Expression of Granzyme B in viral hepatitis in patients with ALPS
Authors: Nicolas André, Bertrand Roquelaure, Isabelle Thuret, Marianne Ziol, Frédéric Rieux-Laucat, Françoise Le Deist
Journal: Hepatology (2004)

Apoptosis has transformed our understanding of the pathogenesis of several diseases. Thus, autoimmune lymphoproliferative syndrome (ALPS) is a rare disease due to genetic defects in the Fas apoptotic pathway.1-3 Patients with ALPS display several clinical manifestations, including lymphoproliferative disorders and systemic autoimmune manifestations.1-3 Only one case in patients associating liver disease with ALPS syndrome has been reported.4 We report here three patients with APLS and chronic viral hepatitis. As the Fas pathway is critical for the genesis of hepatitis,5 the occurrence of hepatitis in a context of Fas deficiency raises the question of the existence of an alternative lymphocytic-mediated apoptotic pathway. To investigate this eventuality, we performed a case control qualitative and quantitative study of Granzyme B (GzB) in intrahepatic cytotoxic lymphocytes in 3 patients with APLS and chronic viral hepatitis. Data concerning clinical presentations and main laboratory findings are reported in Figure 1A. No antibody reflecting the existence of autoimmune hepatitis6 was found. Fas deficiency diagnosis was based on defective Fas-induced apoptosis assay as previously described.3 Sequencing of the Fas-encoding gene confirmed the diagnosis by showing the presence of a heterozygous mutation as previously described.3 The paraffin-embedded formalin-fixed liver biopsies of the 3 patients with ALPS were quantitatively analyzed for CD8 and GzB expression and compared to 3 liver biopsies from patients with chronic hepatitis B matched for histological activity index. Briefly, after heat antigen retrieval, slides were incubated with anti-GzB (Monosan, Uden, The Netherlands) and anti-CD8 (Dako, Glostrup, Denmark) monoclonal antibodies. Standard biotin-avidin complex immunoperoxidase was used with diaminobenzidine as chromogen for revelation. Quantification of CD8 and GzB were performed as follows: for each liver biopsy slide, 8 randomly selected ×400 magnification fields (0.2 mm2) out of portal tracts were analyzed for the number of CD8- and GzB-positive cells. Mean and standard deviation of the number of GzB- and CD8-positive cells per field for each biopsy and the ratio of the number of GzB+ to CD8+ cells was calculated for each case. (A) Clinical and laboratory findings. AN, autoantibodies to nuclear antigens; Farr, autoantibodies to DNA; AP, autoantibodies to phospholipids; AR, autoantibodies to erythrocytes; AP, autoantibodies to platelets; APMN, autoantibody to PMN; ASM, autoantibody to smooth muscle; I, increased; N, normal; ND, not determined. CD4-CD8-/TcR α/β: Percentage of TcR α/β (+) lymphocytes expressing neither CD4 nor CD8 detected by cytometry. CD95 MoAb-induced apoptosis (%): Percentage of apoptotic cells. (B) Microphotographs of Granzyme B and CD8 immunostaining on serial sections of liver biopsy in Patient 3 with ALPS and chronic active hepatitis B (a,b) compared to a control chronic active hepatitis B (c,d) without Fas deficiency. Arrows indicate granular and polarized granzyme B staining in lymphocyte cytoplasm in patients with ALPS (a), or in control patients with chronic hepatitis B (c). (C) Granzyme B/ CD8+ cells in ALPS patients and control patients. The number of CD8+ lymphocyte was not statistically different in ALPS cases compared to control cases (21 ± 10 vs. 24 ± 14; Wilcoxon rank test: P > 0.4). A 10 ± 5-fold increase in the GzB-to-CD8 ratio was found (Wilcoxon rank test: P < 0.05). Details of the results are presented in Figure 1C. We show that when viral hepatitis occurs in a patient with Fas deficiency, GzB-positive cells are overrepresented in the liver lymphocytic infiltrate compared to a control population, while there was no difference in the number of CD8+ lymphocytes in the infiltrate. Even if Fas is essential in the development of hepatitis,5 hepatitis can occur in human even in the case of a defect in the Fas pathway. The hyperactivation of GzB may counterbalance Fas deficiency and explain, at least partially, the mechanism of viral hepatitis in patients with ALPS. Nevertheless, GzB is activated during T-cell activation, and is therefore an excellent marker of an activated T-cell phenotype. Thus, other quantitative methods of GzB activity in the liver but also in circulating lymphocytes are required to confirm the functional role of GzB, and whether or not GzB overexpression is constitutive or depends on certain stimuli such as viral infections. Further studies are also required to confirm that overexpression of GzB can be met in patients with other mutations than the one we describe here. Nicolas André M.D., Ph.D.*, Bertrand Roquelaure M.D. , Isabelle Thuret M.D. , Marianne Ziol M.D. , Frédéric Rieux-Laucat Ph.D.?, Françoise Le Deist M.D., Ph.D.?, * UMR-CNRS 6032, UFR Pharmacy, Marseille, France, Multidisciplinary Department of Pediatrics, Children Hospital of "La Timone", Marseille, France, Department of Pathology, J. Verdier Hospital, Bondy, France, ? Inserm 429, Necker-Enfants-Malades, Paris, France, ? Laboratory of Pediatric Immunology, Necker-Enfants-Malades, Paris, France.