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Keywords:

  • chronic hepatitis C;
  • intrahepatic lymphocytes;
  • liver histological lesions;
  • natural T cells;
  • Vα24

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Intrahepatic lymphocytes are believed to be involved in the immunopathogenesis of hepatitis C virus (HCV) infection and the evolution of HCV-induced hepatitis. In the present study, we examined the three main intrahepatic lymphocyte subsets, namely CD3+CD56 conventional T lymphocytes, CD3+CD56+ natural T (NT) lymphocytes and CD3CD56+ natural killer (NK) lymphocytes in HCV-infected patients. The proportion of each lymphocyte subset was evaluated both in liver biopsies and in samples of peripheral blood lymphocytes (PBL) by flow cytometry in 21 patients with histologically proven chronic hepatitis C. Simultaneously, alanine aminotransferase (ALT) levels, viral load and histological lesions were assessed. Neither NT nor NK populations correlated with any biochemical, viral or histological parameters. Furthermore, Vα24+ NT lymphocytes showed no preferential enrichment in the liver of HCV-infected patients. Regarding conventional T lymphocytes, a highly significant linear correlation was found between intrahepatic CD3+CD56 T lymphocytes and the Knodell score, a numerical score for assessing histological activity and fibrosis (r = 0·715, P < 0·0001) and more specifically with the periportal necrosis parameter, which is the main lesion of chronic hepatitis C. In addition, analysis of the peripheral compartment revealed a high correlation between values of CD3+CD56 lymphocytes and both Knodell score (r = 0·624, P = 0·003) and serum ALT levels and again with periportal necrosis. The strong correlation between the proportion of peripheral CD3+CD56 conventional T lymphocytes and the severity of hepatic lesions leads us to propose that evaluation of this accessible peripheral population could be used as an indicator test for the severity of histological lesions in chronic hepatitis C.

Abbreviations:


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Infection by hepatitis C virus (HCV) is a major medical problem and it has been estimated that 170 million people are infected worldwide [1,2]. The hallmark of this infection is its chronicity: 80% of infected patients develop chronic hepatitis [3]. Despite development of antiviral immunity by the host, in approximately 70–80% of cases the virus is not eradicated leading to chronicity and the failure to cure infection [4–7]. Moreover, there is growing evidence that lymphocytes recruited into the liver are responsible for liver damage [3,8–11].

From the point of view of immunity, the role of the liver is complex due to its anatomical location, its lymphocyte populations and the existence of tolerance in this organ [12,13]. Phenotypically, intrahepatic lymphocytes are distinct from those present in peripheral blood. The three main intrahepatic populations are: first, conventional CD3+CD4+ or CD3+CD8+ T lymphocytes with a reversed CD4 : CD8 ratio; secondly, non-conventional T lymphocytes co-expressing CD3 and CD56, called natural T lymphocytes (NT), which are particularly abundant in this organ; and lastly CD3CD56+ natural killer (NK) lymphocytes [12,14,15]. In humans, some CD3+CD56+ NT lymphocytes expressing a Vα24JαQ invariant T cell receptor (TCR) α chain are considered equivalent to the Vα14Jα281 mouse population [16,17]. Mouse NT cells are abundant in the liver (30% of T lymphocytes) and have important immunoregulatory properties in relation to cytokine production or cytotoxicity [18]. It has been postulated that in man, NT cells could lead to immunological tolerance by modulating the fate of activated T lymphocytes, which normally undergo apoptosis in the liver [12]. In the case of hepatitis C virus, such apoptosis may prevent establishment of specific immunity and could explain the absence of complete virus eradication [19]. The outcome of hepatitis C infection would depend upon the balance between development of immunity (leading to virus elimination) and tolerance (leading to chronic viral persistence).

