By
§
From the * Department of Microbiology and Immunology, and Cardiovascular Research Institute,
University of California San Francisco, San Francisco, California 94143; § Centenary Institute of
Cancer Medicine and Cell Biology, Sydney, 2050 New South Wales, Australia;
Howard Hughes
Medical Institute, University of Alabama at Birmingham, Birmingham, Alabama 35294; and ¶ Biogen Inc., Cambridge, Massachusetts 02142
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Abstract |
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Mice deficient in the cytokines tumor necrosis factor (TNF) or lymphotoxin (LT) /
lack
polarized B cell follicles in the spleen. Deficiency in CXC chemokine receptor 5 (CXCR5), a
receptor for B lymphocyte chemoattractant (BLC), also causes loss of splenic follicles. Here we
report that BLC expression by follicular stromal cells is defective in TNF-, TNF receptor 1 (TNFR1)-, LT
- and LT
-deficient mice. Treatment of adult mice with antagonists of LT
1
2 also leads to decreased BLC expression. These findings indicate that LT
1
2 and
TNF have a role upstream of BLC/CXCR5 in the process of follicle formation. In addition to
disrupted follicles, LT-deficient animals have disorganized T zones. Expression of the T cell attractant, secondary lymphoid tissue chemokine (SLC), by T zone stromal cells is found to be
markedly depressed in LT
-, and LT
-deficient mice. Expression of the SLC-related chemokine, Epstein Barr virus-induced molecule 1 ligand chemokine (ELC), is also reduced. Exploring the basis for the reduced SLC expression led to identification of further disruptions in T
zone stromal cells. Together these findings indicate that LT
1
2 and TNF are required for the
development and function of B and T zone stromal cells that make chemokines necessary for
lymphocyte compartmentalization in the spleen.
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Introduction |
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Genetic studies in mice have established that the cytokines TNF, lymphotoxin (LT
),1 and LT
, and the
receptors TNFR1 and LT
R, are required for normal compartmentalization of lymphocytes in the spleen (1, 2). In TNF- and TNFR1-deficient mice, follicular dendritic
cells (FDCs) are lacking and B cells fail to form follicular
clusters, instead appearing in a ring at the T zone periphery
(3). Mice deficient in LT
, which are deficient in membrane LT
1
2 heterotrimers and soluble LT
3 complexes
(7), show a distinct phenotype that includes absence of
lymph nodes and Peyer's patches, and loss of marginal zone
cells, FDCs, and normal B/T segregation in white pulp
cords of the spleen (6, 8). LT
/
mice, which cannot
produce membrane forms of LT that bind LT
R but continue to express LT
, exhibit a similar phenotype except that some lymph nodes are retained and splenic architecture
is somewhat less disturbed than in LT
/
mice (11, 12;
Korner, H., and J.D. Sedgwick, unpublished observations).
In parallel studies an understanding has begun to develop
of the role played by chemokine receptors and chemokines
in controlling cell movements within lymphoid tissues.
Mice lacking CXCR5 (formerly Burkitt's lymphoma receptor 1 [BLR1]), a chemokine receptor expressed by mature B cells (13, 14), lack polarized follicles in the spleen
and B cells appear as a ring at the boundary of the T zone (5). Recently, a CXCR5 ligand, termed B lymphocyte
chemoattractant (BLC) or B cell attracting chemokine
(BCA)-1 (15, 16), has been found constitutively expressed
by stromal cells in lymphoid follicles and has been proposed
to act as a B cell homing chemokine (15). Three other
chemokines have been identified that are constitutively expressed in lymphoid tissues and that are efficacious attractants of resting lymphocytes: stromal cell-derived factor 1 (SDF1 [17-19]), secondary lymphoid tissue chemokine
(SLC)/6Ckine (20), and EBV-induced molecule 1 ligand chemokine (ELC)/macrophage inflammatory protein (MIP)-3 (25). SDF1 is an efficacious attractant of
mature lymphocytes (19), although its pattern of expression
in lymphoid tissues is not well characterized. SLC and ELC
are related chemokines that strongly attract naive T cells
and more weakly attract B cells. SLC is expressed by high
endothelial venules (HEVs) in lymph nodes and Peyer's
patches and by stromal cells in the T zone of spleen, lymph
nodes, and Peyer's patches (21, 22, 24), whereas ELC is expressed by T zone DCs (27). The strong chemotactic activity of SLC and ELC combined with their compartmentalized expression pattern has led to the suggestion that these
molecules function in lymphocyte homing to the T zone
of peripheral lymphoid tissues (21, 27).
