Elsevier

Journal of Hepatology

Volume 51, Issue 3, September 2009, Pages 458-467
Journal of Hepatology

Fine characterization of intrahepatic NK cells expressing natural killer receptors in chronic hepatitis B and C

https://doi.org/10.1016/j.jhep.2009.05.030Get rights and content

Background/Aims

The fate of intrahepatic NK cell subsets in the course of HCV and HBV infections is not clearly understood.

Methods

Blood and intrahepatic CD56+ NK cell subsets (expressing NKG2A, CD158a,h or CD158b,j receptors) from HCV or HBV patients were quantified by flow cytometry and localized by immunohistochemistry in liver biopsies.

Results

A significant reduction in NK cell frequency and a quantitative imbalance between CD56bright and CD56dim subsets were observed in chronic HCV patients as compared to HBV patients, underlining that the inflammatory environment is not the only cause of these phenomena. The proportions of intrahepatic NK cells expressing either NKG2A, and/or CD158a,h, CD158b,j differed significantly between HCV and HBV patients. A higher frequency of perforin among intrahepatic CD56+CD3 cells was observed in HCV compared to HBV patients. Double immunohistochemical staining showed that CD56+CD3 cells were localized within necrotic areas. Immune monitoring of circulating CD56 subsets revealed that CD3CD56brightNKG2A+ and CD3CD56dimNKG2A+ cells were positively correlated with the necroinflammatory score and inversely correlated with viral load, respectively, in HCV patients.

Conclusions

HCV and HBV affect NK cell subsets according to the status of the diseases, especially CD3CD56dimNKG2A+ and CD3CD56brightNKG2A+ cells, may be of interest for disease monitoring.

Introduction

Hepatitis C virus (HCV) and hepatitis B virus (HBV) are preferentially hepatotropic, not directly cytopathic, capable of provoking acute and chronic necroinflammatory liver injury and are the most common causes of liver disease worldwide [1].

The various strategies that many viruses have evolved to evade natural killer (NK) cell effector functions illustrate the central role for these cells in early host defense against viral infections [2], [3]. It is known that both HCV and HBV impair human and murine NK cell activity [4], [5]. HCV may inhibit the activation of Interferon Regulatory Factor (IRF) [6] and can interfere with PKR kinase function [7] which can activate NK cells. In the case of HBV, both IRF and PKR appear to mediate signals that modulate viral replication in IRF(−/−) or PKR(−/−) transgenic mice [8]. In contrast to other viruses, HCV and HBV induce sustained HLA class I expression on infected cells [9], [10]. The inhibitory effect of NK cells on HCV replication in cultured human hepatocytes [11] suggests that NK cells might be able to contribute to the control of these infections. In the early phase of HBV infection, the host’s response to HBV mediates the activation of NK cells which produce IFN-α and are able to lyse infected cells by TRAIL-mediated death of hepatocytes [12].

Despite multiple studies on the functional potential of NK cells in persistent HCV and HBV infections [13], [14], [15], evidence of a role for NK cell defects in these chronic infections remains open to debate. It is established that NK cell frequencies in HCV-infected patients are lower than those observed in the uninfected population [14], [15], [16]. According to the surface expression level of CD56, NK cells can be divided into two subsets with important functional distinctions. The vast majority of NK cells displays a moderate level of CD56 (CD56dim) with a marked cytotoxicity potential, while, a small proportion harbors a high CD56 expression level (CD56bright) having a greater capacity to secrete cytokines [17], [18]. Recently, it was reported that the frequency of peripheral blood cytolytic CD56dim NK cells is decreased in HCV-infected patients [15]. Less is known about expression of HLA class I specific NK receptors (NKRs). NKRs are subdivided into two major families. The first family is composed of killer immunoglobulin-like receptors (KIR), such as CD158 molecules and LIR1/ILT2 (leukocyte Ig-like receptor 1/Ig-like transcript 2) which recognize classical HLA-A, -B and -C class I molecules. The second family includes C type lectin-like molecules, such as, NKG2A that associate with CD94 to form inhibitory heterodimer that recognize non-classical HLA-E molecules.

Here, we have performed flow cytometry analyses and detailed histological studies on liver biopsies to quantify and localize the CD56+ cells in the different histological areas. Furthermore, we investigated the perforin contents of different NK cells subset as markers of their cytotoxicity. Then, we searched for relationship between NK cells subset distribution and the clinical parameters.

Section snippets

Patients

Chronic hepatitis C (n = 28) and B patients (n = 19) seen in the Grenoble University Hospital between 2005 and 2007, were included in this study. Their main characteristics were analyzed as previously described [19] and are summarised in Table 1. For immunohistochemical studies, HCV (n = 6) and HBV (n = 6) patients were classed as have low necroinflammatory grading (Metavir-A1, 3 patients) and high necroinflammatory grading (Metavir-A3, 3 patients) by the French Metavir Scoring System. Blood samples

Decreased frequency of blood and intrahepatic NK cells in HCV patients

The flow cytometric gating strategy used to identify the different subsets of NK cells is depicted in Fig. 1. Phenotypic analysis of PBMCs from HCV patients demonstrated that the proportion of CD3CD56+ NK cells relative to lymphocyte populations was significantly decreased as compared to healthy donors (8.6% vs 13.3%, p < 0.05) (Fig. 2A). Examination of the CD3CD56dim and CD3CD56bright cells distribution revealed that the frequency of CD3CD56bright cells was significantly increased (10.0% vs

Discussion

Our data confirm a significant reduction in NK cell frequency and a quantitative imbalance of the CD56bright and CD56dim subsets within the total NK cell population in HCV- and HBV-infected patients [14], [16]. In HCV infections, under proinflammatory conditions, it has also been reported that CD3CD56dim NK cells are more susceptible to apoptosis compared to CD3CD56bright NK cells, under inflammatory conditions and that this can contribute to altered maintenance of CD3CD56dim NK cells in

Acknowledgements

We are grateful to the patients who enrolled in this study for their cooperation. This work was supported by grants from INSERM, from the Agence Nationale pour la Recherche sur le Sida (ANRS) and from Pole de compétitivité LyonBiopole. P.B. was supported by ANRS; M.R. from Higher Education Commission of Pakistan and X.C. by Région-Rhône Alpes “Cluster 10”. The authors thank Dr. Mary Callanan for reading this manuscript.

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    The authors who have taken part in this study declared that they do not have anything to disclose regarding funding from industry or conflict of interest with respect to this manuscript.

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    These authors contributed equally to this work.

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