The aim of the work reported herein was, first, to evaluate the proportion of effector and immunoregulatory lymphocytes which seem to play an important role in the pathogenicity and development of chronic hepatitis C. To do this, we used flow cytometry to evaluate the proportion of conventional T lymphocytes, NT lymphocytes and NK cells in infected liver and also in peripheral blood. Conventional T lymphocytes were defined as CD3+CD56, NT cells as CD3+CD56+ and NK cells as CD3CD56+. The proportion of Vα24+CD3+CD56+ lymphocytes was also evaluated by flow cytometry. Secondly, histological parameters, alanine aminotransferase (ALT) levels and viral load were evaluated in order to determine whether a relationship existed between the abundance of the studied lymphocyte populations and the biochemical, virological or histological parameters measured. Finally, we propose that evaluation of a particular circulating lymphocyte subpopulation in HCV-infected patients could be used as an indicator of the severity of histological lesions and therefore be a useful parameter in patient follow-up.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients

Twenty-one unselected and untreated patients (11 males and 10 females, age range: 32–71 years) with histologically proven chronic hepatitis C seen in the gastroenterology department of the University Hospital of Grenoble were studied. All patients were anti-HCV antibody positive (ELISA 3, Ortho Diagnostic Systems, Raritan, NJ, USA) and HCV-RNA positive by PCR (Amplicor HCV, Roche Diagnostic Systems, Meylan, France). All patients were hepatitis B surface (HBs) antigen and anti-HIV antibody negative. No patient had biochemical markers of autoimmune hepatitis (autoantibodies ≤1/80). All patients had alcohol consumption lower than 30 g/day. α1 antitrypsin, ferritin and ceruloplasmin concentrations were normal in all patients. The local ethics committee approved the study and all patients gave their written informed consent for liver biopsy. They all underwent needle liver biopsy as part of their diagnostic evaluation before treatment. No patient received antiviral therapy prior the day of the biopsy. At the time of liver biopsy, blood samples were drawn for peripheral blood lymphocyte (PBL) immunophenotyping, ALT and HCV RNA (viraemia) assays and HCV genotyping. ALT levels were expressed as multiples of normal value (×N). HCV RNA concentration was determined by quantitative RT-PCR (Amplicor Monitor HCV, Roche Diagnostic Systems, Meylan, France) and genotype by Inno-Lipa technology (Innogenetics, Zwijnaarde, Belgium). Patient characteristics are shown in Table 1. As controls, 17 age-matched healthy volunteers provided samples for PBL immunophenotyping.

Table 1.  Clinical, epidemiological and viral characteristics of chronically HCV-infected patients
Patient no.SexAge (years)EpidemiologyDuration of HCV infection (years)ALAT (XN)Knodell scoreKnodell score: activity index*Knodell score: fibrosis indexViraemia (copies/ml)HCV genotype
  1.  M/F, Male/female. IVDA, intravenous drug abuse; n.d.: not determined. *Activity index of the Knodell score is given, periportal necrosis, focal necrosis and portal inflammation parameters, respectively, are added in brackets.

 1M36IVDA171·68 9 8 (1 + 3 + 4)1 365 3914d
 2F71unknownunknown2·9711 7 (3 + 1 + 3)4 517 9291b
 3F43IVDA260·97 4 3 (1 + 1 + 1)1 140 2221
 4M44tranfusionunknown1·73 8 7 (3 + 1 + 3)1  17 5344c
 5M36IVDA173·931310 (3 + 3 + 4)3 417 3431a
 6F35iatrogenic 82·48 2 2 (1 + 0 + 1)01190 0903a
 7F57unknownunknown0·6310 9 (3 + 3 + 3)1 788 0004d
 8F62unknownunknown1·03 7 4 (1 + 0 + 3)3 278 7372
 9F48Needle stickunknown1·44 6 5 (1 + 1 + 3)1 322 5271b
10M32IVDA101·22 8 7 (3 + 1 + 3)1   3 7903a
11M34Transfusion19  1·8 5 4 (1 + 0 + 3)1 532 7951b
12F28transfusion151·14 4 4 (0 + 1 + 3)03730 0001
13F51unknownunknown1 5 4 (1 + 0 + 3)1 265 0001b
14M43Needle stick220·39 1 1 (0 + 0 + 1)02300 0002
15F39IVDA190·93 3 3 (1 + 1 + 1)0 319 0004d
16F39Needle stick22 1·5 6 5 (1 + 1 + 3)1 572 0003a
17M32IVDA 5 0·2 1 1 (0 + 0 + 1)0   1 000nd
18M62unknownunknown 1·513 9 (4 + 1 + 4)41040 0001b
19M44unknown131·58 8 7 (3 + 1 + 3)11180 0001b
20M43IVDA141·34 8 7 (3 + 3 + 1)11820 0004a
21M35unknownunknown1·05 4 4 (0 + 1 + 3)08150 0001b

