1. Volume 121, Issue 6, Pages 1460-1472 (December 2001)
Kupffer cell–mediated recruitment of rat dendritic cells to the liver: Roles of N- acetylgalactosamine–specific sugar receptors 
Ryosuke Uwatoku, Makoto Suematsu, Taichi Ezaki, Takahito Saiki, Makoto Tsuiji, Tatsuro Irimura, Norifumi Kawada, Tatsuo Suganuma, Makoto Naito, Masayasu Ando, Kenjiro Matsuno 
Gastroenterology 
Volume 121, Issue 6, Pages (December 2001)
DOI: /gast
Copyright © 2001 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Recruitment of DC precursors defined as BrdU+OX62+ cells. Total OX62+ cells/mm2 (□), the number of OX62+ cells associated with KC/mm2 (▩), and the number of BrdU+OX62+ cells/mm2 (■) were counted at 0, 6, and 12 hours after latex injection. Note a significant increase of all the parameters at 12 hours. *P = and **P = 0.02 when compared with control. In addition, approximately 50% of BrdU+OX62+ cells were associated with KC (○) by 12 hours. Data are expressed as mean ± SD (n = 3).
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
A representative picture showing a close association of a recruited DC precursor, defined as a small round cell double-positive for BrdU (red nucleus) and OX62 (blue), with a latex-laden Kupffer cell (arrowhead).Scale bar, 20 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Representative pictures showing the behavior of CFSE-labeled DCs in rat hepatic microcirculation. The cells labeled with CFSE were injected into circulation, and their trafficking was visualized by intravital confocal microscopy. (A) Capture of the fluorescent DCs in sinusoids in a control rat. (B) A free-flowing DC in sinusoids of the KC-depleted rat (arrow). Open scale bar, 40 μm. (C and D) Intralobular distribution of DCs in the control and KC-depleted rats, respectively. Dark scale bar, 150 μm. In the KC-depleted group, free-flowing DCs were observed occasionally in sinusoids. (E–G) Serial pictures showing a CFSE-labeled DC extravasated into the space of Disse. Three video frames were captured with approximately a 10-μm z-axis difference in the focusing plane. Closed scale bar, 60 μm. P and C denote periportal and pericentral regions, respectively, throughout the figure.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Immunostaining of liver sections for donor DCs (OX76+; blue) and KCs (ED2+; brown) 30 minutes after cell transfer. Note that most donor DCs in the sinusoids are colocalized with host KCs (arrowheads). Scale bar, 20 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Immunoelectron micrograph of an association of DC and KC in the sinusoids. An OX76+ donor DC (D) forms an intimate cellular contact with a host KC (K) in the sinusoids of a Lewis rat. Note that the KC has distinct phagosomes (arrowheads), various vesicles containing electron-lucent materials, and irregular projections of cell membrane, some portions of which are facing directly the Disse's space (asterisk), because the endothelial cell lining (arrows) is often missing underneath KCs. H, hepatocyte. Scale bar, 2.5 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

7. Fig. 6
In situ cell binding assay in various tissues. DCs (black dots) showed preferential binding to the (A) liver compared with other tissues such as the (B) tongue. DCs also bound to mucous epithelial cells in the (C) colon, and a (D) mucous film. The binding of DCs to the (A) liver was suppressed by pretreatment of (E) GalNAc monosaccharide. Same magnification except D. Scale bars, 200 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Inhibition of cell binding by monosaccharides in vitro. DCs were pretreated with various monosaccharides. The relative number of DCs bound to the liver sections compared with control binding without sugar addition was estimated. Note that GalNAc showed a stronger inhibition compared with all other sugars at 25 mmol/L (■, *P < 0.05), and with other sugars except D-fucose at 50 mmol/L (□, **P < 0.05). At 100 mmol/L (▩), GalNAc and D-fucose showed a strong binding inhibition. Data are expressed as mean ± SD.
Gastroenterology  , DOI: ( /gast )
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9. Fig. 8
Physicochemical characteristics of DC-KC binding concerning temperature, energy, or Ca++ dependency, and effects of various mAbs. Liver sections were also pretreated with glycosidase, with 100 mmol/L GalNAc, or depleted of KCs in vivo. The relative number of DCs bound to the liver sections compared with nontreatment control binding was estimated. Data of 3–5 experiments in duplicate are expressed as mean ± SD.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Receptor specificity for endocytic activity of DCs. Proportions of DCs ingested either (A) GalNAc or (B) L-fucose polymers under the presence of GalNAc, or D-mannose monosaccharides were counted. The GalNAc polymer ingestion was specifically suppressed by GalNAc monosaccharide (*P = 0.007). Data of 3 experiments in duplicate are expressed as mean ± SD.
Gastroenterology  , DOI: ( /gast )
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11. Fig. 10
Endocytic activity of isolated DCs for GalNAc polymers (A, green fluorescence; and B, blue cytoplasmic staining). A granular fluorescent profile of GalNAc polymers in the cytoplasm (arrowheads) indicates endocytosis by DCs. Note a subpopulation of OX62+ cells (brown) ingested GalNAc polymers (arrowheads) in B. Scale bar, 10 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

12. Fig. 11
Reverse-transcription PCR analysis of rat DCs, KCs, thioglycollate-induced peritoneal macrophages (TG), and myeloma cell line, Y3Ag1.2.3 (Y3), for expression of rMgl, rKcr, rAsgr1, and rAsgr2 genes. Note expression of rMgl mRNA by DCs. (A) Ethidium bromide-stained agarose gels of rMgl (700 bp), rKcr (943 bp), rAsgr1 (702 bp), rAsgr2 (839 bp), and rG3PDH (983 bp) products. (B) Quantification of gene expressions was compared with G3PDH in the each cell.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

13. Fig. 12
Diagram illustrating the probable molecular mechanisms for the DC recruitment to the liver: 1, capture of antigens by KCs; 2, activation of KCs and secretion of chemokines; 3, DC migration to the liver in response to chemokines; 4, adherence of DCs to the sinusoidal wall through binding to KCs via interactions between GalNAc-specific receptors and sugar residues; 5, endocytosis of antigens by DCs during migration; and 6, extravasation of bound DCs into the space of Disse, thus achieving the recruitment to the liver.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions