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Pathology International 2015; 65: 335–337
doi:10.1111/pin.12271
Letter to the Editor CD56+ lymphocyte counts remain low in the livers of human immunodeficiency virus/hepatitis C virus patients commencing ART To the Editor: Hepatitis C virus (HCV) infects more than 170 million people worldwide and is a major cause of chronic liver disease. Co-infection with human immunodeficiency virus (HIV) is common due to shared routes of transmission.1 Inflammatory cells found in the liver include natural killer (NK) and natural killer T (NKT) cells which have important roles in host defence through inflammatory cytokines and via hepatic stellate cells.2 NK cells have the phenotype CD56+CD3-, whereas NKT cells are CD56+CD3+. Dysfunction of NK and NKT cells has been reported in patients with chronic HCV infection, with numbers in the liver decreasing as hepatitis progresses to cirrhosis.3 Circulating NK cell numbers increase as patients begin antiretroviral therapy (ART), so HIV replication may have a role in NK cell dysfunction.4 However both T-cell and NK cell dysfunction influence the anti-fibrotic response of NK cells in co-infected patients.5 Meier et al.6 also demonstrated reduced numbers of NK cells, shifts between NK cells subsets and decreased capacity of NK cells to produce IFN-γ in HIV/HCV co-infection. Intrahepatic CD56+ NK/NKT cells are reduced in chronic hepatitis,3 but have not been studied systematically in HIV/ HCV patients. We present a longitudinal study of intrahepatic NK/NKT cells from co-infected patients beginning ART. 10–30% of patients with HIV/HCV co-infection commencing ART experience immune restoration disease (IRD), with increasing alanine transaminase (ALT) levels as circulating CD4+ T cell counts rise when patients begin ART.7 Two reports of a prospective cohort of HIV/HCV patients treated in Jakarta, Indonesia, examined the effects ART and IRD.8,9 Yunihastuti et al.8 showed that a plasma marker of T-cell activation (soluble CD26) rose during HCV IRD. Gani et al.9 showed that intrahepatic CD4+ cells increased while CD8+ cells diminished after ART, but these were not predictive of IRD. Here we quantitate CD56+ NK/NKT cells in liver biopsies of the same patients. The sample set is unique in that patients with early stage liver disease were biopsied twice at predetermined intervals relative to the start of the ART. HIV/HCV patients (n = 48) were recruited consecutively at the HIV/AIDS clinic of Cipto Mangunkusumo Hospital, Jakarta.8 Inclusion criteria were HCV seropositivity (Elecsys anti-HCV, Roche Diagnostics, Pleasanton, CA, USA), aged 17–50 years, no previous ART or therapy for HCV disease and 50g/week. Liver biopsy was accepted by all patients at baseline and 34 patients at week 48.8,9 Embedded tissues were retained, but 9 baseline samples and 8 samples from week 48 contained insufficient tissue for further immunostaining. Antiretroviral drugs comprised two nucleoside reverse trancriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) (zidovudine, stavudine, lamivudine, nevirapine, efavirenz). The study was approved by the Ethics Committee for Research on Human Subjects, Medical Faculty, University of Indonesia. Separate written informed consent was given for each liver biopsy. ALT levels were determined using an ADVIA 1800 Clinical Chemistry Analyser (Bayer, Leverkusen, Germany). Plasma HIV RNA and HCV RNA levels were measured using a Cobas Amplicore Monitor (Roche Molecular Systems). Lower limits of detection were 400 copies/mL and 200 HCV RNA copies/mL. Modified Menghini needles (Hepafix, B-Braun, Melsungen, Germany) were used to collect 2 cm specimens of liver tissue, which were fixed with formalin and embedded in paraffin. To assess fibrosis and necroinflammation, specimens were stained with Massontrichrome and hematoxylin-eosin. A pathologist read the specimens without knowing the patient’s condition. Necroinflammation was evaluated using Ishak’s scale (scored 0–18) and fibrosis (scored 0–6).9 CD56+ lymphocytes (identified by morphology and brown staining) were counted over 3–5 portal areas in sections stained with antihuman CD56 clone CMSSB (eBioscience, San Diego, CA, USA). Some cases showed positivity in biliary duct epithelia, so this was used as an internal control (Fig. 1a). Positive control slides included in each run were derived from a neuroendocrine carcinoma, and negative controls were liver biopsies stained without primary antibodies. Data were tabulated as medians and interquartile ranges, and compared using nonparametric Mann-Whitney tests. Results were noted when the differences achieved a P value of 2.7 in 3 patients 5.6 (5.6–6.0) 40.5 (27.2–67.5)
5 (4–7) 1 (1–1) 29 (24–32) 46 (39–55) 0.75 (0.30–1.5)
5 (4–6) 1 (1–2) 28 (24–31) 44 (38–53) 0.4 (0.25–1)
4 (3–6) 1 (1–2) 55 (45–70) 33 (30–42) 0.67 (0.3–1.35)
†Numbers of lymphocytes per field, averaged over 3–5 fields.
