The Role of Liver Stromal Cells in Dendritic Cells
Development in Mice
C.-C. Hsieh, H.-S. Chou, J.J. Fung, S. Qian, and L. Lu
ABSTRACT
The inherent tolerogenicity of liver allografts may be due to tolerogenic dendritic cells
(DC) therein. It is not clear whether the unique antigen-presenting function of liver DC is
intrinsic or whether it is altered by microenvironmental factors in the liver. In the present
study, we investigated the effect of hepatic stellate cells (HSC) on the development and
function of DC propagated from bone marrow. DC exposed to HSC or HSC supernates
expressed low CD11c, CD86, and major histocompatibility complex class II and elicited
inferior allostimulatory function compared with conventional DC. These results suggested
that soluble factor(s) secreted from HSC influence DC development.
T
OLEROGENIC PROPERTIES of the liver have been
demonstrated by spontaneous acceptance of allografts
in many species, as well as induction of tolerance to
antigens delivered via the portal vein.
1–3
The mechanisms
are not completely understood. Hepatic stellate cells (HSC)
are the major cell effectors of liver fibrogenesis, producing
large amounts of extracellular matrix proteins and cytokines
and engaging in tissue repair.
4
We have reported that
activated HSC possess strong immunoinhibitory activity to
suppress allogeneic T-cell immune responses.
5
Dendritic cells (DC) are professional antigen-presenting
elements with the potential to either stimulate or inhibit
immune responses. The inherent tolerogenicity of the liver
has been linked to tolerogenic antigen-presenting cells,
especially DC, therein.
7
It is not clear whether the tolero-
genic antigen-presenting function of liver DC is intrinsic or
whether it is altered by microenvironmental factors within
the liver. In the present study, we investigated the effect of
HSC on the generation and function of bone marrow
(BM)-derived DC.
MATERIALS AND METHODS
Mice and Preparation of HSC
Male C57BL/6 (B6; H-2
b
) and BALB/c (H-2
d
) mice were pur-
chased from The Jackson Laboratory (Bar Harbor, ME). All
animals were maintained in a specific pathogen-free facility. HSC
isolated from mouse liver nonparenchymal cells as previously
described
5,6
were cultured (10
5
/mL) in 25 cm
2
surface area flasks
(Nunclon, Roskilde, Denmark) at 37°C for 7 to 14 days in Roswell
Park Memorial Institute (RPMI)-1640 (Mediatech Inc, Herndon,
VA) media supplemented with 20% fetal calf serum under 5% CO
2
in air. HSC purity was determined by desmin immunostaining and
by their typical light microscopic appearance of lipid droplets.
Culture of DC
BM cells isolated from the femurs of B6 mice were cultured for 5
days in RPMI 1640 medium (2 10
6
/well) in the presence of
mouse recombinant granulocyte-macrophage colony-stimulating
factor (GM-CSF, 8 ng/mL; Schering-Plough, Kenilworth, NJ) as
previously described.
7
To test the influence of HSC on DC
development, the former was added at the beginning of culture
at a HSC:BM cell ratio of 1:80 (hereafter referred as HSC-
conditioned DC [HDC]).
Antibodies
Monoclonal antibodies specific for CD86, CD11b, CD11c, and
major histocompatibility complex (MHC) class II (IA
b
) were
purchased from BD PharMingen (San Diego, CA). Appropriate
isotype control reagents were used in all experiments. The
expression of cell surface antigens was evaluated using a BD
FACSCalibur flow cytometer (BD Biosciences, San Diego, CA).
Mixed Leukocyte Reaction
One-way mixed leukocyte reaction assays were performed in
triplicate in 96-well, round-bottom microculture plates (Corning,
Corning, NY) with BALB/c or B6 (syngeneic) spleen T cells as
From the Department of Immunology (C.-C.H., H.-S.C., S.Q.,
L.L.), Lerner Research Institute, and Department of General
Surgery (J.J.F., S.Q., L.L.), Cleveland Clinic, Cleveland, Ohio.
Ching-Chuan Hsieh is a Research Fellow from Department of
General Surgery, Chang Gung Memorial Hospital-Chiayi, Grad-
uate Institute of Clinical Medical Sciences, Chang Gung Univer-
sity, Taoyuan, Taiwan.
Address correspondence to Lina Lu, MD, Department of
Immunology, Lerner Research Institute, Cleveland Clinic, 9500
Euclid Ave., NB30, Cleveland, OH 44195. E-mail: lul2@ccf.org
© 2010 by Elsevier Inc. All rights reserved. 0041-1345/–see front matter
360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2010.09.139
Transplantation Proceedings, 42, 4279 – 4281 (2010) 4279