annexin V but negative for PI with respect to staining. However, the majority of apoptotic cells was positive for PI and Annexin V 12 h after incubation. In addition, apoptosis of the cells was characterized by two additional methods, light microscope assessment with trypan blue staining and fluorescence microscopy after staining with the nuclear dye Hoechst 33342 (not shown), as reported previously [22 ].



View larger version (55K):
[in this window]
[in a new window]
  		Figure 1. Isolation of CD4+ and CD8+ T lymphocyte subsets and CD14+ monocyte subset and detection of apoptosis induction. PBMC were isolated from heparinized venous blood of a nonC control using Ficoll-Hypaque density gradient centrifugation (upper panel). Subsets were subsequently separated, CD4+ and CD8+ T lymphocytes and CD14+ monocytes, by positive selection using MACS (middle panel). Double staining with the indicated mAb was performed and quantitated by flow cytometric analysis on an EPICS® Elite. Each PBMC subset exhibiting purity in excess of 96% was obtained. Following an 18 h incubation in complete RPMI culture medium, the subsets were individually incubated in RPMI medium containing 10% FCS for 12 h. Apoptotic cells of each subset were identified by double-staining with PI and FITC-labeled Annexin V and quantitated by flow cytometric analysis (lower panel). Each quadrant label shows the percentage of cells analyzed in each subset.

The proportion (%) of PI-positive cells in each subset was determined under apoptotic stimuli of serum starvation and incubation with PMA and anti-CD3 (Fig. 2 ). The mean cell mortalities of all three subsets increased significantly when apoptosis was induced by serum starvation in patients displaying LC and HCC in comparison with those of nonC controls. In contrast, no difference in mean percent mortalities of PBMC subsets incubated in media in the absence of apoptotic stimuli or in culture with PMA and anti-CD3 was observed. In addition, the results obtained after 12 h apoptosis induction (Fig. 2) were comparable to those after 24 h (not shown), suggesting the differences in mortality of PBMC subsets shown in Figure 2 were not a result of insufficiency of the apoptosis stimuli. The data indicate that the susceptibility of PBMC from patients exhibiting LC and HCC to apoptosis increased under apoptotic stimulation by serum starvation. This observation was suggestive of the immune dysfunction of CD4+ and CD8+ T lymphocytes and monocytes with increasing severity of liver disease.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2. Induction of apoptosis in freshly isolated PBMC subsets. PBMC subsets (pre-induction) obtained from 64 patients, 48 positive for anti-HCV (14 CH, 14 LC, 20 HCC) and 16 controls, were incubated separately at 2.5 x 105 cells/ml in 96-well culture plates at 37°C. Apoptosis was induced by serum starvation or by incubation with 1 µg/ml anti-CD3 antibody or with 10 ng/ml PMA. Apoptotic cells from each subset were detected by staining with PI, 0 (pre), 12, and 24 h following incubation, and were quantitated by flow cytometric analysis on an EPICS® Elite. The data presented are the means of cell mortality (%) at 12 h following apoptosis induction. *, P < 0.05 when compared with the nonC group by the Mann-Whitney U test. Mean cell mortality of all three subsets increased significantly when apoptosis was induced by serum starvation in patients with LC [mean percent mortality (±SD) of CD4+, CD8+, and CD14+ cell subsets: 53.8±4.0%, 65.9±12.3%, 69.8±8.8%, respectively] and HCC (53.6±10.5%, 66.3±14.0%, 70.1±7.8%, respectively) in comparison with nonC controls (38.2±5.6%, 38.5±7.3%, 61.4±2.7%, respectively).

Role of TNF-{alpha} and FasL in increased susceptibility to apoptosis
PBMC subsets were incubated with anti-TNF-{alpha}- and anti-FasL-neutralizing antibodies and control IgG to examine the molecular basis of stimulus-induced apoptosis (not shown). Mean cell mortalities were virtually identical, irrespective of the presence or absence of neutralizing antibodies. This finding suggests that the increased susceptibility of the cell subsets as a result of serum starvation is not a consequence of the differences of apoptotic stimuli through the FasL/Fas and TNF-{alpha} death pathways.