Reagents and monoclonal antibodies

Fluorescein isothiocyanate (FITC)-labelled anti-CD45, FITC-labelled anti-CD8, phycoerythrin (PE)-labelled anti-CD56 and peridinin chlorophyll protein (PerCP)-labelled anti-CD3 antibodies were purchased from Becton Dickinson (Le Pont-de Claix, France). FITC-labelled anti-Vα24 was purchased from Immunotech (Marseille, France) and FACS lysing solution from Becton Dickinson.

Extraction of intrahepatic lymphocytes (IHL)

Liver biopsy specimens were obtained using 1·5 mm diameter disposable biopsy needles with a length range 10–20 mm. One part of the biopsy was fixed in Bouin's solution and processed for histopathological examination, and a second part was collected in 1·5 ml RPMI-1640 medium containing 10% fetal calf serum (FCS) for immunological analysis. When the biopsy took longer than 15 mm, part of it was frozen rapidly in liquid nitrogen and stored at −80°C. The isolation method of IHL has been described previously [20]. Briefly, fresh liver samples were washed twice in fresh medium and shaken gently to avoid blood contamination. Liver specimens were disrupted mechanically into small fragments in RPMI−10% FCS medium using forceps and a scalpel, and then the fragments were homogenized in glass mortars. The resulting cell suspension was washed and resuspended in 2% FCS-Hanks's medium.

PBL preparation

Blood samples were collected in heparinized vacutainer tubes. PBL were isolated by density centrifugation over Ficoll gradients.

Immunostaining

For all 21 hepatic samples 100 µl of cell suspension was incubated with 10 µl PerCP conjugated anti-CD3 MoAb and 10 µl PE conjugated anti-CD56 for 30 min at 4°C. With three of the samples, an additional staining with FITC conjugated anti-CD45 MoAb was carried out. For 14 other samples, additional staining was performed with 10µl FITC conjugated anti-Vα24 MoAb and for four samples 10 µl FITC conjugated anti-CD8 MoAb was used. After immunostaining, 1 ml of 1× FACS lysing solution was added to the samples and incubated for 10 min at room temperature. After washing in 0·5% BSA-PBS and resuspension in PBS, samples were ready for FACS analysis.

In parallel, 0·5 × 106 PBL from each HCV-infected patient were labelled with the same set of antibody combinations used for hepatic immunophenotyping and processed, as described above.

Flow cytometry analysis

Samples were analysed with a FacsScan cytometer (Becton-Dickinson) using CellQuest software. Lymphocytes were analysed using a gate set on forward scatter versus side scatter. For hepatic samples, all cells selected with this gate − representing 4000–60 000 events − were analysed. In the case of PBL, 20 000 gated events were analysed.

Histological examination

Liver biopsies were processed for histological examination. Biopsy specimens were assessed histologically by the pathologist who performs liver biopsy examinations routinely at the University Hospital of Grenoble. This pathologist is extremely experienced, reviewing 300 liver biopsies per year for 5 years, and had no knowledge of the clinical and biological characteristics of the patients at the time of biopsy. The well-established scoring system proposed by Knodell et al. was used [21]. The Knodell score is composed of two items: first, an activity index obtained by the addition of periportal necrosis, focal necrosis and portal inflammation scores (reported as necrotico-inflammatory lesions) and secondly, a fibrosis index. The overall Knodell score combines both activity and fibrosis indices.