HIV RNA levels at 48 weeks correlated with initial CD56+ lymphocyte counts (r = 0.35, P = 0.03). CD56+ lymphocyte counts did not correlate with fibrosis at baseline or at week 48 (r = −0.08, P = 0.63 and r = −0.03, P = 0.86; respectively). There was a weak correlation between CD56+ and CD4+ lymphocytes in the portal area (r = 0.30, P = 0.06) at baseline but not at week 48 (r = −0.09, P = 0.70). There were no correlations between CD56+ and CD8+ lymphocytes in portal area at baseline (r = −0.05, P = 0.78) or week 48 (r = 0.00, P = 1.00). Five patients experienced HCV IRD within 12 weeks on ART. At baseline, there was no difference between numbers of CD56+ lymphocytes in the liver in patients with and without IRD [median (interquartile range) 0.3 (0–1) vs 0.9 (0.3–1.7) respectively; P = 0.12)]. Similar CD56 + lymphocyte numbers in patients with and without HCV IRD is consistent with the lack of any association between IRD and hepatic CD4+ and CD8+ cells in this cohort.9 This study showed that CD56+ lymphocytes were rare or absent in the livers of HIV/HCV co-infected patients and their numbers did not rise during ART. NK and NKT cells can produce IFN-γ and inhibit fibrogenesis.2 Their numbers in blood and in the liver can decrease in chronic HCV monoinfection with a negative correlation with fibrosis.10 We found no correlation between numbers of CD56+ lymphocytes in the liver and fibrosis score. This may reflect the influence of HIV or the low levels of fibrosis present in the patients. At baseline, there was possible correlation between
CD56+ and CD4+ lymphocytes in the portal area, so local CD4+ T cells in the liver may have a larger influence than cells in the blood. However, there was no clear effect of ART on CD56+ lymphocytes whilst numbers of CD4+ cells in the liver increased on ART.9 Hence, there is no clear role for CD56+ lymphocytes in the evolution of HCV disease on ART. ACKNOWLEDGMENT We thank all patients who participated in this study. FINANCIAL SUPPORT The work was supported by the Strategic Research Scheme of the University of Indonesia and by Abbvie Pty. Ltd. DISCLOSURE No authors report conflicts in relation to this work. Yosephine G. Susufi,1 Ening Krisnuhoni,1 Rino A. Gani,2 Evy Yunihastuti,2 Silvia Lee3 and Patricia Price4 1
Departments of Anatomical Pathology, 2 Internal Medicine, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia Hospital, University of Indonesia, Jakarta, Indonesia, 3Department of Microbiology, Royal Perth Hospital, Perth and 4Curtin University of Technology, Bentley, Western Australia, Australia
© 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Letter to the Editor
REFERENCES 1 Blackard JT, Sherman KE. HCV/HIV co-infection: Time to re-evaluate the role of HIV in the liver? J Viral Hepat 2008; 15: 323–30. 2 Gao B, Radaeva S, Park O. Liver natural killer and natural killer T cells: Immunobiology and emerging roles in liver diseases. J Leukoc Biol 2009; 86: 513–28. 3 Kawarabayashi N, Seki S, Hatsuse K et al. Decrease of CD56+ T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma. Hepatology 2002; 32: 962–9. 4 Scott-Algara D, Paul P. NK cells and HIV infection: Lessons from other viruses. Curr Mol Med 2002; 2: 757–68. 5 Glassner A, Eisenhardt M, Kokordelis P et al. Impaired CD4+ T cell stimulation of NK cell anti-fibrotic activity may contribute to accelerated liver fibrosis progression in HIV/HCV patients. J Hepatol 2013; 59: 427–33.
