CCR5 antagonists seem to be a new class of potent anti-HIV drugs.1 Because approximately one quarter to one third of HIV-infected subjects are coinfected with hepatitis C virus (HCV),2 the potential effect of CCR5 antagonists on HCV infection must be considered. This concern has recently been emphasized by studies reporting an increased prevalence of hepatitis C as well as an increase in viral load,3 serum alanine aminotransferase (ALT) levels,4 and liver fibrosis5 during chronic hepatitis C in subjects homozygous for a 32-base deleted CCR5 gene, Δ32CCR5, which encodes a truncated CCR5 molecule that is not expressed at the cell surface.6
Actually, various factors argue for a role of CCR5 in the pathobiology of HCV infection. Chemokines and chemokine receptors are likely to be essential in the recruitment of activated T lymphocytes in the inflamed HCV-infected liver, especially CCR5, which is a receptor for the C-C chemokines macrophage inflammatory protein (MIP)-1α (CCL-3) and MIP-1β (CCL-4) and regulated on activation of normal T-cell expression and secretion (regulated on activation, normal T expressed and secreted [RANTES], CCL-5). These proinflammatory chemokines are able to attract and activate CCR5-expressing T lymphocytes.7 MIP-1α and MIP-1β are expressed by vascular endothelium in portal tracts in the normal and HCV-infected liver.8 RANTES, whose plasma level is elevated in viral hepatitis,9 is produced in the portal area and at sites of piecemeal necrosis in HCV-infected liver, where its local expression level is correlated with serum ALT levels10 and liver inflammatory activity.11 Furthermore, CCR5 chemokine receptor is overexpressed on liver-infiltrating T lymphocytes.8,11 Finally, MIP-1α, MIP-1β, and RANTES are released by CD8+ cytotoxic T cells during their effector functions,12 allowing the recruitment of additional CCR5+ T cells. All these data suggest that CCR5 might be important for T-cell recruitment in the inflamed HCV-infected liver and might thus be involved in viral clearance in acute HCV infection and in the generation of hepatic lesions during chronic hepatitis C.
We have previously shown that CCR5 density varies in each individual but is stable over time for a given individual.13 Moreover, we have also shown that the level of CD4+ as well as CD8+ T-cell surface expression of CCR5 determines the level of cellular response to CCR5-binding chemokines, so that T cells with high CCR5 densities migrate more efficiently toward these chemokines than cells with low CCR5 densities (personal data, Pierre Corbeau, 2004). Therefore, subjects expressing high CCR5 densities might fight HCV infection more vigorously than low CCR5 expressers. If this hypothesis proves to be correct, cell surface expression of CCR5 might become a new prognostic factor in HCV infection and might be helpful in therapeutic decision making during chronic hepatitis C. Moreover, if low CCR5 density is associated with a bad prognosis in chronic HCV infection, CCR5 antagonists administrated as anti-HIV agents to HIV-infected subjects might be contraindicated in case of HIV/HCV coinfection. Therefore, it is important to elucidate the role of CCR5 in HCV infection. For this purpose, we have determined by flow cytometry the mean number of CCR5 molecules at the surface of T lymphocytes in 51 HIV-1/HCV-coinfected patients and looked for a correlation with the main parameters of liver damage and HCV replication.
MATERIALS AND METHODS
Pathologic findings of the liver were classified using the standard METAVIR scoring system14 for necroinflammatory grade of activity (0, no histologic activity; 1, slight activity; 2, moderate activity; and 3, severe activity) and stage of fibrosis (0, no fibrosis; 1, portal fibrosis without septa; 2, portal fibrosis with rare septa; 3, numerous septa with bridging fibrosis without cirrhosis; and 4, cirrhosis). Knodell's numeric scoring system was also used for assessing liver histologic activity.15
Fifty-one HCV/HIV-1-coinfected patients (33 men, 18 women) were recruited at the University Hospital of Montpellier, France. They ranged in age from 29 to 53 years (median = 42 years). Most of the patients (80%) were intravenous drug users. None of them had received anti-HCV treatment before the study. The median duration of HCV infection, estimated according to serology and history of injection drug use, was 18 years (range: 9-23 years). The median HCV RNA concentration, as determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR; Amplicor Monitor HCV; Roche Diagnostic Systems, Meylan, France), was 7.9 × 105 IU/mL (range: 4100-6.3 × 106 IU/mL). Liver tissue was obtained from 49 patients. Four percent had no histologic activity, 51% had slight activity, 23% had moderate activity, and 22% had severe activity. Sixteen percent had no fibrosis, 43% had fibrosis stage 1, 23% had fibrosis stage 2, 12% had fibrosis stage 3, and 6% had cirrhosis. The CD4 counts of the 51 patients ranged from 47 to 746 CD4+ T cells/μL (median = 329 cells/μL). Their median HIV RNA plasma level was 101 copies/mL (range: <20-750,000 copies/mL). Forty-four (86%) of 51 patients received antiretroviral multitherapy.
