B cell depletion therapy exacerbates murine primary biliary cirrhosis

Abstract

Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of antimitochondrial antibodies (AMAs). Indeed, the presence of AMAs represents the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear. Therefore, although AMAs appear to interact with the biliary cell apotope and contribute to biliary pathology, there is no correlation of disease severity and titer of AMAs. The recent development of well-characterized monoclonal antibodies specific for the B cell populations, anti-CD20 and anti-CD79, and the development of a well-defined xenobiotic-induced model of autoimmune cholangitis prompted us to use these reagents and the model to address the contribution of B cells in the pathogenesis of murine PBC. Prior to the induction of autoimmune cholangitis, mice were treated with either anti-CD20, anti-CD79, or isotype-matched control monoclonal antibody and followed for B cell development, the appearance of AMAs, liver pathology, and cytokine production. Results of the studies reported herein show that the in vivo depletion of B cells using either anti-CD20 or anti-CD79 led to the development of a more severe form of cholangitis than observed in control mice, which is in contrast with results from several other autoimmune models that have documented an important therapeutic role of B cell-specific depletion. Anti-CD20/CD79-treated mice had increased liver T cell infiltrates and higher levels of proinflammatory cytokines.

Conclusion: Our results reflect a novel disease-protective role of B cells in PBC and suggest that B cell depletion therapy in humans with PBC should be approached with caution.

Figure 1

Time course of anti-CD20 (A) and anti CD-79 (B) induced B cell depletion in peripheral blood. Representative MNC gated plots are shown for both CD20 mAb (C) and CD79 mAb (D) and respective control mAbs after 4 weeks of treatment. B cell frequencies were determined by immunofluorescence staining with flow cytometry analysis. Line and dot graphs indicate mean ± SEM. Significant differences were determined with Mann-Whitney test and are indicated as ***p < 0.0001.

Figure 2

Serum reactivity to PDC-E2 was quantified by ELISA before immunization with 2OA in both anti-CD20 (A) and anti CD79 treated mice (B). All values were corrected with baseline to eliminate background noise; significant differences between treatment antibodies and their controls were evaluated with Mann-Whitney test and are indicated as ***p < 0.001.

Figure 3

(A) Hematoxylin-eosin-stained tissue sections of livers from anti-CD20 -mAb-treated mice (right) demonstrate greater levels of lymphoid infiltration in the portal tract (black arrows) than controls (left). Bile duct damage is indicated with red arrows and portal space lacking bile ducts with an asterisk; two asterisks highlight a granuloma. Black scale bars = 100 µm. (B) The portal inflammation score, percentage of portal granulomas and parenchymal granulomas are presented for each sample. **p < 0.01, * p<0.05 in Mann-Whitney test.

Figure 4

(A) Histological evidence of cholangitis in representative hematoxylin-eosin-stained liver sections; tissue sections from anti-CD79 -mAb-treated mice demonstrate increase of cellular infiltrates around portal spaces (black arrows) and interlobular bile duct damage (red arrows). Portal space lacking bile ducts are indicated with an asterisk; two asterisks highlight a granuloma. Black scale bars = 100 µm. (B) The portal inflammation score, percentage of portal granulomas and parenchymal granulomas are presented for each sample. ** p<0.001, *p < 0.05 in Mann-Whitney test.

Figure 5

(A, C) Bile duct damage was studied by immunostaining with anti-CK22. Black arrow indicates interlobular bile duct damage, and the red arrow chronic non suppurative destructive cholangitis. Portal spaces with bile duct loss are indicated with an asterisk. Scale bars = 100 µm. (B, D) A quantitative evaluation of bile duct damage (percentage of portal spaces without interlobular ducts) is presented for each sample. *p < 0.05 in Mann-Whitney test

Figure 6

(A) Serum concentration of INF-γ and MCP-1 was significantly higher in B cell depleted mice than controls, whereas (B) IL-10 was lower. The bar graphs indicate mean ± SEM. Significant differences are indicated as **p < 0.01, *p < 0.05.