Effects of costimulation on intrahepatic immunopathogenesis in patients with chronic HBV infection

Abstract

Objective: Chronic HBV infection can lead to "immune tolerance" in asymptomatic carriers (ACs), "immune injury" in active chronic hepatitis (ACH) patients or "immune abnormality" in cirrhosis (Cir) and hepatocellular carcinoma (HCC) patients. Previous investigations reported that chronic hepatitis presented abnormal expression of costimulatory molecules. We investigated the costimulation profile in the liver of ACs and patients with ACH, Cir and HCC.

Methods: Patients with ACH, Cir and HCC, ACs and normal controls were recruited into the present study. The costimulation profiles and cytokines in the liver of patients were investigated by Western blotting, immunohistochemistry and real-time quantitative PCR. Correlations between serum alanime aminotransferase (ALT) levels, necroinflammation scores, cytokines and costimulatory proteins were assessed.

Results: The ACs presented decreased inflammatory and increased inhibitory costimulation, which was negatively correlated with inflammatory costimulatory proteins and ALT, whereas the ACH patients exhibited increased inflammatory costimulation and decreased inhibitory costimulation, which was correlated with increased ALT. The Cir patients showed both increased inhibitory and inflammatory costimulation. The HCC patients exhibited both decreased inhibitory and inflammatory costimulation.

Conclusion: Costimulation participates in intrahepatic immune responses, and plays important roles in immune tolerance, immune injury and immune abnormalities in patients with chronic HBV infection.

Fig. 1

Correlation between intrahepatic scores of necroinflammation and serum ALT, TB, HBsAg or HBV-DNA load. The histological examination of liver tissues was performed in 24 CHB patients, 6 patients in each group. The intrahepatic scores of necroinflammation were graded by the system of Ishak modified HAI. The correlation coefficient between the scores of necroinflammation and ALT, TB, HBsAg or HBV-DNA load was analyzed. The scores of necroinflammation in liver were positively correlated with ALT and TB, but negatively correlated with HBsAg and HBVDNA load in all the CHB patients

Fig. 2

Quantitative detection of costimulatory proteins or cytokine mRNA in liver. a Identification of CD80 and CD86 in liver. Western blotting was performed using a specific antibody against human costimulatory protein. Recombinant human CD80 and CD86 proteins, positive control CD80 protein (separate Ramos cell) and CD86 protein (separate Jurkat cell), negative control and liver homogenates of human were applied to determination of CD80 and CD86 in liver tissue. In the same western blotting, recombinant protein (Rec), positive control (Pos), negative control (Neg) and liver homogenates of human (Liver) were subjected to electrophoresis. In the photograph of CD86 protein, the molecular weight of recombinant CD86 protein was 90 kDa, and that of CD86 protein in human liver was 80 kDa. In the photograph of CD80, the molecular weight of recombinant CD80 protein was 76 kDa, and that of CD80 protein in human liver was 60 kDa. b Representative western blotting of costimulatory proteins in livers from six separate experiments. Homogenates obtained from the livers in the five groups were subjected to electrophoresis. β-Actin protein served as a protein loading standard. The molecular weight of CD86, CD80, CD83, CD28, CTLA-4, CD40, ICAM-1 and β-actin was 80, 60, 45, 44, 33, 48, 100 and 42 kDa, respectively. The CD80 stains in the ACH or Cir patient were darker than those in the HD, AC or HCC subject. The CD86 stains showed that the stains in the ACH or Cir patient were also darker than those in the HD, AC or HCC subject. In CD83 stains, the photograph showed dark stains of CD83 in the Cir patient. The photograph showed dark stains of CD28 and weak stains of CTLA-4 in the ACH patient. The stains of CTLA-4 in the AC or Cir patient were darker than those in the ACH, HD or HCC patient, whereas the stains in the ACH patient were weaker than those in other subjects. As the CD40 photograph showed, the dark stains of CD40 presented in the ACH or Cir patient. The ICAM-1 stains showed that the stains in the ACH and Cir patients were darker than those in the HD, AC and HCC subjects. c Relative quantity of costimulatory proteins in livers of the five groups (n = 6, in each group). The images on X-ray membrane were scanned, and the relative quantity of protein was normalized to the protein quantity for each sample using β-actin protein. All the parameters were shown in this figure. The data showed increased CD80, CD86, CD83, CD28 and CD40 and decreased CTLA-4 in the ACH patient, increased CD80, CD86, CD83, CD28, CD40 and CTLA-4 in the Cir patient, increased CTLA-4 and decreased CD80, CD86, CD28 and CD40 in the AC subject, and decreased CD80, CD86, CD28, CD40 and CTLA-4 in the HCC patient. The ICAM-1 levels in the five groups were gradually declined in the order of the ACH group, the Cir group, the HCC group, the AC group and the HD group. d Quantitative detection of cytokines mRNA in liver. The relative quantity of the INFγmRNA, IL-6 mRNA, IL-18 mRNA and IL-10 mRNA in liver in all the five groups was detected by real-time quantitative PCR. The data showed unchanged INFγmRNA, IL-6 mRNA and IL18 mRNA in all the five groups, and increased IL-10 mRNA in the AC or HCC patient

Fig. 3

Distribution of intrahepatic costimulatory proteins by immunochemical staining. Immunohistochemical staining was performed using a specific antibody against human costimulatory protein. In this figure, seven costimulation stains in liver of the ACH patient were shown. a Distribution of CD80 protein in liver. The hepatocytes were surrounded by CD80⁺ cells and inflammatory cells, ×400 magnification. b Distribution of CD86. CD86⁺ cells were localized in the inflammatory zone and they surrounded the hepatocytes, ×400 magnification. c Distribution of CD83 protein by immunochemical stains. CD83 stains mainly appeared in the inflammatory cells, and the CD83⁺ cells were localized in the inflammatory-necrotic zone, ×400 magnification. d Distribution of CD28 protein in the liver. CD28⁺ cells were enriched in the inflammatory zone, ×100 magnification. e CTLA-4 distribution in liver. Few CTLA-4 stains were found in the inflammatory zone in the liver of the ACH patient, ×400 magnification. f Distribution CD40 in the liver. CD40 ⁺ cells were enriched in the necrotic zone, and CD40 stains surrounded the hepatocytes, ×100 magnification. g ICAM-1 distribution in liver. Most ICAM-1 stains appeared in the hepatic sinus, ×400 magnification