The strikingly similar disruption of splenic B cell distribution in TNF- and CXCR5-deficient mice suggested that
these molecules act in a common pathway to maintain follicular organization (29). The more severe splenic disruptions in LT/
-deficient mice suggest LT may function
upstream of molecules that help organize cells into both
follicles and T zones. Here we report that expression of the
CXCR5 ligand, BLC, is substantially reduced in TNF-,
TNFR1-, LT
-, and LT
-deficient mice. Expression of
SLC and ELC is also reduced, whereas SDF1 is unaffected.
Antagonism of LT
1
2 function in the adult by treatment
with soluble LT
R-Ig or anti-LT
antibody caused reductions in BLC and SLC expression. We also observed that in
addition to defects in follicular stromal cells, the LT- and
TNF-deficient mice had disruptions in T zone stromal
cells. To identify which cell types may act as sources of the
LT
/
and TNF required for upregulating BLC expression, mice lacking subpopulations of hematopoietic cells
were studied. Mice deficient in B cells, which also lack follicular stromal cells, had reduced BLC expression, whereas
T cell and marginal zone macrophage (MZM)-deficient
mice were unaffected. These findings suggest that TNF and
membrane LT
/
heterotrimer transmit signals required
for the development and function of stromal cells that produce chemokines essential for normal organization of lymphoid tissue compartments.
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Materials and Methods |
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Animals.
TNFNorthern Blot Analysis.
10-15 µg of total RNA from mouse spleens was subjected to gel electrophoresis, transferred to Hybond N+ membranes (Amersham Pharmacia Biotech), and probed using randomly primed 32P-labeled mouse cDNA probes of the following types: BLC, bases 10-532 (15); SLC, bases 1-848 (21); ELC, bases 1-755 (27); and SDF1In Situ Hybridization.
For in situ hybridizations, frozen sections (6 µm) were treated as described (15). In brief, sections were fixed in 4% paraformaldehyde, washed in PBS, prehybridized for 1-3 h, and hybridized overnight at 60°C with sense or antisense digoxigenin-labeled riboprobes in hybridization solution. After washing at high stringency, sections were incubated with sheep antidigoxigenin antibody (Boehringer Mannheim) followed by alkaline phosphatase-coupled donkey anti-sheep antibody (Jackson ImmunoResearch Laboratories) and developed with NBT (Bio-Rad) and BCIP (Sigma).Immunohistochemistry.
Cryostat sections (6-7 µm) were fixed and stained as described previously (27) using the following mAbs: rat anti-B220 (RA3-6B2); rat anti-CD4 and -CD8 (Caltag); rat anti-CD35 (8C12; PharMingen); rat anti-MOMA1 (provided by Georg Kraal, Free University, Amsterdam, The Netherlands); and biotinylated mouse anti-BP-3 (38). Rat IgG antibodies were detected with goat anti-rat-conjugated horseradish peroxidase or alkaline phosphatase (Southern Biotechnology Associates) and biotinylated antibodies with avidin-alkaline phosphatase (Sigma Chemical Co.). Enzyme reactions were developed with conventional substrates for peroxidases (diaminobenzidine/H2O2 [Sigma]) and alkaline phosphatase (FAST RED/Naphthol AS-MX [Sigma] or NBT/BCIP). In some cases, sections were counterstained with hematoxylin (Fisher Scientific Co.). Sections were mounted in crystal mount (Biomeda Corp.) and viewed with a Leica DMRL microscope. Images were acquired on an Optronics MDEI850 cooled CCD video camera (Optronics Engineering) and were processed with Photoshop software (Adobe Systems, Inc.). ![]() |
Results |
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To explore whether TNF/TNFR1
and CXCR5 function in a common pathway of follicular
organization, we measured CXCR5 expression in TNFR1-
and TNF-deficient mice by flow cytometry. Splenic B cells
from TNF- and TNFR1-deficient mice expressed levels of
CXCR5 that were slightly elevated compared with wild-type controls (39; Ansel, K.M., and J.G. Cyster, data not
shown). Increased CXCR5 expression seemed unlikely to
account for the disrupted organization of B cells in TNF-
or TNFR1-deficient animals, but could result from reduced
expression of ligands that normally engage and downregulate CXCR5. Therefore, we tested whether TNF/TNFR1
regulated CXCR5 ligand expression by measuring BLC
RNA levels in TNF- and TNFR1-deficient mouse spleens
(Fig. 1, A and B). BLC expression was reduced approximately threefold in both types of mutant mice compared
with wild-type littermates. In situ hybridization analysis of
TNFR1-deficient spleen confirmed the reduced expression
of BLC by follicular stromal cells (Fig. 1 C). Animals deficient in LT or LT
also lack follicles and follicular stromal cells, although the absence of MZMs and the severely
disrupted B/T boundary make the splenic phenotype of
these mice distinct. BLC expression was reduced even
more severely in spleens from LT
- and LT
-deficient animals than from TNF-deficient mice (Fig. 1, A and B), and
the residual expression was too low to be detected in in situ
hybridization analysis (Fig. 1 C). In mice deficient in both
LT
and TNF, BLC expression was reduced to an extent
similar to LT
single mutants (Fig. 1, A and B), consistent
with the possibility that these cytokines function in a common pathway leading to BLC expression.