Role of Bcl-2 family in increased susceptibility to apoptosis
Bcl-2 is known to be a critical factor involved in the regulation of PBMC apoptosis under various conditions including chronic viral infections [26 27 28 29 30 ]. The levels of bcl-2 family gene expression were compared by RNase protection assay using the multiprobe for the expression of the proapoptotic members, bad, bik, bak, and bax, and the antiapoptotic members, bcl-W, bcl-X(L), bfl-1, and bcl-2 (Fig. 3A ). Relative expression levels were calculated in comparison with that of L32 (Fig. 3B) . With regard to freshly isolated PBMC, levels of bcl-W and bcl-X(L) mRNA expression were virtually identical in the patients; however, levels of bfl-1 bad, bak, bax, and bcl-2 expression varied. It is interesting that bcl-2 mRNA levels were higher in nonC and CH patients than that in LC and HCC patients. Furthermore, to understand the molecular changes in PBMC to influence the susceptibility to apoptosis, the kinetics of bcl-2 family gene expression was monitored during apoptosis induction. bcl-2 expression in nonC and CH patients decreased to levels similar to those displayed by LC and HCC patients after 9 h of apoptosis induction as a result of serum starvation. This result was consistent with studies noting that bcl-2 mRNA levels decrease because of growth factor deprivation [31 ].



View larger version (84K):
[in this window]
[in a new window]
  		Figure 3. Analysis of bcl-2 family gene expression in freshly isolated PBMC (P) and in PBMC serum-starved for 9 h (S). (A) Total RNA (5–10 µg) was extracted from 5 x 106 PBMC and subjected to RNase protection analysis to monitor the expression of bcl-2 family genes bcl-W, bcl-X(L), bfl-1, bad, bik, bak, bax, and bcl-2, as well as housekeeping gene L32. The multiprobe hAPO-2c (PharMingen) was used. Patients (Pt): 1, nonC; 2 and 3, CH; 4 and 5, LC; 6 and 7, HCC. Po, 2 µg HeLa control RNA; N, 4 µg yeast tRNA. Autoradiography was conducted using a BAS 2000 image analyzer. (B) Each band corresponding to a bcl-2 family gene was quantitated and expressed as a percentage of the L32 band. The data indicate means ± SD of three independent experiments. *, P < 0.05 when compared with freshly isolated PBMC of the nonC group by the Mann-Whitney U test.

Intracellular Bcl-2 protein was detected by specific mAb following cytoplasmic-membrane permeabilization so as to determine Bcl-2 expression at the protein level (Fig. 4A ). Bcl-2 expression of each cell subset was analyzed by double staining with PE-conjugated antibodies specific for cell surface molecules, CD4, CD8, or CD14, and FITC-conjugated antibody against Bcl-2. Expression levels were quantitated as mean fluorescence intensities (MFI) of gated dots positive for the cell surface molecules. Bcl-2 expression levels decreased in all PBMC subsets in a HCC patient (Fig. 4C) in comparison with the PBMC obtained from a CH patient (Fig. 4B) . In addition, differences in the expression level were confirmed by immunoblotting (Fig. 4D) in which the levels of Bcl-2 were decreased in the HCC patient (lane 2) compared with the CH patient (lane 1). Bcl-2 expression levels in each PBMC subset were quantitated, and the MFI in CH, LC, and HCC patients were compared with nonC controls using double staining methodology (Fig. 5 ). The levels of intracellular Bcl-2 were reduced in all subsets of LC and HCC patients (P<0.05). Consequently, this result may correlate with increased susceptibility of all subsets to apoptosis in the patients.