Statistical analysis

Results are presented as means ± one standard deviation (s.d.). Statistical analysis was performed with Statview software version 4·5. Non-parametric tests: Mann–Whitney U-test, Wilcoxon test and Spearman test were used. Values of P < 0·05 were considered significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cytometric analysis of intrahepatic lymphocytes

Typically 50 000–150 000 mononuclear cells were recovered per liver biopsy. Following the use of FACS Lysing Solution, which induces erythrocyte lysis, the lymphocyte populations in the liver biopsy cell suspensions could readily be identified (Fig. 1). The fact that we found 88–96% CD45+ lymphocytes in the CD45-labelled samples confirmed the quality of our gating (data not shown).

image

Figure 1. Characteristic flow cytometric analysis of (a) intrahepatic and (b) peripheral blood lymphocytes from a patient suffering from chronic hepatitis C. A lymphocyte gate was set on the basis of size and granulometry (left). Then, gated lymphocytes were examined for their expression of CD56 and CD3 markers (right). The percentages of cells with the CD3+CD56, CD3+CD56+, CD3CD56 and CD3CD56+ phenotypes are mentioned in the appropriate quadrants.

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PBL subset composition in HCV-infected patients and normal controls

The proportions of conventional T lymphocytes (CD3+CD56), NT lymphocytes (CD3+CD56+) and NK lymphocytes (CD56+CD3) were evaluated using lymphocyte gating on PBL suspensions. There were no statistical differences in each of the three subsets between normal controls and patients suffering from chronic hepatitis C (P > 0·05) (Table 2).

Table 2.  Lymphocyte subset composition in the peripheral blood and the liver of HCV-infected patients and in the peripheral blood from normal controls
 CD3+CD56CD3+CD56+CD3CD56+CD56+ (CD3+)
  1.  Results are expressed as mean of percentages ± s.d.; they were obtained with 17 normal controls and 21 HCV-infected patients. CD56+ (CD3+) means that CD56+ were evaluated in the CD3+ population.

PBL (controls)66·9 ± 6·9 6·3 ± 3·810·8 ± 5·99·0 ± 5·2
PBL (HCV)65·9 ± 7·4 6·9 ± 4·511·4 ± 6·59·6 ± 6·3
IHL (HCV) 60·4 ± 12·8 14·5 ± 8·014·7 ± 9·819·8 ± 11·4

Characteristics of intrahepatic lymphocytes

Analysis of intrahepatic lymphocytes in HCV-infected patients showed a statistically significant increase in the percentage of CD56+ cells among the CD3+ population compared with that in the periphery (19·8 ± 11·4%versus 9·6 ± 6·3%, P < 0·0001). Previous results obtained with normal non-infected human liver have also reported an elevated proportion of CD56+CD3+ lymphocytes [14,22]. Thus, our result show that the same is true in HCV-infected liver.

In four biopsy samples, the proportion of CD3+ lymphocytes expressing CD8 (mean 61·2 ± 4·9%) was considerably higher than in PBL from the same patient (mean 22·5 ± 5·7). Similar results have been described previously in hepatitis C-infected liver and, although the CD4/CD8 ratio is reversed, it remains higher than in normal liver [10,20,23].

Finally, in terms of CD56 and CD8 staining, the distinct phenotype of intrahepatic lymphocytes indicates that they were not derived from PBL contaminants.

Moreover, 18 frozen biopsies were processed for T cell receptor (TCR) α transcript evaluation by real-time quantitative RT-PCR (Light Cycler, Roche Diagnostic Systems, Meylan, France) as described by Jouvin-Marche et al. [24]. We found a significant correlation between TCR α transcripts and CD3+CD56 T cells as evaluated by cytometry (r = 0·51, P = 0·049) (data not shown). This correlation allows us to conclude that cytometric analysis of intrahepatic cellular suspensions gives a reliable estimate of intrahepatic T lymphocyte numbers.

