PMC

Colocalization of CXCL12 and biliary epithelial cells (BECs) in human and murine livers. A: Frozen sections from cirrhotic human livers show similar expression patterns of CXCL12 (green, clone 79081) and cholangiocytes, identified by expression of cytokeratin 19 (CK19; magenta). B and C: Uninjured (B) and bile duct–ligated (C) murine livers costained with CXCL12 (green) and BEC marker, MIC1-1C3 (magenta), show BEC expression of CXCL12 (white). n = 5 (A); n = 6 (B and C). Original magnification, ×400.

Time course of CXCL12 expression from murine and human cholangiocyte cell lines. Cell culture supernatants from both murine (A, 603b) and human (B, H69 and MMNK-1) cholangiocyte cell lines show no CXCL12 expression over a 96-hour time period. Conversely, murine (JS1) and human (LX2) stellate cell lines demonstrate increasing CXCL12 secretion between 0 and 96 hours. n = 3 independent experiments. ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Antibody confirmation of CXCL12 epitope expression in CXCL12–green fluorescent protein (GFP) reporter-positive cells. Dual-immunofluorescence staining on liver sections from mice expressing Gfp in the Cxcl12 locus with either CXCL12-ab25117 or clone 79081. A and B: CXCL12-GFP reporter cells appear green (A, white), with CXCL12-ab25117–positive cells appearing magenta (B). C: Overlap of GFP and CXCL12-ab25117 confirms CXCL12 epitope expression in GFP-positive cells, which appears white. D and E: Similarly, CXCL12 clone 79081 (D, red) also identifies CXCL12 epitope in GFP-positive cells (E, yellow). Arrowheads identify CXCL12-positive cells. Original magnification, ×400.

Cxcl12-GFP reporter mice show no green fluorescent protein (GFP) expression in biliary epithelial cells (BECs). Confocal images of liver sections from mice expressing Gfp in the Cxcl12 locus show no GFP expression in BECs. A and B: Cxcl12-expressing cells appear green (A), with MIC1-1C3–expressing cholangiocytes in magenta (B). C: Expression of Cxcl12 by BECs would appear white, which is notably absent. There is, however, an intimate association between Cxcl12-expressing cells and BECs (arrowheads). D and E: These Cxcl12-expressing cells surrounding the bile duct can be identified as hepatic stellate cells given their coexpression of desmin (arrowheads). n = 5. Original magnification, ×630.

Lack of CXCL12 expression in biliary epithelial cells (BECs) with alternate CXCL12 antibodies. CXCL12 staining on liver sections from cirrhotic human livers and mice with ligated bile ducts shows no CXCL12 expression using three alternate commercially available CXCL12 antibodies. Frozen sections were costained with anti-CXCL12 clone 79018 (A, C, E, G, and I, green) and either Santa Cruz Biotechnology Inc. rabbit anti-human/mouse CXCL12 (B and F, red), Abcam rabbit anti-human/mouse CXCL12 (D and J, red), or eBiosciences rabbit anti-mouse CXCL12 (H, red). Liver sections show bright fluorescence in BECs stained with CXCL12 antibody clone 79018 but not with the three alternate antibodies. Original magnification, ×400.

Increased number of Cxcl12-expressing cells in central vein (CV) region compared with the portal tract (PT) are hepatic stellate cells. A and B: Cxcl12-expressing cells (green) can be seen directly adjacent to bile ducts in the PT (arrowheads pointing to bile duct) and around a CV, with more Cxcl12-expressing cells seen in the vicinity of the CV compared with the PT. C: Quantification of green fluorescent protein (GFP)–positive cells shows threefold more cells located in the proximity of the CV, defined as 50% to 100% of PT to CV distance. D–F: Coimmunostaining for stellate cell marker desmin (D, red) shows that many of these CXCL12-expressing cells (E, green) surrounding the CV are hepatic stellate cells (F, yellow). n = 5. ^∗∗∗P < 0.001.

Murine and human cholangiocytes do not express CXCL12. Enzyme-linked immunosorbent assay (ELISA) from cell culture supernatants collected at 24 hours from murine (A) and human (B) cholangiocytes shows no significant CXCL12 secretion compared with serum-free media (SFM). Murine (JS1) and human (LX2) hepatic stellate cell lines, used as positive controls, show robust CXCL12 secretion. Immunoblots from murine (C) and human (D) cholangiocytes demonstrate no band at the expected location for CXCL12, whereas strong bands are seen in stellate cell protein lysates. Absolute quantification of CXCL12 copy number per microgram of total mRNA in murine (E) and human (F) cholangiocytes shows minimal CXCL12 transcript compared with stellate cells. G: Treatment of a murine cholangiocyte cell line (603b) or primary murine cholangiocytes with 500 U/mL tumor necrosis factor-α (TNF-α) or 100 ng/mL lipopolysaccharide (LPS), factors known to stimulate cholangiocytes, does not lead to any CXCL12 production, whereas JS1 murine hepatic stellate cell secretion is increased by 1.8- and 3.5-fold, respectively. n = 3 independent experiments for ELISA, immunoblot assays, and real-time quantitative PCR. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. BEC, biliary epithelial cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.