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Intrahepatic CD4+ Cell Depletion in Hepatitis C Virus/HIV-Coinfected Patients
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JAIDS Journal of Acquired Immune Deficiency Syndromes: Volume 37(1) 1 September 2004
Canchis, P Wilfredo MD*; Yee, Herman T PhD, MD†; Fiel, M Isabel MD‡; Dieterich, Douglas T MD§; Liu, Ruei-Che PhD?; Chiriboga, Luis PhD†; Jacobson, Ira M MD*; Edlin, Brian R MD*; Talal, Andrew H MD, MPH*
From the *Center for the Study of Hepatitis C, Department of Medicine, Weill Medical College of Cornell University, New York, NY; †Department of Pathology, New York University School of Medicine, New York, NY; ‡Department of Pathology, Mount Sinai School of Medicine, New York, NY; §Department of Medicine, Mount Sinai School of Medicine, New York, NY; and ?Department of Biostatistics, Columbia University, New York, NY.
"...In this investigation, we found that HCV/HIV-coinfected patients had decreased portal CD4+ cells, increased lobular lymphocyte apoptosis, and increased periportal hepatocyte proliferation compared with HCV-monoinfected patients. The relative decrease in the number of intrahepatic CD4+ cells may contribute to increased levels of HCV RNA observed in coinfected patients. In addition, HIV RNA may promote hepatocyte proliferation, possibly contributing to more advanced hepatic injury frequently observed in coinfected patients. Further investigation will be required to discern the functional significance of portal CD4+ cell decreases and their antigenic specificity in coinfected patients...As a result of HIV infection, intrahepatic lymphocyte phenotypes may be altered, which may impact on the natural history and resolution of HCV infection...An increase in lobular CD4+ cell apoptosis may contribute to the decreased number of portal CD4+ cells...In the chimpanzee model, intrahepatic CD4+ cells are important in spontaneous HCV clearance and are necessary for adequate control of HCV replication. In humans, intrahepatic CD4+ cells may be important in HCV clearance during acute infection and in response to anti-HCV treatment..."
Abstract
Summary: Coinfection with HIV and hepatitis C virus (HCV)-specific immune responses, increases hepatic inflammation, accelerates hepatic fibrosis, and is associated with deceased treatment responses.
We quantified intrahepatic lymphocyte and hepatocyte phenotypes in HCV-infected patients with (n = 38) and without (n = 41) HIV infection. A single pathologist counted positive cells in 5 portal and 5 lobular areas.
Coinfected patients had 6.81 ± 1.9 fewer CD4+ cells per portal field (10.58 ± 1.12 vs. 4.97 ± 1.09 cells/high-power field [HPF]; P < 0.001) and 0.48 ± 0.15 more apoptotic lymphocytes per lobular field (0.16 ± 0.06 vs. 0.64 ± 0.15 cell/HPF; P = 0.002) than monoinfected patients.
The number of portal CD4+ cells was not associated with the peripheral CD4+ cell number. Portal and lobular CD8+ cells did not differ between the 2 groups. Portal proliferative hepatocytes were increased in coinfected patients with HIV RNA levels of >400 copies/mL (1.13 ± 0.32 cells/HPF; P = 0.01) compared with those with undetectable HIV RNA (0.46 ± 0.09 cell/HPF) and monoinfected patients (0.45 ± 0.08 cell/HPF).
Portal Lymphocytes
The mean number of portal CD4+ cells was significantly decreased in coinfected patients compared with monoinfected patients. Multivariate analysis revealed that coinfected patients had 6.8 ± 1.9 fewer CD4+ cells per portal field than monoinfected patients. Coinfection was also associated with fewer portal CD4+ cells in analyses restricted to patients with grade 2 or higher (5.7 vs. 11.0 cells/HPF; P = 0.005) and stage 2 or higher (5.6 vs. 9.8 cells/HPF; P = 0.01) disease. Multivariate analysis demonstrated that stage, grade, HCV genotype, race, and HCV RNA levels were not significantly associated with CD4+ cell number in either monoinfected or coinfected patients. Interestingly, portal CD4+ cell number was not associated with either peripheral CD4+ cell number (P = 0.35) or peripheral HIV RNA levels (P = 0.78). Portal CD3+ cells were also decreased in coinfected patients, although the difference did not reach statistical significance (P = 0.06); however, the mean numbers of portal CD8+ cells and proliferative and apoptotic lymphocytes in the portal region were similar in coinfected and monoinfected patients.
In conclusion, HIV coinfection is associated with fewer portal CD4+ cells and increased lobular lymphocyte apoptosis that may impact on the natural history of HCV infection.
INTRODUCTION
End-stage liver disease has emerged as a major contributor to morbidity and mortality in hepatitis C virus (HCV)/HIV-infected patients.1 Epidemiologic studies have suggested that HCV/HIV-coinfected patients have increased hepatic inflammation and fibrosis compared with HCV-monoinfected patients.