337
6 Meier UC, Owen R, Taylor E et al. Shared alteration in NK cell frequency, phenotype and function in chronic human immunodeficiency virus and hepatitis C virus infection. J Virol 2005; 79: 12365–74. 7 Crane M, Matthews G, Lewin SR. Hepatitis virus immune restoration disease of the liver. Curr Opin HIV AIDS 2008; 3: 446–52. 8 Yunihastuti E, Lee S, Gani RA et al. Antibody and markers of T-cell activation illuminate the pathogenesis of HCV immune restoration disease in HIV/HCV co-infected patients commencing ART. Clin Immunol 2011; 139: 32–9. 9 Gani RA, Yunihastuti E, Krisnuhoni E et al. Periportal CD4+ cell infiltration increases in HIV/Hepatitis C virus-coinfected patients commencing ART, whereas CD8+ cells clear from the liver. J Infect Dis 2014; 210: 405–9. 10 El Aggan HA, Mohamed FAS, El Deeb N et al. Peripheral blood and intrahepatic natural killer cells in chronic hepatitis C: Relation to disease activity and hepatic fibrosis. J Med Res Inst 2008; 29: 28–39.
© 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Pathology International 2015; 65: 335–337
doi:10.1111/pin.12271
Letter to the Editor CD56+ lymphocyte counts remain low in the livers of human immunodeficiency virus/hepatitis C virus patients commencing ART To the Editor: Hepatitis C virus (HCV) infects more than 170 million people worldwide and is a major cause of chronic liver disease. Co-infection with human immunodeficiency virus (HIV) is common due to shared routes of transmission.1 Inflammatory cells found in the liver include natural killer (NK) and natural killer T (NKT) cells which have important roles in host defence through inflammatory cytokines and via hepatic stellate cells.2 NK cells have the phenotype CD56+CD3-, whereas NKT cells are CD56+CD3+. Dysfunction of NK and NKT cells has been reported in patients with chronic HCV infection, with numbers in the liver decreasing as hepatitis progresses to cirrhosis.3 Circulating NK cell numbers increase as patients begin antiretroviral therapy (ART), so HIV replication may have a role in NK cell dysfunction.4 However both T-cell and NK cell dysfunction influence the anti-fibrotic response of NK cells in co-infected patients.5 Meier et al.6 also demonstrated reduced numbers of NK cells, shifts between NK cells subsets and decreased capacity of NK cells to produce IFN-γ in HIV/HCV co-infection. Intrahepatic CD56+ NK/NKT cells are reduced in chronic hepatitis,3 but have not been studied systematically in HIV/ HCV patients. We present a longitudinal study of intrahepatic NK/NKT cells from co-infected patients beginning ART. 10–30% of patients with HIV/HCV co-infection commencing ART experience immune restoration disease (IRD), with increasing alanine transaminase (ALT) levels as circulating CD4+ T cell counts rise when patients begin ART.7 Two reports of a prospective cohort of HIV/HCV patients treated in Jakarta, Indonesia, examined the effects ART and IRD.8,9 Yunihastuti et al.8 showed that