Tonsil Stromal-Cell Lines Expressing FDC-Like Properties: Isolation, Characterization, and Interaction with B Lymphocytes
Journal of
Tonsil Stromal-Cell Lines Expressing FDC-Like Properties: Isolation, Characterization, and Interaction with B Lymphocytes
GRZEGORZ SKIBINSKI 0
ANNA SKIBINSKA 0
MARTINE DECKERS 0
KEITH JAMES 0
0 Lister Research Laboratories, Department of Surgery, University of Edinburgh, Royal Infirmary , Lauriston Place, Edinburgh EH3 9YW, Scotland , UK
The microenvironment of secondary lymphoid organs consists of two major populations of cells, the lymphoid cells and a population of stromal cells that contribute to both tissue architecture and function. Interactions of both populations are essential for the development and control of humoral immune responses. In this study, stromal-cell preparations were obtained by a multistage process. This involved culturing 300-400-/zm slices of human tonsil for 6-8 days at 25C, trypsin digestion of the residual explant, followed by CD45-positive-cell depletion using magnetic beads, and a final period of culture for 4 days to remove remaining nonadherent cells. Phenotypifig with a panel of monoclonal antibodies revealed that the cells express HLA-DR, CD54 (ICAM-1), CD44, but no CD45 nor a range of other markers for epithelial and endothelial cells. Immunoassays of supernatants from stromal cells revealed that IL-6 was produced constitutively, and its production was increased by treatment with TNF-ce and IFN-y. In contrast IL-1, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12, TNF-ce, and IFN y were not produced. Functional tests showed that these cells express follicular dendritic cell-like properties. Coculturing of tonsilar B cells with stromal cells resulted in enhanced proliferation and also led to increased production of immunoglobulins and IL-6, suggesting crucial signaling between these populations.
FDC-like cell lines; FDC/B lymphocyte interactions; human tonsil
INTRODUCTION
During antigen-specific T-cell-dependent immune
al., 1990; M611er, 1992). This maturation process is
highly controlled and depends on numerous
interactions with interdigidating dendritic cells, T cells,
responses in secondary lymphoid tissues,
antigenand stromal cells, including highly specialized
follispecific naive B cells undergo a series of events
cular dendritic cells (Kosco and Gray, 1992). The
including activation, expansion, somatic mutations,
crosstalk between B-cell subsets and their
microisotype switch, selection, and differentiation into
environment is controlled by membrane/ligand
recepeither antibody-secreting or memory cells (Kroese et
tor interactions (MacLennan, 1994) and signals from
*Corresponding author.
the cytokine network (Banchereau and Rousset,
1992).
Most of the studies on follicular dendritic cells in
vitro have been hampered by the fact that it is very
difficult to isolate pure FDC with reasonable yields.
Tsunoda et al. (1990) first reported isolation and
maintenance of human FDC in long-term culture;
these cells, however, did not grow in vitro. Lindhout
et al. (1994) obtained EBV-transformed human FDC
lines, but their long doubling time makes their
cultivation in vitro difficult. Clark et al. (1995) and
Kim et al. (1995) described cell lines with FDC-like
properties that grew in vitro without exogenous
growth factors.
In this paper, we followed the idea used by
Jenkinson et al. (1992) in the mouse and by Gady et
al. (1993) in human systems to obtain thymic stromal
elements. We cut tonsil tissue into 300-400-/zm thick
slices and then cultured them at 25C in order to
deplete them of CD45-positive cells. CD45-negative
cells obtained in this way were cultured in vitro.
Stromal elements thus obtained were
enzymedigested and expanded in vitro. In this study, we show
that the stromal-cell lines (SCL) obtained by this
procedure exhibit FDC-like properties. Furthermore,
we have characterized their phenotype, cytokine
secretion, and their interaction with B lymphocytes.
Isolation of Human Tonsil Stromal Cells
Tonsils were cut into 300-400-#m thick slices and
incubated in RPMI containing 1% FBS for 6-8 days at
25C with gentle rocking. During this time, most of
the lymphocytes were shed into the medium. At the
end of the culture period, the tissue slices were
washed and digested with 0.25% trypsin. The cells
obtained still contained a variable proportion of
CD45-positive cells (5-20%, depending on the
experiment); it was therefore further subjected to a depletion
step with the aid of magnetic beads. The resulting cell
population contained less then 2% of CD45-positive
cells and was seeded into culture flasks. After 24 hr
incubation, large adherent cells appeared. During the
first 3-4 days of culture, the few remaining lymphoid
cells degenerated. The remaining cells were adherent
and uniform, grew in the absence of exogenous
cytokines, and exhibited characteristic
fibroblastoidlike morphology (Fig. 1). Confluent cells were split
using 0.25% trypsin to detach them from plastic. The
results presented here were obtained from tonsils of
seven different donors. The cells were passaged u (...truncated)