As a result of HIV infection, intrahepatic lymphocyte phenotypes may be altered, which may impact on the natural history and resolution of HCV infection.
The cellular composition of the liver consists of parenchymal (hepatocytes) and nonparenchymal cells of which intrahepatic lymphocytes account for 16%-22%. The adult liver contains ~109-1010 lymphocytes, most of which are located in the portal region. A predominantly portal T-cell infiltrate is a characteristic finding in HCV infection, while the hepatic lobule is a proposed site of activated CD8+ cell apoptosis. The ratio of CD4+:CD8+ cells in the liver (1:3.5) is the inverse of that found in lymph nodes.
In the chimpanzee model, intrahepatic CD4+ cells are important in spontaneous HCV clearance and are necessary for adequate control of HCV replication. In humans, intrahepatic CD4+ cells may be important in HCV clearance during acute infection and in response to anti-HCV treatment. Although coinfected patients may have increased hepatic inflammation compared with monoinfected patients, the effect of HIV on T-cell subsets in the liver in HCV-infected patients is not known. We investigated whether HIV infection leads to a decrease in intrahepatic CD4+ cells and whether coinfection increases hepatocyte proliferation. We performed immunohistochemical staining on liver sections from HCV-monoinfected and HCV/HIV-coinfected patients to phenotype intrahepatic T cells and to enumerate proliferative and apoptotic lymphocytes and hepatocytes.
AUTHOR DISCUSSION
In this study, we phenotyped and enumerated proliferative and apoptotic intrahepatic T cells and hepatocytes within the portal and lobular regions, areas of the liver with distinct microanatomic, immunologic, and functional characteristics. We found that portal CD4+ cells were decreased in coinfected compared with monoinfected patients, while the number of lobular CD4+ cells did not differ significantly. We also found that the number of apoptotic lymphocytes in the liver lobule was increased in HCV/HIV-coinfected patients compared with monoinfected patients. In addition, periportal hepatocyte proliferation was increased in coinfected patients with detectable HIV RNA levels.
Portal CD4+ cells were decreased and lobular apoptotic lymphocytes were increased in coinfected compared with monoinfected patients. Lobular apoptotic lymphocytes may be either CD4+ or CD8+ cells. An increase in lobular CD4+ cell apoptosis may contribute to the decreased number of portal CD4+ cells if T cells migrate from the portal to the lobular region. Alternatively, activated CD8+ cells may migrate to the liver lobule where they undergo apoptosis.
The decrease in intrahepatic CD4+ cells, as a consequence of HIV-associated cell death, may partially account for increased HCV RNA levels that have been observed in coinfected patients. In chimpanzees and in humans, resolution of HCV infection requires strong and multispecific intrahepatic CD4+ cell responses, while patients with weak responses are likely to develop chronic infection. In the absence of adequate CD4+ cell priming, memory CD8+ cells in the chimpanzee are unable to completely suppress HCV replication. Consequently, HIV-associated decreases in intrahepatic CD4+ cells may weaken HCV-specific cytotoxic T-lymphocyte suppression of HCV replication, resulting in increased HCV RNA levels frequently observed in coinfected patients.
We found increased periportal hepatocyte proliferation in coinfected patients with detectable HIV RNA levels and an inverse association between periportal hepatocyte proliferation and elevated HCV RNA levels. Hepatocyte proliferation was significantly increased in patients with HIV RNA levels of >400 copies/mL compared with patients with HIV RNA levels of <=400 copies/mL. A hypothesis deserving further investigation is whether HIV-1 augments hepatocyte proliferation. The explanation for the inverse association between hepatocyte proliferation and HCV RNA levels is not clear; however, the level of HCV RNA is not associated with the extent of hepatic inflammation or fibrosis in untreated patients. In another study, we recently found that hepatocyte proliferation increased with increasing severity of liver inflammation and fibrosis in HCV infection, a process that is evidently independent of the level of peripheral blood HCV RNA. Consequently, both viruses, HIV and HCV, may contribute to periportal hepatocyte proliferation. The interaction between these 2 viruses at the level of the hepatocyte is deserving of further study.
Although several investigators have noted that hepatic inflammation is increased in coinfected patients, we observed that intrahepatic T cells were decreased in these patients. T cells, however, are only one component of the cells that comprise the necroinflammatory grade assessed on liver biopsy specimens. Other cell types that are also included in the assessment of necroinflammatory grade are B cells, macrophages, natural killer cells, and neutrophils. Immunohistochemical staining of hepatic sections would permit enumeration of each cell type and their localization within the liver (ie, portal or lobular).
