Emperipolesis mediated by CD8+ T cells correlates with biliary epithelia cell injury in primary biliary cholangitis

Abstract Primary biliary cholangitis (PBC) is an autoimmune disease characterized by chronic destruction of the bile ducts. A major unanswered question regarding the pathogenesis of PBC is the precise mechanisms of small bile duct injury. Emperipolesis is one of cell‐in‐cell structures that is a potential histological hallmark associated with chronic hepatitis B. This study aimed to clarify the pathogenesis and characteristics of emperipolesis in PBC liver injury. Sixty‐six PBC patients, diagnosed by liver biopsy combined with laboratory test, were divided into early‐stage PBC (stages I and II, n = 39) and late‐stage PBC (stages III and IV, n = 27). Emperipolesis was measured in liver sections stained with haematoxylin‐eosin. The expressions of CK19, CD3, CD4, CD8, CD20, Ki67 and apoptosis of BECs were evaluated by immunohistochemistry or immunofluorescence double labelling. Emperipolesis was observed in 62.1% of patients with PBC, and BECs were predominantly host cells. The number of infiltrating CD3+ and CD8+ T cells correlated with the advancement of emperipolesis (R 2 = 0.318, P < .001; R 2 = 0.060, P < .05). The cell numbers of TUNEL‐positive BECs and double staining for CK19 and Ki67 showed a significant positive correlation with emperipolesis degree (R 2 = 0.236, P < .001; R 2 = 0.267, P < .001). We conclude that emperipolesis mediated by CD8+ T cells appears to be relevant to apoptosis of BEC and thus may aggravate the further injury of interlobular bile ducts.

Emperipolesis is one of cell-in-cell structures that have been observed in chronic viral hepatitis. 5 Recently, a study on auto-immune hepatitis showed emperipolesis to be predominantly mediated by CD8 + T cells, which appeared to induce apoptosis of hepatocytes and thus revealing another potential mechanism of autoimmune hepatocyte injury. 6 In our previous study, we had shown that emperipolesis was frequently seen in PBC, while BEC was predominantly host cells. 7 However, it remains unknown whether emperipolesis is involved in BEC injury. Our present study aims to define the role of emperipolesis in bile duct injury in PBC and to identify the cell types entering BEC concerning the chronic nonsuppurative destructive cholangitis by analysing the infiltrating cells around the damaged bile duct in PBC patient samples.

| Subjects
Liver biopsy specimens were obtained from all patients histologically characterized as early PBC (E-PBC, stages I and II, n = 39) and  (Table 1). These patients, whose average ages were 43.5 ± 7.1 years, included 59 women and 7 men. With approval from our institutional review board, all subjects provided written informed consent prior to enrollment in the study. The diagnosis was made by serum biochemistry and clinical features according to EASL and AASLD guidance, and all subjects were double-checked by liver biopsy. 1,8 Liver specimens fulfilled the needle biopsy standard (1.5 cm length, portal tract number no <8), and the evaluations were undergone blinded. All other infections or liver diseases were excluded from the selected type of disease. All patients were provided informed consent according to a protocol approved by the Fifth Medical Center, General Hospital of PLA and Hebei Medical University. Demographic and biochemistry data of all cases were shown in Table 1.

| Immunohistochemistry
Rabbit polyclonal antibodies against human CD3, CD4, CD8 and CD20 were used for immunohistochemical staining of liver. Tissue sections were cut by 4 μm from paraffin-embedded tissue blocks and placed on slides. After deparaffinization, slides were exposed to microwave pretreatment in 10 mmol/L sodium citrate buffer (pH 6.0) at 98°C for 15 minutes for antigen retrieval. Endogenous After benzidine reaction, sections were counterstained with haematoxylin. Negative controls were run by replacing the primary antibody with PBS.  Semi-quantitative analysis was performed for immunoreactivity.

TA B L E 1 Demographic characteristics and laboratory parameters of patients with PBC and controls
Five representative hepatic lobules and portal tracts were randomly selected in each section. The populations of CD3, CD4, CD8 and CD20 positive cells were assessed by counting five random fields (magnification, ×200) using a standard grid. Values are given as the mean ± standard deviation (SD).

| IHC double staining and IF staining
Samples were pretreated suitably for each antibody, respectively.

| TUNEL apoptosis detection
The sections were deparaffinized with routine process. The specimens were then rinsed in PBS for 5 minutes and incubated with proteinase K (20 μm/mL) for 15 minutes at 37°C. The slides were then rinsed in PBS 3 times for 5 minutes each. DNA fragmentation was detected using TUNEL Apoptosis Detection Kit (Merck Millipore, German). DAPI was used to stain the nucleus.

| Liver histopathological assessment
Liver biopsies, performed using a 16-gauge needle, were fixed in 10% neutral buffered formalin and embedded in paraffin before 4-μm-thick sections were cut. Sections were stained with haematoxylin and eosin (H&E) and Masson methods. All liver tissue specimens were independently reviewed blindly by two liver pathologists. were considered statistically significant, as the P value was <.05. The pathological diagnoses were triply verified by 3 hepato-pathologists.

| The clinical characteristics of PBC patients
Totally, 66 patients were enrolled in our study, which were divided into  (Table 1).

| Histological features of liver tissues from PBC patients and emperipolesis
The main morphology changes of PBC were in portal tracts. The liver tissues in early stage showed the damage of bile duct and obvious proliferation of small bile ducts. The granulomas and lymphoid follicles were found in the liver tissues of PBC ( Figure 1). There was significant difference between early stage and late stage in the presence of granulomas, lymphoid follicles, damage and proliferation of bile ducts in portal areas ( Table 2). Severity of fibrosis was demon- In PBC patients with emperipolesis, there were more severe changes including portal inflammation, lymphoid follicles, damage and proliferation of bile ducts. PBC patients without emperipolesis also had more advanced fibrosis than those with emperipolesis.