a plasma marker of T-cell activation (soluble CD26) rose during HCV IRD. Gani et al.9 showed that intrahepatic CD4+ cells increased while CD8+ cells diminished after ART, but these were not predictive of IRD. Here we quantitate CD56+ NK/NKT cells in liver biopsies of the same patients. The sample set is unique in that patients with early stage liver disease were biopsied twice at predetermined intervals relative to the start of the ART. HIV/HCV patients (n = 48) were recruited consecutively at the HIV/AIDS clinic of Cipto Mangunkusumo Hospital, Jakarta.8 Inclusion criteria were HCV seropositivity (Elecsys anti-HCV, Roche Diagnostics, Pleasanton, CA, USA), aged 17–50 years, no previous ART or therapy for HCV disease and 50g/week. Liver biopsy was accepted by all patients at baseline and 34 patients at week 48.8,9 Embedded tissues were retained, but 9 baseline samples and 8 samples from week 48 contained insufficient tissue for further immunostaining. Antiretroviral drugs comprised two nucleoside reverse trancriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) (zidovudine, stavudine, lamivudine, nevirapine, efavirenz). The study was approved by the Ethics Committee for Research on Human Subjects, Medical Faculty, University of Indonesia. Separate written informed consent was given for each liver biopsy. ALT levels were determined using an ADVIA 1800 Clinical Chemistry Analyser (Bayer, Leverkusen, Germany). Plasma HIV RNA and HCV RNA levels were measured using a Cobas Amplicore Monitor (Roche Molecular Systems). Lower limits of detection were 400 copies/mL and 200 HCV RNA copies/mL. Modified Menghini needles (Hepafix, B-Braun, Melsungen, Germany) were used to collect 2 cm specimens of liver tissue, which were fixed with formalin and embedded in paraffin. To assess fibrosis and necroinflammation, specimens were stained with Massontrichrome and hematoxylin-eosin. A pathologist read the specimens without knowing the patient’s condition. Necroinflammation was evaluated using Ishak’s scale (scored 0–18) and fibrosis (scored 0–6).9 CD56+ lymphocytes (identified by morphology and brown staining) were counted over 3–5 portal areas in sections stained with antihuman CD56 clone CMSSB (eBioscience, San Diego, CA, USA). Some cases showed positivity in biliary duct epithelia, so this was used as an internal control (Fig. 1a). Positive control slides included in each run were derived from a neuroendocrine carcinoma, and negative controls were liver biopsies stained without primary antibodies. Data were tabulated as medians and interquartile ranges, and compared using nonparametric Mann-Whitney tests. Results were noted when the differences achieved a P value of 2.7 in 3 patients 5.6 (5.6–6.0) 40.5 (27.2–67.5)
5 (4–7) 1 (1–1) 29 (24–32) 46 (39–55) 0.75 (0.30–1.5)
5 (4–6) 1 (1–2) 28 (24–31) 44 (38–53) 0.4 (0.25–1)
4 (3–6) 1 (1–2) 55 (45–70) 33 (30–42) 0.67 (0.3–1.35)
†Numbers of lymphocytes per field, averaged over 3–5 fields.