View larger version (61K):
[in this window]
[in a new window]
  		Figure 4. Analysis of intracellular Bcl-2 protein expression in freshly isolated PBMC subsets. (A) Intracellular Bcl-2 protein was stained following cytoplasmic-membrane permeabilization. Intracellular Bcl-2 was detected specifically by the mAb against Bcl-2. (B and C) PBMC isolated from patients with CH and HCC, respectively, were stained with PE-conjugated mAb specific for CD4, CD8, or CD14 or control IgG. After fixing/permeabilization, cells were incubated with FITC-conjugated anti-Bcl-2 mAb and analyzed on an EPICS® Elite. Bcl-2 expression was quantitated as MFI of gated dots positive for the indicated cell surface molecule. (D) Immunoblotting for Bcl-2 and ß-actin in 20 µg PBMC lysates from patients b and c (lanes 1 and 2) described in panels B and C, respectively. On the same gel, the indicated amounts of Jurkat cell lysates were loaded and analyzed as standards (lanes 3–6).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 5. Intracellular Bcl-2 protein expression in PBMC subsets freshly isolated from CH, LC, and HCC patients and nonC controls. MFI of intracellular Bcl-2 expression were calculated as shown in Figure 4 . Means ± SD of the MFI in each subset were denoted by vertical lines. *, P < 0.05 when compared with the nonC group by the Mann-Whitney U test.


arrow
DISCUSSION
 
The current study indicates that in chronic HCV infection, the susceptibility of isolated CD4+ and CD8+ T lymphocyte and CD14+ monocyte subsets to apoptosis is elevated in patients with advanced CH compared with that observed in HCV-negative controls under the apoptotic stimulus of serum starvation, despite the absence of differences in mean percent mortality of PBMC subsets incubated in media lacking apoptotic stimuli and in culture with anti-CD3 or PMA. It is interesting that intracellular expression of the antiapoptotic protein Bcl-2 was diminished in patients with advanced CH. This observation is suggestive of a molecular mechanism in which the PBMC subsets become susceptible to apoptosis presumably because of the down-regulation of Bcl-2 expression during persistent viral infection.

The mechanisms responsible for viral persistence and disease progression in HCV infection are poorly understood. However, several hypotheses have been proposed [3 ]. Recent studies have indicated that some viral infections influence the susceptibility of PBMC to apoptosis. Dysfunction of the FasL/Fas and TNF-{alpha} death pathways and the Bcl-2 family, which are directly related to viral persistence and disease progression, was noted [14 15 16 17 18 19 20 21 ]. To our knowledge, extensive studies have not been conducted with respect to the susceptibilities of PBMC subsets to apoptosis in patients exhibiting chronic HCV infection. We predicted that PBMC abnormalities in undergoing apoptosis would contribute to disease pathogenesis during chronic HCV infection. The results indicate that CD4+ and CD8+ T lymphocytes and CD14+ monocytes were highly susceptible to apoptosis under apoptotic stimulus by serum starvation. As CD4+ and CD8+ T lymphocytes and monocytes are known to be critically involved in virus clearance and eradication of infected cells in patients presenting with chronic HCV infection [9 10 11 12 13 ], dysfunction of these cell subsets may result in the insufficient antiviral effect that causes viral persistence.

T lymphocytes undergo physiologic apoptosis to govern immune homeostasis and tolerance via the elimination of excessive, harmful, or useless clonotypes. T lymphocyte apoptosis exists in at least two major forms: antigen-driven (active) and lymphokine withdrawal (passive) [32 ]. Active apoptosis of T lymphocytes occurs indirectly by the antigen-induced expression of death cytokines, chiefly FasL and TNF-{alpha}. The death mechanism entrained to Fas has been implicated in various disease processes. For instance, Fas-mediated apoptosis participates in the depletion of CD4+ T lymphocytes in acquired immunodeficiency syndrome. This effect may involve "bystander" killing of uninfected cells secondary to generalized activation of the immune system [33 ]. TNF-{alpha} may play a similar pathogenetic role in disease [34 35 36 ]. In contrast, passive (lymphokine withdrawal) apoptosis displays no known requirement for death cytokines or their receptors. Instead, it appears to involve the direct cytoplasmic activation of caspases, possibly as a result of mitochondrial damage [37 , 38 ]. Bcl-2 may inhibit this form of apoptosis by binding to the mitochondrial membrane, to the caspase activator apoptotic protease-activating factor-1, or to both; moreover, Bcl-2 may exert inhibitory effects on the active caspase complex [39 40 41 42 43 ].