| Inflammatory cells infiltration around the injured bile ducts and Emperipolesis
In portal area of the liver tissue from patients with PBC, CD3 + , CD4 + , CD8 + and CD20 + positive cell number increased (Table 3; Figure 2).
The numbers of CD3 + , CD4 + and CD8 + cells around the damaged bile duct in the early stage were more than that in the late stage.
Compared with E-PBC, the number of infiltrated lymphocytes in L-PBC decreased, especially CD3 + and CD8 + positive cells. There was significant difference between two groups (all P < .05).
We performed immunohistochemistry double labelling staining for CD3, CD4 and CD8 T cells, CD20 B cells, and the biliary epithelial cells marker CK19 in the liver sections which showed emperipolesis in H&E stained slides from PBC patients. BEC was stained red, and invading cells were stained brown in IHC ( Figure 2B). BEC was stained blue, and invading cells were stained red in IHC ( Figure 2C).
CD8 + positive cells were stained green, and BEC was stained red, F I G U R E 1 Types of infiltrating immune cells in the portal area from patients with PBC. Representative images of CD3 + , CD4 + and CD8 + T cell and B cell (CD20) staining of the portal area from liver biopsies (n = 66) by immunohistochemistry. Dark brown colour indicates positive staining, whereas the cellular structure is visualized by haematoxylin counterstaining. Note the higher number of infiltrating cells in E-PBC compared to L-PBC. Magnification ×200 and the invading cells were stained yellow in IF ( Figure 2D). Contact between CD8 T cells and CK19 was frequently detected, while entry of CD8 T cells into BECs was occasionally observed (Figure 2). The entry of CD4 and CD20 cells into BECs was not detected. CD3 and CD8 T cells correlated with emperipolesis process (R 2 = 0.318, P < .001; R 2 = 0.060, P < .05; Figure 4).

| Emperipolesis correlated with apoptosis and proliferation of BECs
To illustrate the consequence of emperipolesis of CD8 T cells, There was significant difference between the two groups (P < .01, P < .05). TUNEL-positive BEC were correlated with emperipolesis (R 2 = 0.236, P < .001; Figure 4). Double staining cells of CK19 and Ki67 were correlated with emperipolesis (R 2 = 0.267, P < .001; Figure 4). In early stage of PBC, the entry of CD8 + T cells into BECs was observed and correlated with TUNEL and Ki67-positive BECs but was not found in late stage.

| D ISCUSS I ON
In our study, emperipolesis of lymphocytes in BECs was more likely to be observed in E-PBC with lymphoid follicles, damage and proliferation of bile ducts, and inflammatory cell infiltration. Emperipolesis is associated with inflammation and reduced in the repair of fibrosis.
These features may be closely related to injuries of bile duct and may eventually be followed by the development of fibrosis and cirrhosis in PBC. Our previous study had shown emperipolesis was accompanied with severe inflammation of CHB including interface hepatitis, confluent necrosis, focal lytic necrosis and portal inflammation. 7 So, emperipolesis may be one of the initiating factors of the disease.
Lymphocytic infiltration around interlobular bile duct in liver portal area is one of the histological features of PBC. Our results showed that most of the infiltrating lymphocytes were T lymphocytes which were consistent with the findings of Ichik. 9 In our study, the proportion of CD4 + cell in the portal area is relatively high in Our study showed that CD20 + lymphocytes increased significantly in PBC. We speculate that humoural immunity may also involve in the inflammatory reaction of PBC. The study showed BECs invasion by T cells might be promoted by the CD5-B cell population in liver tissues of PBC patients. 13 Ballot and others' studies also support our results. 14  Cell-in-cell phenomena have gained more attention over the recent years after being ignored for almost a century. Their biological mechanisms and pathogenic roles are starting to emerge. Some findings suggested that lymphocytes may use the alternative cell-in-cell phenomenon to kill target tumour cells. 15,16 Alternatively, tumour cells may destroy invading lymphocytes as a mechanism to escape immune surveillance. 17 The consequences of emperipolesis were also debatable. We found that CD8 T cells were the major cell type of lymphocytes infiltrating BECs in PBC.
The fate of inner cells in cell-in-cell structures had been reported to be partially dependent on the properties of the invading cells. 18,19 We found emperipolesis of CD8 T cells appeared to be related with apoptosis and proliferation of BECs. In addition, the up-regulation of WAF1 and p53 related to biliary apoptosis is found in cholangiocytes of PBC, 4 and significantly greater apoptosis and correlated with TUNEL-positive BECs but was not found in late stage. In early stage of PBC, the biliary epithelial cells were damaged by apoptosis and proliferation. The injured cells were eliminated, and the cells proliferated in order to replace the apoptotic cells simultaneously.

| CON CLUS ION
In conclusion, the main immune cells in liver tissues were CD3+, CD4+, CD8 + lymphocytes, and CD8 + T cells were predominant around the damaged interlobular bile ducts in early stage of PBC.
However, in the late stage of PBC, all the cell number decreased.
According to our results, the changes of number, proportion and distribution of immune cells in PBC liver tissues showed the damage of intrahepatic bile ducts was mediated mainly by cell immunologic injury through Th1 way. In early stage of PBC, emperipolesis mediated by CD8 + T cells appeared to be relevant to apoptosis of BECs and further led to the injury of interlobular bile ducts.

CO N FLI C T O F I NTE R E S T
No conflict of interest claimed.