|
Splenic T zone organization is also disrupted in the cytokine- and cytokine receptor-deficient animals, ranging
from subtle changes in TNF- and TNFR1-deficient mice
to almost complete loss of T zones in LT, LT
, and
LT
/TNF double mutant mice. To determine whether
LT
/
and TNF also functioned in a pathway leading to T zone chemokine expression, we measured the expression of SLC and ELC, related T cell attracting chemokines
that are made in the T zone. We also measured expression
of the more broadly distributed chemokine, SDF1, which
is an efficacious attractant of both B and T cells. SLC was
reduced in expression ~2-fold in TNFR1- and TNF-
deficient animals and >20-fold in LT
, LT
, and LT
/
TNF double mutant animals (Fig. 1, A and B). By in situ
hybridization analysis, the network of SLC expressing
stromal cells remained visible in the TNFR1 mutant mice
but could not be detected in LT
-deficient animals (Fig. 1
C). Expression of ELC was also reduced in all of the mutant strains, although less severely than SLC (Fig. 1, A and
B). By contrast, SDF1 expression was not significantly reduced in any of the mutant animals (Fig. 1, A and B), indicating that the reductions in BLC, SLC, and ELC are
physiologically relevant and not the result of an overall
decrease in chemokine gene expression. It should be emphasized that the TNFR1 mutant and the four cytokine mutants used in this study (6, 30, 32; Korner, H., D.S.
Riminton, F.A. Lemckert, and J.D. Sedgwick, unpublished) were all generated using C57BL/6 ES cells and
maintained on a C57BL/6 background, making it unlikely
that any of the differences we observed in chemokine
expression are due to linked genetic differences. Therefore, these experiments demonstrate that TNF/TNFR1
and LT
/
are required for normal expression of BLC,
SLC, and ELC in the spleen.
To determine whether the requirement for LT and LT
in the expression of BLC and
SLC was developmental or constitutive, we treated adult
mice for various time periods with soluble LT
R-Ig fusion
protein (8, 40), an antagonist of LT
1
2, and a related molecule, LIGHT (41). Control mice were treated for
equal periods of time with an LFA3-Ig fusion protein (8).
After 1 wk of LT
R-Ig treatment, splenic BLC expression
was reduced twofold compared with the controls (Fig. 2
A). A further reduction in BLC expression occurred after 2 wk of treatment and did not become more severe after 3 or
4 wk of treatment (Fig. 2 A). 2 wk of treatment also lead to
decreased BLC levels in mesenteric lymph nodes (Fig. 2 B).