In this investigation, we found that HCV/HIV-coinfected patients had decreased portal CD4+ cells, increased lobular lymphocyte apoptosis, and increased periportal hepatocyte proliferation compared with HCV-monoinfected patients. The relative decrease in the number of intrahepatic CD4+ cells may contribute to increased levels of HCV RNA observed in coinfected patients. In addition, HIV RNA may promote hepatocyte proliferation, possibly contributing to more advanced hepatic injury frequently observed in coinfected patients. Further investigation will be required to discern the functional significance of portal CD4+ cell decreases and their antigenic specificity in coinfected patients.
METHODS
Study Subjects
Liver biopsy specimens were obtained from 38 HCV/HIV- coinfected and 41 HCV-monoinfected patients for assessment of the severity of liver disease before initiating anti-HCV therapy. Clinical information including age, sex, ethnicity, and antiretroviral medication use was obtained through retrospective review of medical records. Laboratory data including alanine aminotransferase levels, HCV RNA levels, HCV genotypes, HIV RNA levels, and peripheral CD4+ cell counts were also collected. Informed consent was obtained from each patient. The Institutional Review Boards at Weill Medical College of Cornell University, New York University School of Medicine, and Cabrini Medical Center approved the study protocol.
Histologic Evaluation
At the time of biopsy, liver tissue was placed in 10% neutral buffered formalin, processed overnight, and embedded in paraffin. Routine histopathologic assessment included hematoxylin-eosin, periodic acid-Schiff (with and without diastase digestion), reticulin, and Masson trichrome. A pathologist (M.I.F.), blind to the subjects' clinical diagnoses, evaluated the grade of necroinflammatory activity (0-4) and the stage of fibrosis (0-4) in each liver section according to the Metavir system.
HCV Genotyping and HCV and HIV-1 RNA Quantitation
HCV genotypes were determined by sequencing the NS5B region and classified according to the method of Simmonds et al. HCV RNA and HIV RNA levels were determined by the Roche Amplicor assay (Version 2.0; Roche Diagnostics, Branchburg, NJ) with linear ranges from 103 to 107 HCV RNA copies/mL and 400 to 750,000 HIV RNA copies/mL.
RESULTS
Thirty-eight HCV/HIV-coinfected and 41 HCV-monoinfected patients were included in this study. The monoinfected patients were predominately caucasian, while the coinfected patients were predominantly non-caucasian. HCV RNA levels were significantly increased in coinfected compared with monoinfected patients. There were more Caucasians in coinfection group (55% vs 82%). Liver biopsy grade: <2, 23% in coinfection group vs 41% in monoinfection; >=2, 81% in coinfection & 75% in monoinfection, NS.
Lobular Lymphocytes
The mean number of CD3+, CD4+, CD8+, and proliferative lymphocytes in the lobular region did not differ between coinfected and monoinfected patients. In contrast, coinfected patients had 0.48 ± 0.15 more apoptotic lymphocytes per lobular field than monoinfected patients (P = 0.002). Portal CD4+ cells were also significantly associated with increased lobular apoptotic lymphocytes (P = 0.005). Both of these associations persisted in multivariate analysis. Coinfection was also associated with increased lobular apoptotic lymphocytes in analyses restricted to patients with grade 2 or higher (0.7 vs. 0.2 cell/HPF; P < 0.01) or stage 2 or higher (0.7 vs. 0.2 cell/HPF; P = 0.003) disease. In addition, lobular proliferative lymphocytes were increased in coinfected patients with HIV RNA levels of >400 copies/mL (1.5 cells/HPF) compared with those with HIV RNA levels of <=400 copies/mL (0.72 cell/HPF; P = 0.013).
Portal Hepatocytes
Periportal proliferative hepatocytes tended to be increased in coinfected (0.72 ± 0.13) compared with monoinfected (0.45 ± 0.08; P = 0.07) patients. This difference, however, was entirely explained by increased numbers of portal proliferative hepatocytes in the 13 patients with detectable HIV RNA (1.13 ± 0.32; P = 0.01). Portal proliferative hepatocytes were similar in HIV-infected patients with undetectable HIV RNA (0.46 ± 0.09) and in those without HIV infection (0.45 ± 0.08). By multivariate modeling, we found that portal proliferative hepatocytes were increased in coinfected patients with detectable HIV RNA levels (>400 copies/mL) and in those with low HCV RNA levels (<1 x 106 copies/mL), while patients with HCV RNA levels of >1 ¥ 106 copies/mL had significantly fewer periportal proliferative hepatocytes (P = 0.02).
Lobular Hepatocytes
The number of lobular proliferative hepatocytes was similar in coinfected and monoinfected patients, but it was increased in those with elevated HCV RNA levels and age older than 40 years. The number of apoptotic hepatocytes in either portal (P = 0.19) or lobular (P = 0.34) areas did not differ significantly between coinfected and monoinfected patients.
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