Finally, for all three lymphocyte populations analysed (Fig. 2), we found a significant correlation between the percentages in the liver and PBL.

image

Figure 2. Correlations between the liver and the periphery for the percentages of the different lymphocytes subsets in HCV-infected patients. Conventional T lymphocytes (CD3+CD56), NT lymphocytes (CD3+CD56+) and NK lymphocytes (CD3CD56+) were evaluated both in the liver and periphery (n = 21). The results of the statistical analysis show significant correlations between the liver and periphery for the three populations studied.

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The Vα24-positive lymphocyte population is very low in the liver of HCV-infected patients

As expected, we found an extremely low percentage of Vα24 positive lymphocytes in the peripheral CD3+CD56+ NT lymphocyte population of patients (mean 1·2 ± 1·5). There was no statistical difference between the percentages of these Vα24-positive lymphocytes observed in PBL of chronic hepatitis C patients and those of normal controls (P > 0·05). More surprisingly, as in the periphery, we found that the percentage of Vα24-positive cells among the intrahepatic CD3+CD56+ subset of HCV-infected patients was very low and highly variable (mean 1·6 ± 0.1·4). Lastly, there was no statistical difference between peripheral and intrahepatic Vα24-positive NT cell percentages in HCV-infected patients (P > 0·05) (Fig. 3).

image

Figure 3. Repartition of Vα24+ cells in the CD3+CD56+ NT lymphocyte population in HCV-infected patients and normal controls. Vα24+ lymphocytes were evaluated in the CD3+CD56+ NT population (mentioned as Vα24+ (CD3+CD56+)) in PBL of 17 controls and in both PBL and liver of 17 patients. No statistical difference was observed between either normal PBL and HCV-infected patients PBL (Mann–Whitney U-test), or between periphery and liver in HCV-infected patients (Wilcoxon test); n.s.: non-significant.

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High correlation between the severity of hepatic damage and the proportion of intrahepatic CD3+CD56  T lymphocytes

As shown in Fig. 4, we found an extremely significant correlation between the proportion of CD3+CD56- intrahepatic conventional T lymphocytes and the Knodell score (r = 0·715, P < 0·0001). The same correlation was observed between these CD3+CD56 lymphocytes and the inflammatory activity index of the Knodell score (r = 0·671, P = 0·003) and more specifically with the periportal necrosis parameter (r = 0·759, P = 0·0007). No such correlation was seen with either the focal necrosis or portal inflammation parameters. Finally, CD3+CD56 lymphocyte numbers were also found to correlate with the fibrosis index (r = 0·62, P = 0·055) but not with serum ALT concentrations. No significant correlation was found between the proportions of the other two intrahepatic populations we studied, namely NT cells and NK lymphocytes, and the Knodell score (data not shown).

image

Figure 4. Correlations between CD3+CD56- intrahepatic or peripheral conventional T lymphocyte numbers and the Knodell score or ALT levels in HCV-infected patients. The percentages of CD3+CD56- lymphocytes were evaluated in the 21 chronically HCV-infected patients and a correlation with both the histological Knodell score (activity index + fibrosis index) and ALT levels (hepatocyte lysis) was searched. In the liver, the correlation with the Knodell score is very strong and the correlation with ALT levels is not significant. In the periphery, the correlation with Knodell score is still high and the correlation with ALT levels is significant.

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Correlation between severity of hepatic damage and the proportion of peripheral CD3+CD56 T lymphocytes

We also found that CD3+CD56 conventional peripheral T lymphocyte numbers correlated highly with the Knodell score (r = 0·624, P = 0·003) and with both activity (r = 0·583, P = 0·009) and fibrosis indices (r = 0·588, P = 0·008). A significant correlation was also found between conventional T cell numbers and periportal necrosis (r = 0·530, P = 0·0177) but not with the other two activity index parameters mentioned above. Serum levels of the ALT enzyme, the most important marker of hepatocyte lysis, correlated with the percentages of CD3+56 T lymphocytes present in the periphery (r = 0·499, P = 0·021) (Fig. 4).