Immunofluorescent Staining and Flow Cytometry Analysis
CCR5 density at the surface of D-related (DR)−CD4+ T cells was measured by a commonly used quantitative flow cytometry assay as described previously.16 Briefly, cells were indirectly labeled with the anti-CCR5 antibody 2D7 (Pharmingen, San Diego, CA) and fluorescein isothiocyanate (FITC)-conjugated anti-immunoglobulin (Ig) and directly labeled with phycoerythrin (PE)-conjugated anti-CD4 antibody and PE-Cy-5 anti-human leukocyte antigen (HLA)-DR antibody (Beckman-Coulter, Margency, France). CCR5 expression was analyzed after gating on DR−CD4+ T cells. CCR5 density was calculated by converting FITC fluorescence intensity into antibody-binding capacity, which corresponds, at the saturating concentrations we used, to the number of CCR5 molecules present on the cell surface. Fluorescence intensity was converted to CCR5 density according to a calibration curve obtained with standard microbeads (DAKO QIFIKIT; DAKO, Glostrup, Denmark) precoated by the manufacturer with various densities of monoclonal antibody (mAb) and subsequently labeled with the FITC-conjugated anti-Ig probe. Intra-assay variability was less than 5%.
Spearman rank correlations were used as a measure of association between T-cell surface CCR5 density and the other factors. CCR5 expression on CD4+ T cells from HCV-infected individuals with or without hepatitis was compared by the Mann-Whitney U test.
CCR5 Expression on Peripheral CD4+ and CD8+ T Lymphocytes
Because CD4+ T cells and CD8+ T cells are both involved in anti-HCV immunity, we wondered whether CCR5 expression on these 2 lymphocyte subpopulations was correlated. To address this question, we measured CCR5 membrane expression on CD4+ and CD8+ T lymphocytes of 72 healthy volunteers by quantitative flow cytometry. Because CCR5 is modulated by T-cell activation, all CCR5 densities were determined on nonactivated HLA-DR− T cells. We observed a correlation between DR−CD4+ T-cell and DR−CD8+ T-cell CCR5 densities (r = 0.71, P < 0.01; Fig. 1A). This correlation allowed us to extrapolate CCR5 density measured on DR−CD4+ T cells to DR−CD8+ T lymphocytes. Second, we checked that DR−CD4+ T-cell surface CCR5 expression was stable over time in HIV/HCV-coinfected patients, because we have shown that it is stable in HIV-positive patients.13 For this purpose, we followed the DR−CD4+ T-lymphocyte CCR5 density of 10 HCV/HIV-coinfected patients for 7 to 60 months. As shown in Figure 1B, CCR5 density was stable during this period.
T-Cell Surface CCR5 Density Does Not Correlate With Hepatitis C Virus Load, Serum Transaminase Levels, or Histologic Parameters
Based on the hypothesis that high CCR5 expression could strengthen anti-HCV immunity, we first looked for an inverse correlation between DR−CD4+ T-cell surface CCR5 density and HCV load. As shown in Figure 2A, we did not find any link between CCR5 density and HCV load (r = 0.12, P = 0.39). The average serum ALT and aspartate aminotransferase (AST) levels of the 51 HCV/HIV-coinfected patients were calculated from independent blood samples drawn over a period of 4 to 24 months and compared with cell surface CCR5 density measured on DR−CD4+ T lymphocytes. Here again, no correlation was found between DR−CD4+ T-cell surface CCR5 density and either AST (r = −0.04, P = 0.75; see Fig. 2B) or ALT (r = −0.08, P = 0.57; see Fig. 2C) serum level. Histologic analysis was performed in 49 patients and compared with CCR5 density on DR−CD4+ T lymphocytes. Using the METAVIR score, we observed no correlation between DR−CD4+ T-cell surface CCR5 expression and liver inflammatory activity (r = −0.07, P = 0.62; see Fig. 2D) or fibrosis (r = 0.01, P = 0.97; see Fig. 2E). Likewise, Knodell's numeric scoring system did not reveal any correlation between DR−CD4+ T-cell surface CCR5 density and histologic activity (r = −0.21, P = 0.17; data not shown). To better appreciate liver damage progression, we calculated the fibrosis evolution over time (METAVIR fibrosis score/years of HCV infection) in 37 patients for whom the duration of HCV infection was documented. As shown in Figure 2F, we did not find any correlation between DR−CD4+ T-cell surface CCR5 density and the rate of liver fibrosis progression (r = −0.05, P = 0.97). Sorting of the patients according to sex or HCV genotype did not improve the correlations (data not shown). The percentage of DR−CD4+ T cells expressing CCR5 was not correlated with virus load, aminotransferase levels, or histologic activity and fibrosis (data not shown).
