The immunopathology of liver granulomas in primary biliary cirrhosis

Abstract

Liver granulomas and elevated serum IgM are commonly observed in patients with primary biliary cirrhosis (PBC) but their pathogenetic significance remains largely unknown. To address this issue we performed an extensive immunostaining and colocalization study of markers associated with dendritic cells and IgM in a large cohort of tissue samples from PBC and control livers as well as from non-hepatic granulomatous diseases. First, the classical dendritic cell CD11c marker is highly expressed and more sensitive than classical hematoxylin-eosin staining in detecting granulomas associated with PBC and other conditions. Second, PBC cases with CD11c-positive granulomas have significantly higher serum IgM levels and earlier disease stages. Third, granulomas from PBC and other diseases demonstrate markers of dendritic cell immaturity, i.e. CD11b, reduced MHC II, IL-23, CCR7 and CD83 expression, and elevated C1q expression. Lastly, B cells and IgM-positive plasma cells are largely represented around PBC granulomas along with macrophages. In conclusion, our comprehensive immunohistochemical study suggests that dendritic cells are key to the pathogenesis of granulomas, regardless of their origin. More specifically, PBC liver granulomas may result from the interaction between immature dendritic cells and IgM.

Figure 1

Panel A: CD11c immunostain clearly define hepatic granulomas in PBC patients. (A) Representative immunohistochemical staining for CD11c in liver sections from PBC (n=51), AIH (n=37) and CHB (n= 30) (magnification 400×). Five fields were randomly selected and the area of CD11c deposition scored on a 0-4 scale to be compared between PBC, AIH and CHB patients (*p<0.05, ** p<0.01). Panel B: Representative hematoxylin-eosin staining and CD11c immunostaining of hepatic granulomas in portal tracts illustrating the vicinity with injured bile ducts (magnification 400×). Panel C: Representative CD11c immunostaining of small granulomas in hepatic lobules (Left) (magnification 400×), which in some cases are located in the vicinity of the portal tracts germinal center (Right) (magnification 200×).

Figure 2

Immature dendritic cell properties in PBC granulomas. Representative immunohistochemical staining for CD11b, PU.1, MHC II, C1q and IL-23 expression (magnification 400×). Confocal staining and imaging were performed for CD11c and MHC-II.

Figure 3

Presence of macrophages in PBC granulomas. Panel A: Representative immunohistochemical staining for CD68, CD163, as classical cellular markers of macrophages (magnification 400×). Panel B: Confocal staining analysis of CD11c and CD68 (magnification 400×).

Figure 4

Presence of B cells and IgM in PBC granulomas. Panel A: Representative immunohistochemical staining for CD19 (magnification 400×) and IgM (magnification 400×) in liver samples from PBC (n=51), AIH (n=37) and CHB (n=30). Five fields were randomly selected for observation in every sample and CD19 and IgM-positive areas scored on a 0-4 point scale for comparison between diseases (** p<0.01, respectively). Panel B: double immunohistochemical staining performed for CD11c (Red) and IgM (Black). Panel C: CD19 and IgM scores in the portal tracts were compared between granuloma positive (n=29) and granuloma negative (n=22) patients with PBC (* p<0.05).

Figure 5

IgM inhibition of the maturation of bone marrow-derived DC. Myeloid DC were pretreated with IgM (20 µg/ml) for an hour, then stimulated with LPS (1µg/ml) for 48 hours. Flow cytometry ananlysis was performed to observe the expression of MHC II, CD80 and CD86 in immature (left) and mature (Right) myeloid DC treated with (Blue) or without (Red) IgM (* p<0.05).

Figure 6

IgM inhibition of DC phagocytosis. Panel A: myeloid DC were pretreated with IgM (20 µg/ml) then incubated with apoptotic cells (CSFE stained). The percentages of CD11c and CSFE double positive cells were compared between groups treated with or without IgM (* p<0.05). Panel B: DC were pretreated with IgM (20 µg/ml) for 1 hour, then using LPS stimulated for 6 hours. RT-PCR was used to analyze the expression of IL-12p40, IL-6, IL-23p19, TNF-α, IFN-β, C1q, and β-actin (* p<0.05). Panel C: DC were pretreated with IgM (20 µg/ml) for an hour, then total protein was extracted from DCs after stimulated with LPS (1µg/ml) for 30 min. Immunoblots were performed with primary antibodies against phosphorylated or total ERK1/2, JNK p38, IKB-α, IRF3. α-Tubulin expression was determined as the loading control.