Both viral load and genotype do not correlate with intrahepatic CD3+CD56 lymphocyte numbers

We did not find any statistically significant correlation between any lymphocyte subpopulation in either the liver or the periphery and the HCV RNA level. However, conventional intrahepatic T lymphocytes tended to correlate inversely with viral load (r =−0·338, P > 0·05) (data not shown). Lastly, due probably to the small number of patients, no correlation was observed between any parameter and viral genotype.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this report, studying patients suffering from chronic hepatitis C, we looked for a correlation between the three major intrahepatic lymphocyte populations, namely conventional T (CD3+CD56), NT (CD3+CD56+) and NK (CD3CD56+) cells, and biochemical, virological or histological parameters. These different lymphocyte subsets are presumed to display effector and/or immunoregulatory roles as part of the immune response developed by the host against HCV. We also analysed these populations in periphery.

Regarding the NT and NK lymphocyte subsets, there were no significant correlations between their proportions and either histological parameters or ALT levels, suggesting that these populations are not directly implicated in the pathology. In a previous study, the same lack of correlation was observed when intrahepatic NK cells were studied, although there was no information about the NKT population [25]. Regarding this NKT population, another study showed that the CD56+ T lymphocyte ratio in the liver of patients with a high Knodell score was higher than that of patients with a low Knodell score [26]. However, there was no linear correlation between these two parameters. Finally, the absence of evidence for a linear correlation between these populations and histological lesions could be explained by the fact that both human NK and NT cells are heterogeneous and express different NK receptors [12,27]. For this reason, we explored more precisely NT lymphocytes expressing a Vα24 TCR α chain which are thought, like their mouse equivalent, to be extremely abundant in human liver [16,17]. Surprisingly, we found extremely low percentages (with large variations in the percentages) of this population in the liver. Others have found in a previous study that in HCV, but also in HBV-infected patients, the total CD3+ population of intrahepatic lymphocytes was enriched in Vα24+ lymphocytes in comparison with peripheral lymphocytes, but neither CD3+CD56+ lymphocytes nor normal liver were studied [28]. The fact that we found a low proportion of Vα24+ cells in HCV-infected liver is not surprising because, in another study, the proportion of Vα24+ NT lymphocytes was found to be extremely low in normal human liver [29]. Finally, there was no correlation between this population in either the liver or periphery and other disease parameters.

The most convincing results were obtained with conventional T lymphocytes. They were identified as CD3+CD56 because this allows a more accurate analysis, particularly in the liver, where the relative proportion of CD3+CD56+ lymphocytes is high. For this reason, the correlation between overall CD3+ lymphocyte numbers and parameters reflecting hepatic injury were either low or not significant (data not shown). When we analysed the results of lymphocyte phenotyping in the liver of the 21 HCV-infected patients, we found an important and significant linear correlation between the percentages of CD3+ CD56 T lymphocytes and the Knodell score, a well-established histological index of disease activity (r = 0·715, P < 0·0001). Because this correlation is high compared to those published previously, our data reinforce the involvement of conventional T lymphocytes in the generation of hepatic lesions [8,23,25].