Bcl-2 expression was reduced at the mRNA and protein levels in LC and HCC patients. These patients demonstrated PBMC subsets, which were more susceptible to apoptosis under apoptotic stimulus by serum starvation than that displayed by CH patients and nonC controls. Down-regulation of Bcl-2 expression may provide a mechanism that accounts for the increase in apoptosis as a result of serum starvation. Bcl-2 expression is regulated independently of the FasL/Fas death pathway in lymphocytes [44 , 45 ]. Reduction in Bcl-2 expression has been observed in activated lymphocytes from patients with viral infection [46 , 47 ]. More interestingly, decreased expression of bcl-2 and the increased susceptibility to apoptosis were observed in T lymphocyte subsets from aging humans. These processes may contribute to increased frequency of infection and increased incidence of cancer [23 ]. Consistent with these observations, decreased bcl-2 expression and increased susceptibility to apoptosis in PBMC subsets from LC and HCC patients may reflect prolonged, in vivo activation as a consequence of persistent viral infection. Conversely, changes in the level of Bcl-2 expression and in the susceptibility to apoptosis may contribute to the progression of liver disease.

Finally, current observations suggest an additional immunological basis of the insufficient antiviral responses leading to persistent viral infection and disease progression in CH C. Further studies are needed to determine the reversion of changes in the Bcl-2 expression and in the susceptibility to apoptosis in PBMC subsets from patients who recovered from the disease and cleared the viral infection following interferon treatment.


arrow
ACKNOWLEDGEMENTS
 
This work was supported by the Suzuken Memorial Foundation and by the Foundation for Advancement of International Science (FAIS). We thank Barbara Rehermann for critiquing this manuscript before submission.

Received June 22, 2001; revised December 5, 2001; accepted February 20, 2002.