Expression of SLC was reduced in spleen and mesenteric
lymph nodes of mice given LT
R-Ig, although the degree
of inhibition was variable and less severe than the reduction
in BLC (Fig. 2, A and B). To distinguish the possible contribution of LIGHT from that of LT
1
2, mice were
treated for 1 or 2 wk with an anti-LT
mAb that specifically blocks LT
/
heterotrimer function (37, 40). Analysis of splenic RNA showed that BLC and SLC expression were both reduced after 2 wk of anti-LT
mAb treatment
(Fig. 2 A). These results establish a key role for LT
1
2 in
maintaining normal chemokine expression. Although the
lesser effect of the antibody treatment compared with
LT
R-Ig treatment (Fig. 2 A) suggests that LIGHT might
also contribute, the results may equally be explained by the
mAb causing less complete inhibition of LT
1
2 function,
as has been observed in in vitro studies (40). To explore
further the relationship noted in the mutant mice between chemokine deficiency and loss of follicular organization,
spleen sections from LT
R-Ig-treated mice were stained
for B cell markers as well as FDCs and marginal zone markers. As observed previously (36), expression of mucosal addressin cell adhesion molecule (MAdCAM)-1 and FDC
markers were reduced after 1 wk of treatment and were
undetectable by 2 wk, whereas loss of the marginal metallophilic macrophage (MMM) marker MOMA1 was more
gradual (data not shown). Changes in B cell follicular organization were also observed after 1 wk of treatment (Fig. 2
C) and were maximal after 2 wk of treatment (Fig. 2 C),
paralleling the decrease in BLC expression. These results
establish a constitutive requirement for LT
1
2 in maintaining normal levels of BLC and SLC. The more modest
decrease in BLC and SLC expression in LT
R-Ig-treated
mice compared with LT
/
or LT
/
mice could reflect incomplete blocking of LT
1
2 function but is also
consistent with a role for LT
1
2 in development that does not continue in the adult mouse.
|
The above experiments demonstrated that TNF,
LT, and LT
are required for normal expression of the
chemokines BLC and SLC by stromal cells in the spleen.
Several studies have established that the organization of
FDCs is disrupted in TNF-, LT
-, and LT
-deficient
mice (1), indicating that the cell type normally producing
BLC might be disrupted. However, disruption of FDC organization could not account for the decreased SLC expression, since FDCs do not extend into the T zone. To
test whether changes in addition to reduced SLC expression could be detected in T zone stromal cells, we examined expression of BP-3, a marker for an extensive network
of stromal cells in both the T zone and follicles (38, 42).
Strikingly, the network of BP-3+ cells was greatly reduced
in both the B and T zones of TNF- and TNFR1-deficient
mice (Fig. 3, and data not shown) and was undetectable in
LT
- and LT
-deficient mice except for a small number of
cells with altered morphology that were occasionally observed (Fig. 3). The disruption of BP-3-expressing stromal
cells in both TNF- and LT
/
-deficient spleens appeared
more severe than in lymphocyte-deficient (RAG-1
/
)
spleens (Fig. 3). BP-3 expression in T zone and follicles was also markedly disrupted after 1 wk of treatment with
LT
R-Ig or anti-LT
antibody and was almost undetectable after 2 wk of treatment (Fig. 3, and data not shown).
This period of treatment is also sufficient to disrupt staining
for MAdCAM-1 and FDC markers (36, 43). To determine
the relationship between BP-3-expressing cells in follicles
and the cell types previously defined as TNF- and LT
and
-dependent, sections from wild-type mice were double stained for MAdCAM-1 or CR1 (CD35) and BP-3 (Fig. 4
A). BP-3-expressing cells in the outer follicle appeared to
line the marginal sinus, and in some cases these cells costained for MAdCAM-1 (Fig. 4 A). Many of the BP-3+
cells located in the center of the follicle costained with
CD35 (Fig. 4 A), whereas BP-3+ cells in other parts of
the follicle, especially cells near the marginal sinus, were
CD35-low or -negative (Fig. 4 A). Therefore, the BP-3-
expressing stromal cell population includes T zone stromal cells (Fig. 3 and Fig. 4 A), FDCs, marginal sinus lining cells, and follicular stromal cells that are low or negative for FDC markers (Fig. 4 A). The severe disruption of BP-3 expression in the mutant mice, together with the reduced BLC
and SLC expression and the loss of FDCs, indicates that
TNF, LT
, and LT
have a broad role in inducing and
maintaining stromal cell integrity in T zones and B zones of
lymphoid tissues.
|
|
In addition
to defects in FDCs, MAdCAM-1+ cells, and BP-3+ cells,
LT- and LT
-deficient mice also lack MZMs and
MMMs (1, 11, 12). To test the possibility that the deficiency in these macrophage populations in LT
/
and
LT
/
mice contributed to the greatly reduced BLC expression and loss of follicular organization, we characterized
spleens from op/op mice, a strain that is deficient in MMMs
and MZMs due to a mutation in the colony stimulating
factor 1 gene (44, 45). Organization of B cell follicles appeared normal in op/op spleen (Fig. 4 B), and BLC expression was not reduced (Fig. 5). Expression of BP-3, MAdCAM-1, and CD35 was also not disrupted (Fig. 4 B, and
data not shown). These findings demonstrate that MZMs
and MMMs do not make a significant contribution to the
constitutive production of BLC, and also establish that
these cells are not required as a source of TNF or LT
1
2
to maintain BLC expression or follicular organization.