Focusing on the different parameters of the Knodell score shows a good correlation between T lymphocyte numbers and the activity inflammatory index, more specifically with the periportal necrosis parameter. Piecemeal necrosis and bridging necrosis in the periportal area are characteristic of chronic hepatitis C [21]. Recently, it has also been reported that active T cells are mainly located at sites of piecemeal necrosis in the periportal area [30]. Finally, the correlation between intrahepatic T lymphocytes and the fibrosis index can probably be explained by the fact that fibrosis is the consequence of necrotico-inflammatory lesions, as there is a clear relationship between activity and fibrosis in our cohort. All these results and more specifically the strong correlation between conventional T lymphocytes and the most specific parameter of chronic hepatitis C evaluation, namely periportal necrosis, suggest that intrahepatic T lymphocytes are directly responsible for hepatic damage and are implicated in disease severity in its chronic stage. These results confirm the hypothesis that HCV virus alone is not cytopathic but that T lymphocytes activated during the host's immune response are responsible for liver cell injury. Regarding viral load, we only found a tendency for an inverse correlation with conventional CD3+ CD56 T lymphocyte percentages. This suggests that hepatic lymphocytes are able to mediate a certain degree of control over viral spread but that, at least at the chronic stage, this control is insufficient to eradicate the virus, leading to establishment of a balance between viral load and the immune response.

In addition to the analysis of intrahepatic lymphocytes, we performed the same immune phenotyping on the peripheral lymphocyte compartment. Comparison of PBL subsets between HCV-infected patients and healthy controls showed no significant differences. Interestingly, as with hepatic lymphocytes, we found a good correlation in the periphery between CD3+CD56- T lymphocyte numbers and the Knodell score, particularly with the hepatitis C characteristic periportal necrosis lesions, but the correlation was less significant than in the liver. The correlation can be explained by the fact that a clear correlation between intrahepatic and peripheral lymphocyte subsets was observed. Finally, we can conclude from these results that there is an exchange between intrahepatic and peripheral compartments. Similarly, two recent works have shown that the same specific T lymphocytes can be isolated from either the liver or PBL of chronically HCV-infected patients [31,32]. For this reason, the authors of one of these works proposed a characterization of the lymphocyte response to HCV in the peripheral blood instead of in the liver compartment [31]. We can also hypothesize that T lymphocytes leave the liver after encountering antigen there, although it has recently been shown that the majority of activated CD8+ T cells entering the liver undergo apoptosis within the liver environment [33,34]. On the other hand, numerous reports have suggested that HCV may infect peripheral blood mononuclear cells [35]. Nuti et al. suggest that intrahepatic lymphocytes do not undergo clonal expansion within the liver but migrate there, from extrahepatic sites to the chronically infected liver, where they display effector function and subsequently die [28]. If correct, this implies that maintenance of the intrahepatic lymphocyte pool depends on continuous lymphocyte immigration.

The fact that we found that the proportion of CD3+CD56 T lymphocytes correlates with the extent of hepatic injury for both hepatic and peripheral lymphocytes does not mean that all these lymphocytes are HCV specific. From recent work using MHC class I-peptide tetramers, it is now clear that these intrahepatic CD8+ lymphocytes are not all HCV specific because only 1–2% were found to bind tetramers containing two different epitopes of the HCV NS3 protein [36]. Although tetramer technology allows the study of a limited specific population and so underestimates the frequency of specific T cells, many lymphocytes in inflammatory lesions are certainly activated by non-specific mechanisms. The continued presentation of HCV antigens to antigen-specific T lymphocytes in lymphoid organs or in the liver probably induces cytokines, which activate HCV-non-specific bystander cells.

In conclusion, in this study we found that the proportion of CD3+CD56- T lymphocytes not only in the liver but also in peripheral blood significantly correlates with the degree of histological damage. This information is of great interest because, first, it will be possible to predict liver injury in the future by detecting CD3+CD56- lymphocyte percentages in HCV-infected patients and secondly, this parameter can be monitored in blood. Therefore, the detection of CD3+D56- cells in the peripheral blood of patients suffering from chronic hepatitis C could represent a simple parameter for the measurement of disease progression.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors wish to thank Raphaëlle Barnoud for histological examination, Maryline Baud for viral genotyping, Nadine Dutertre for viremia assays, Rhodri Ceredig and Serge Candeias for critical comments on the manuscript. This work was supported by specific grants from MENRT (Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses), ANRS (Agence Nationale de Recherche sur le SIDA). Inès Vigan was supported by Fondation Merieux.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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