arrow
REFERENCES
 
    1
  1. Houghton, M. (1996) Hepatitis C virus Fields, B. N. Knipe, D. M. Howley, P. M. eds. Virology 1,1035-1058 Lippincott-Raven New York.
    2. 2
  2. Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., Ohta, Y., Choo, Q., Houghton, M., Kuo, G. (1990) Hepatitis C virus infection is associated with the development of hepatocellular carcinoma Proc. Natl. Acad. Sci. USA 87,6547-6549[Abstract/Free Full Text]
    3. 3
  3. Cerny, A., Chisari, F. V. (1999) Pathogenesis of chronic hepatitis C: immunological features of hepatic injury and viral persistence Hepatology 30,595-601[Medline]
    4. 4
  4. Weiner, A. J., Geysen, H. M., Christopherson, C., Hall, J. E., Mason, T. J., Saracco, G., Bonino, F., Crawford, K., Marion, C. D., Crawford, K. A., Brunetto, M., Barr, P. J., Miyamura, T., McHutchinson, J., Houghton, M. (1992) Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections Proc. Natl. Acad. Sci. USA 89,3468-3472[Abstract/Free Full Text]
    5. 5
  5. Taniguchi, S., Okamoto, H., Sakamoto, M., Kojima, M., Tsuda, F., Tanaka, T., Munekata, E., Muchmore, E. E., Peterson, D. A., Mishiro, S. (1993) A structurally flexible and antigenically variable N-terminal domain of the hepatitis C virus E2/NS1 protein: implication for an escape from antibody Virology 195,297-301[Medline]
    6. 6
  6. Kato, N., Ootsuyama, Y., Sekiya, H., Ohkoshi, S., Nakazawa, T., Hijikata, M., Shimotohno, K. (1994) Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection J. Virol. 68,4776-4784[Abstract/Free Full Text]
    7. 7
  7. Nakamoto, Y., Kaneko, S., Honda, M., Unoura, M., Cheong, J., Harada, A., Matsushima, K., Kobayashi, K., Murakami, S. (1994) Detection of the putative E2 protein of hepatitis C virus in human liver J. Med. Virol. 42,374-379[Medline]
    8. 8
  8. Nakamoto, Y., Kaneko, S., Ohno, H., Honda, M., Unoura, M., Murakami, S., Kobayashi, K. (1996) B-cell epitopes in hypervariable region 1 of hepatitis C virus obtained from patients with chronic persistent hepatitis J. Med. Virol. 50,35-41[Medline]
    9. 9
  9. Chisari, F. V., Ferrari, C. (1995) Hepatitis B virus immunopathogenesis Annu. Rev. Immunol. 13,29-60[Medline]
    10. 10
  10. Cerny, A., Chisari, F. V. (1994) Immunological aspects of HCV infection Intervirology 37,119-125[Medline]
    11. 11
  11. Chang, K. M., Rehermann, B., Chisari, F. V. (1997) Immunopathology of hepatitis C Springer Semin. Immunopathol. 19,57-68[Medline]
    12. 12
  12. Rehermann, B., Chisari, F. V. (2000) Cell mediated immune response to the hepatitis C virus Curr. Top. Microbiol. Immunol. 242,299-325[Medline]
    13. 13
  13. Ando, K., Moriyama, T., Guidotti, L. G., Wirth, S., Schreiber, R. D., Schlicht, H. J., Huang, S. N., Chisari, F. V. (1993) Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis J. Exp. Med. 178,1541-1554[Abstract/Free Full Text]
    14. 14
  14. Gougeon, M. L., Montagnier, L. (1999) Programmed cell death as a mechanism of CD4 and CD8 T cell deletion in AIDS. Molecular control and effect of highly active anti-retroviral therapy Ann. N. Y. Acad. Sci. 887,199-212[Medline]
    15. 15
  15. Welsh, R. M., McNally, J. M. (1999) Immune deficiency, immune silencing, and clonal exhaustion of T cell responses during viral infections Curr. Opin. Microbiol. 2,382-387[Medline]
    16. 16
  16. Wold, W. S., Doronin, K., Toth, K., Kuppuswamy, M., Lichtenstein, D. L., Tollefson, A. E. (1999) Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic Curr. Opin. Immunol. 11,380-386[Medline]
    17. 17
  17. Kaplan, D., Sieg, S. (1998) Role of the Fas/Fas ligand apoptotic pathway in human immunodeficiency virus type 1 disease J. Virol. 72,6279-6282[Free Full Text]
    18. 18
  18. Berzofsky, J. A., Ahlers, J. D., Derby, M. A., Pendleton, C. D., Arichi, T., Belyakov, I. M. (1999) Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections Immunol. Rev. 170,151-172[Medline]