HIV RNA levels at 48 weeks correlated with initial CD56+ lymphocyte counts (r = 0.35, P = 0.03). CD56+ lymphocyte counts did not correlate with fibrosis at baseline or at week 48 (r = −0.08, P = 0.63 and r = −0.03, P = 0.86; respectively). There was a weak correlation between CD56+ and CD4+ lymphocytes in the portal area (r = 0.30, P = 0.06) at baseline but not at week 48 (r = −0.09, P = 0.70). There were no correlations between CD56+ and CD8+ lymphocytes in portal area at baseline (r = −0.05, P = 0.78) or week 48 (r = 0.00, P = 1.00). Five patients experienced HCV IRD within 12 weeks on ART. At baseline, there was no difference between numbers of CD56+ lymphocytes in the liver in patients with and without IRD [median (interquartile range) 0.3 (0–1) vs 0.9 (0.3–1.7) respectively; P = 0.12)]. Similar CD56 + lymphocyte numbers in patients with and without HCV IRD is consistent with the lack of any association between IRD and hepatic CD4+ and CD8+ cells in this cohort.9 This study showed that CD56+ lymphocytes were rare or absent in the livers of HIV/HCV co-infected patients and their numbers did not rise during ART. NK and NKT cells can produce IFN-γ and inhibit fibrogenesis.2 Their numbers in blood and in the liver can decrease in chronic HCV monoinfection with a negative correlation with fibrosis.10 We found no correlation between numbers of CD56+ lymphocytes in the liver and fibrosis score. This may reflect the influence of HIV or the low levels of fibrosis present in the patients. At baseline, there was possible correlation between
CD56+ and CD4+ lymphocytes in the portal area, so local CD4+ T cells in the liver may have a larger influence than cells in the blood. However, there was no clear effect of ART on CD56+ lymphocytes whilst numbers of CD4+ cells in the liver increased on ART.9 Hence, there is no clear role for CD56+ lymphocytes in the evolution of HCV disease on ART. ACKNOWLEDGMENT We thank all patients who participated in this study. FINANCIAL SUPPORT The work was supported by the Strategic Research Scheme of the University of Indonesia and by Abbvie Pty. Ltd. DISCLOSURE No authors report conflicts in relation to this work. Yosephine G. Susufi,1 Ening Krisnuhoni,1 Rino A. Gani,2 Evy Yunihastuti,2 Silvia Lee3 and Patricia Price4 1
Departments of Anatomical Pathology, 2 Internal Medicine, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia Hospital, University of Indonesia, Jakarta, Indonesia, 3Department of Microbiology, Royal Perth Hospital, Perth and 4Curtin University of Technology, Bentley, Western Australia, Australia
© 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Letter to the Editor
REFERENCES 1 Blackard JT, Sherman KE. HCV/HIV co-infection: Time to re-evaluate the role of HIV in the liver? J Viral Hepat 2008; 15: 323–30. 2 Gao B, Radaeva S, Park O. Liver natural killer and natural killer T cells: Immunobiology and emerging roles in liver diseases. J Leukoc Biol 2009; 86: 513–28. 3 Kawarabayashi N, Seki S, Hatsuse K et al. Decrease of CD56+ T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma. Hepatology 2002; 32: 962–9. 4 Scott-Algara D, Paul P. NK cells and HIV infection: Lessons from other viruses. Curr Mol Med 2002; 2: 757–68. 5 Glassner A, Eisenhardt M, Kokordelis P et al. Impaired CD4+ T cell stimulation of NK cell anti-fibrotic activity may contribute to accelerated liver fibrosis progression in HIV/HCV patients. J Hepatol 2013; 59: 427–33.
337
6 Meier UC, Owen R, Taylor E et al. Shared alteration in NK cell frequency, phenotype and function in chronic human immunodeficiency virus and hepatitis C virus infection. J Virol 2005; 79: 12365–74. 7 Crane M, Matthews G, Lewin SR. Hepatitis virus immune restoration disease of the liver. Curr Opin HIV AIDS 2008; 3: 446–52. 8 Yunihastuti E, Lee S, Gani RA et al. Antibody and markers of T-cell activation illuminate the pathogenesis of HCV immune restoration disease in HIV/HCV co-infected patients commencing ART. Clin Immunol 2011; 139: 32–9. 9 Gani RA, Yunihastuti E, Krisnuhoni E et al. Periportal CD4+ cell infiltration increases in HIV/Hepatitis C virus-coinfected patients commencing ART, whereas CD8+ cells clear from the liver. J Infect Dis 2014; 210: 405–9. 10 El Aggan HA, Mohamed FAS, El Deeb N et al. Peripheral blood and intrahepatic natural killer cells in chronic hepatitis C: Relation to disease activity and hepatic fibrosis. J Med Res Inst 2008; 29: 28–39.
© 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