|
Re cent studies have demonstrated that B lymphocytes are an
essential source of membrane LT1
2 for establishing
FDC networks and follicular organization (46, 47). However, mice congenitally deficient in LT
have a more severe disruption of lymphoid compartmentalization than
mice lacking only in lymphocyte LT
expression, indicating that there is also a nonlymphocyte source of LT
(47,
48). To determine whether either or both sources of LT
were required for induction of BLC, chemokine expression levels in RAG-1
/
, B cell receptor (BCR)
/
, and
TCR
/
mice were compared with levels in LT
/
animals. BLC expression was reduced approximately fivefold
in spleens from lymphocyte-deficient (RAG-1
/
) and B
cell-deficient (µMT) mice, but were not reduced in T
cell-deficient (TCR-
/
/
) mice (Fig. 5), demonstrating that B cells are important for induction of BLC expression, presumably providing LT
1
2 and possibly also
TNF. However, BLC levels in RAG-1
/
and BCR
/
mice were not reduced to the extent of LT
/
or LT
/
mice (Fig. 5, and see Fig. 1), indicating that some BLC expression in the spleen is induced by LT
/
-expressing cells
other than B and T lymphocytes.
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Discussion |
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These studies provide new insight into the mechanism
by which TNF and LT/
promote normal compartmentalization of lymphocytes in the white pulp cords of the
spleen. The findings extend the previously defined requirement for TNF and LT
/
in the development and function of follicular stromal cells to also include stromal cells in
the splenic T zone. The results demonstrate that a key function of the LT
/
- and TNF-dependent stromal cells
is constitutive production of chemokines that strongly attract resting lymphocytes, and they suggest that these
chemokines function with other properties of the stroma to
compartmentalize cells into follicles and T zones.
The chemokine receptor CXCR5 is expressed by all
mature B cells and is the only known receptor for BLC, an
efficacious attractant of resting B cells (13, 15, 16). Since
loss of CXCR5 is sufficient to disrupt organization of
splenic follicles (5), it is reasonable to suggest that the
greatly reduced expression of BLC in TNF-, TNFR1-,
LT-, and LT
-deficient mice directly contributes to the
disrupted organization of splenic follicles in these animals.
Polarized follicles also fail to form in lymph nodes of TNF-deficient mice and in the nodes that develop under some
conditions in LT-deficient mice (6, 11, 12, 37, 49). The
finding that BLC expression is reduced in mesenteric lymph nodes of LT
R-Ig-treated mice indicates that
LT
/
plays a role in directing BLC expression in lymph
nodes. However, whether BLC is likely to contribute to
the organization of B cells into lymph node follicles is presently unclear, since CXCR5 does not appear to be required (5).
SLC and ELC both stimulate cells through CCR7, a receptor expressed by T and B lymphocytes, and these
chemokines are the most efficacious attractants of T cells so
far described (21, 25, 27, 50). We propose that the severe
reduction in T zone SLC expression in LT/
-deficient
mice directly contributes to the loss of normal T cell compartmentalization in these animals. Maturing DCs upregulate CCR7 and have been suggested to migrate to lymphoid tissues in response to CCR7 ligands (51), making it
possible that the decrease in SLC also leads to reduced accumulation of mature DCs in the T zone. Consistent with
this possibility, lymph nodes developing in mice with reduced LT
levels have threefold fewer DCs than controls
(52), and we have observed a similar decrease in DC frequency in LT
-deficient mouse spleens (our unpublished observations). Reduced DC accumulation may be at least
partially responsible for the decreased expression of ELC, a
chemokine made by T zone DCs (27). The lowered ELC
levels are likely to exacerbate the effect of SLC deficiency
and contribute to the loss of T zone organization. TNF and
TNFR1 are also required for maximal SLC and ELC expression. However, mice deficient in TNF or TNFR1 do continue to express significant amounts of SLC and ELC,
and this is consistent with the relatively unaffected T zone
organization in these mutant animals (3, 4, 6, 53). The generally greater reduction of chemokine expression in TNF-deficient compared with TNFR1-deficient mice should
not be due to background gene effects, since all the animals
were generated on the C57BL/6 background; a more
likely possibility is that TNFR2 transmits some of the TNF
signals necessary for chemokine expression. In support of this possibility is the finding that Langerhans cell migration to lymph nodes is depressed in TNFR2-deficient mice
(54). Interestingly, during the analysis of several TNFR1-deficient mice that had been housed in a conventional
animal facility, we found that whereas BLC and SLC levels
remained depressed, ELC expression was equal to the wild-type controls (our unpublished observations). These observations are similar to other findings indicating that some of
the nonredundant functions of TNF in the resting state can be overcome during an immune response (43).