    19. 19
  19. Romero-Alvira, D., Roche, E. (1998) The keys of oxidative stress in acquired immune deficiency syndrome apoptosis Med. Hypotheses 51,169-173[Medline]
    20. 20
  20. Grayson, J. M., Zajac, A. J., Altman, J. D., Ahmed, R. (2000) Cutting edge: increased expression of Bcl-2 in antigen-specific memory CD8+ T cells J. Immunol. 164,3950-3954[Abstract/Free Full Text]
    21. 21
  21. Feuillard, J., Schuhmacher, M., Kohanna, S., Asso-Bonnet, M., Ledeur, F., Joubert-Caron, R., Bissieres, P., Polack, A., Bornkamm, G. W., Raphael, M. (2000) Inducible loss of NF-kappaB activity is associated with apoptosis and Bcl-2 down-regulation in Epstein-Barr virus-transformed B lymphocytes Blood 95,2068-2075[Abstract/Free Full Text]
    22. 22
  22. Nakamoto, Y., Kaneko, S., Buttner, S. W., Matsushita, E., Kobayashi, K. (1999) Inhibition of peripheral blood lymphocyte apoptosis by soluble fas ligand in patients with hepatocellular carcinoma Oncol. Rep. 6,733-739[Medline]
    23. 23
  23. Aggarwal, S., Gupta, S. (1998) Increased apoptosis of T cell subsets in aging humans: altered expression of Fas (CD95), Fas ligand, Bcl-2, and Bax J. Immunol. 160,1627-1637[Abstract/Free Full Text]
    24. 24
  24. Gaubin, M., Autiero, M., Basmaciogullari, S., Metivier, D., Mishal, Z., Culerrier, R., Oudin, A., Guardiola, J., Piatier-Tonneau, D. (1999) Potent inhibition of CD4/TCR-mediated T cell apoptosis by a CD4-binding glycoprotein secreted from breast tumor and seminal vesicle cells J. Immunol. 162,2631-2638[Abstract/Free Full Text]
    25. 25
  25. Hogarth, L. A., Hall, A. G. (1999) Increased BAX expression is associated with an increased risk of relapse in childhood acute lymphocytic leukemia Blood 93,2671-2678[Abstract/Free Full Text]
    26. 26
  26. Boudet, F., Lecoeur, H., Gougeon, M. L. (1996) Apoptosis associated with ex vivo down-regulation of Bcl-2 and up-regulation of Fas in potential cytotoxic CD8+ T lymphocytes during HIV infection J. Immunol. 156,2282-2293[Abstract]
    27. 27
  27. Hashimoto, F., Oyaizu, N., Kalyanaraman, V. S., Pahwa, S. (1997) Modulation of Bcl-2 protein by CD4 cross-linking: a possible mechanism for lymphocyte apoptosis in human immunodeficiency virus infection and for rescue of apoptosis by interleukin-2 Blood 90,745-753[Abstract/Free Full Text]
    28. 28
  28. Conti, L., Rainaldi, G., Matarrese, P., Varano, B., Rivabene, R., Columba, S., Sato, A., Belardelli, F., Malorni, W., Gessani, S. (1998) The HIV-1 vpr protein acts as a negative regulator of apoptosis in a human lymphoblastoid T cell line: possible implications for the pathogenesis of AIDS J. Exp. Med. 187,403-413[Abstract/Free Full Text]
    29. 29
  29. Ledru, E., Lecoeur, H., Garcia, S., Debord, T., Gougeon, M. L. (1998) Differential susceptibility to activation-induced apoptosis among peripheral Th1 subsets: correlation with Bcl-2 expression and consequences for AIDS pathogenesis J. Immunol. 160,3194-3206[Abstract/Free Full Text]
    30. 30
  30. Re, M., Gibellini, D., Aschbacher, R., Vignoli, M., Furlini, G., Ramazzotti, E., Bertolaso, L., La Placa, M. (1998) High levels of HIV-1 replication show a clear correlation with downmodulation of Bcl-2 protein in peripheral blood lymphocytes of HIV- 1-seropositive subjects J. Med. Virol. 56,66-73[Medline]
    31. 31
  31. Otani, H., Erdos, M., Leonard, W. J. (1993) Tyrosine kinase(s) regulate apoptosis and bcl-2 expression in a growth factor-dependent cell line J. Biol. Chem. 268,22733-22736[Abstract/Free Full Text]
    32. 32
  32. Lenardo, M., Chan, K. M., Hornung, F., McFarland, H., Siegel, R., Wang, J., Zheng, L. (1999) Mature T lymphocyte apoptosis—immune regulation in a dynamic and unpredictable antigenic environment Annu. Rev. Immunol. 17,221-253[Medline]
    33. 33