Absence of Correlation Between CCR5 Density and Hepatitis C Virus Clearance
To evaluate the role of CCR5 in the clearance of the virus and resolution of acute HCV infection, we compared DR−CD4+ T-cell surface CCR5 density between 18 patients who have cleared the virus (ie, HCV aviremic patients without biologic sign of hepatitis) and the 51 patients of our cohort, who have chronic HCV infection. As shown in Figure 3, DR−CD4+ T-cell surface CCR5 expression was similar in these 2 groups of patients: 8184 ± 3610 (n = 18) and 7082 ± 2929 (n = 51), respectively (P = 0.13). No difference in the percentage of DR−CD4+ T-cells expressing CCR5 was observed between these 2 groups (data not shown).
In the present study, we tested the hypothesis that the intensity of CCR5 expression at the single T-cell level, which determines the importance of the T-cell chemotactic response to CCR5-binding C-C chemokines, might influence the efficiency of the anti-HCV T-cell response in HCV/HIV-coinfected patients. We failed to find any correlation between CCR5 density on T lymphocytes and the main biochemical, virologic, or histologic markers of chronic hepatitis C. Moreover, we did not detect any difference in CCR5 density between patients who have resolved acute hepatitis and patients who have developed chronic HCV infection. Thus, in these patients, CCR5 cell surface expression does not seem to be decisive for the success of an acute immune response against HCV or for the liver damage caused by HCV-specific T cells.
One explanation for our findings might be that the level of expression of CCR5 does not determine the intensity of the anti-HCV immune response. The simplest explanation for the absence of a role of CCR5 in HCV infection could be the redundancy of the chemokines/chemokine receptors system, with other chemokine receptors like CXCR3 or CCR1 compensating for CCR5 deficiency. The hypothesis that CCR5 is not involved in the anti-HCV immune response is in conflict with the increase in the prevalence of the CCR5 Δ32/Δ32 genotype among HCV-infected patients reported by Woitas et al.3 Yet, various groups have argued that this observation carried out in a population of anti-HCV-positive, anti-HIV-negative hemophiliac patients was the result of a selection bias, with the HIV-protective effect of the CCR5 Δ32/Δ32 mutation having artificially increased the prevalence of this genotype in this restricted group of patients dually exposed to HIV and HCV but monoinfected by HCV.17-21 Nevertheless, in the cohort we have studied, CD4+ T-cell surface CCR5 densities range from 1150 to 18,944 molecules per cell, and we cannot exclude the possibility that the absence of CCR5 expression in CCR5/Δ32 homozygous patients could have a negative impact on HCV disease evolution.
An alternative explanation for our findings is that CCR5 actually plays a role in anti-HCV immunity but that it is compensated by its role in HIV infection. Thus, the deleterious effect on the anti-HCV immune response of low CCR5 expression could be counterbalanced by its protective effect against HIV-1 replication. Indeed, we have previously shown that CD4+ T-cell surface CCR5 density is correlated with HIV disease progression,13 so that in patients with high CCR5 expression, the faster evolution of HIV infection might overcome the HCV-infected hepatocyte destruction by the immune system.
Our data argue against a key role in HCV infection of the variability in CCR5 expression among HCV/HIV-coinfected patients. These findings are reassuring based on the use of CCR5 antagonists to treat HCV/HIV-coinfected patients.
The authors are grateful to Pascal Perney for his critical reading of the manuscript.
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