  33. Muro-Cacho, C. A., Pantaleo, G., Fauci, A. S. (1995) Analysis of apoptosis in lymph nodes of HIV-infected persons. Intensity of apoptosis correlates with the general state of activation of the lymphoid tissue and not with stage of disease or viral burden J. Immunol. 154,5555-5566[Abstract]
    34. 34
  34. Ayyavoo, V., Mahboubi, A., Mahalingam, S., Ramalingam, R., Kudchodkar, S., Williams, W. V., Green, D. R., Weiner, D. B. (1997) HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B Nat. Med. 3,1117-1123[Medline]
    35. 35
  35. Gomez del Moral, M., Ortuno, E., Fernandez-Zapatero, P., Alonso, F., Alonso, C., Ezquerra, A., Dominguez, J. (1999) African swine fever virus infection induces tumor necrosis factor alpha production: implications in pathogenesis J. Virol. 73,2173-2180[Abstract/Free Full Text]
    36. 36
  36. Ledru, E., Christeff, N., Patey, O., de Truchis, P., Melchior, J. C., Gougeon, M. L. (2000) Alteration of tumor necrosis factor-alpha T-cell homeostasis following potent antiretroviral therapy: contribution to the development of human immunodeficiency virus-associated lipodystrophy syndrome Blood 95,3191-3198[Abstract/Free Full Text]
    37. 37
  37. Ohta, T., Kinoshita, T., Naito, M., Nozaki, T., Masutani, M., Tsuruo, T., Miyajima, A. (1997) Requirement of the caspase-3/CPP32 protease cascade for apoptotic death following cytokine deprivation in hematopoietic cells J. Biol. Chem. 272,23111-23116[Abstract/Free Full Text]
    38. 38
  38. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., Newmeyer, D. D. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis Science 275,1132-1136[Abstract/Free Full Text]
    39. 39
  39. Reed, J. C. (1997) Cytochrome c: can’t live with it—can’t live without it Cell 91,559-562[Medline]
    40. 40
  40. Vander Heiden, M. G., Chandel, N. S., Williamson, E. K., Schumacker, P. T., Thompson, C. B. (1997) Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria Cell 91,627-637[Medline]
    41. 41
  41. Hengartner, M. O. (1998) Apoptosis. Death cycle and Swiss army knives Nature 391,441-442[Medline]
    42. 42
  42. Rosse, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B., Borner, C. (1998) Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c Nature 391,496-499[Medline]
    43. 43
  43. Li, F., Srinivasan, A., Wang, Y., Armstrong, R. C., Tomaselli, K. J., Fritz, L. C. (1997) Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release J. Biol. Chem. 272,30299-30305[Abstract/Free Full Text]
    44. 44
  44. Strasser, A., Harris, A. W., Huang, D. C., Krammer, P. H., Cory, S. (1995) Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis EMBO J. 14,6136-6147[Medline]
    45. 45
  45. Debatin, K. M., Krammer, P. H. (1995) Resistance to APO-1 (CD95) induced apoptosis in T-ALL is determined by a BCL-2 independent anti-apoptotic program Leukemia 9,815-820[Medline]
    46. 46
  46. Akbar, A. N., Borthwick, N., Salmon, M., Gombert, W., Bofill, M., Shamsadeen, N., Pilling, D., Pett, S., Grundy, J. E., Janossy, G. (1993) The significance of low bcl-2 expression by CD45RO T cells in normal individuals and patients with acute viral infections. The role of apoptosis in T cell memory J. Exp. Med. 178,427-438[Abstract/Free Full Text]
    47. 47
  47. Yoshino, T., Kondo, E., Cao, L., Takahashi, K., Hayashi, K., Nomura, S., Akagi, T. (1994) Inverse expression of bcl-2 protein and Fas antigen in lymphoblasts in peripheral lymph nodes and activated peripheral blood T and B lymphocytes Blood 83,1856-1861[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
M. W. Cruise, H. M. Melief, J. Lukens, C. Soguero, and Y. S. Hahn
Increased Fas ligand expression of CD4+ T cells by HCV core induces T cell-dependent hepatic inflammation
J. Leukoc. Biol., August 1, 2005; 78(2): 412 - 425.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakamoto, Y.
Right arrow Articles by Kobayashi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakamoto, Y.
Right arrow Articles by Kobayashi, K.