The deficiency of FDCs in LT- and TNF-deficient mice
has been well characterized (1). Ultrastructural studies have
demonstrated that FDCs are part of a broader network of
follicular stromal cells (55), and using the molecule BP-3 as
a marker it has been possible to show that this more extensive stromal cell network is also LT and TNF dependent
(see Fig. 3). Elegant bone marrow chimera and adoptive
transfer experiments have established that FDC development requires TNFR1 and LTR expression by the follicular stroma and cytokine (LT and TNF) expression by hematopoietic cells, in particular B cells (39, 46). The
necessity for B cells in the maximal expression of BLC (see
Fig. 5) is consistent with these results and suggests that a
feedback loop exists which helps to keep the number of
BLC producing follicular stromal cells in proportion to the
number of B cells. The more depressed BLC expression in
LT
- and LT
-deficient mice than in B cell-deficient animals is also in agreement with studies showing that B cells
cannot be the sole source of LT
/
for follicle formation
(47, 48). Perhaps the LT
/
-expressing CD4+CD3
cells
that enter lymphoid tissues early in development (56) induce stromal cells to express BLC. Requirements for development of T zone stromal cells have been less well characterized than for FDCs, but our results indicate they are
similar in being TNF and LT
/
dependent. Experiments
are ongoing to address whether T cells, B cells, or other
cell types must express TNF or LT
/
for induction of
normal SLC expression. At this stage, it has not been possible to determine whether LT and TNF work directly to
induce chemokine expression or whether they function
further upstream, inducing and maintaining the development and viability of chemokine-expressing stromal cells.
Although treatment of adult mice with soluble LT
R-Ig
leads within 1 wk to decreased expression of BLC and
SLC, the treatment also leads to rapid disruption of stromal
cells as defined by a variety of markers (36; and see Fig. 3).
Future studies must define in more detail the subpopulations of LT- and TNF-dependent stromal cells that express
BLC and SLC and characterize the signaling pathways that
control chemokine expression.
The studies in this report have established a major role
for LT/
, and a lesser but significant role for TNF, in
promoting the function of chemokine-expressing stromal
cells in lymphoid areas of the spleen. Analysis of lymph
nodes from LT
R-Ig-treated mice has provided initial evidence that LT
/
is also required for normal chemokine
expression in lymph nodes. Given the requirement for
LT
/
and TNF in normal organization of all peripheral
lymphoid tissues, as well as the ability of ectopically expressed LT
to promote accumulation of B and T lymphocytes in lymphoid aggregates (57), it appears likely that
LT
/
and TNF function broadly in regulating lymphoid
tissue chemokine expression. Accumulation at nonlymphoid sites of cells in follicle- and T zone-like structures also typifies several human diseases, including rheumatoid
arthritis and type I diabetes, and the possibility that locally
produced LT and TNF induce the development of BLC-
and SLC-expressing stromal cells deserves investigation.
![]() |
Footnotes |
---|
Address correspondence to Jason G. Cyster, Department of Microbiology and Immunology, University of California San Francisco, 513 Parnassus St., San Francisco, CA 94143-0414. Phone: 415-502-6427; Fax: 415-502-8424; E-mail: cyster{at}itsa.ucsf.edu
Received for publication 9 September 1998 and in revised form 4 November 1998.
J.D. Sedgwick, H. Korner, and D.S. Riminton were supported by grants from the National Health and Medical Research Council of Australia and the National Multiple Sclerosis Society of Australia. J.G. Cyster is a Pew Scholar in the biomedical sciences. This work was supported in part by National Institutes of Health grant AI40098 to J.G. Cyster and by Howard Hughes Medical Institute grant 76296-549901 to the University of California San Francisco School of Medicine.The authors thank Jacques Peschon (Immunex Corp.) for generously providing TNFR1-deficient spleens
and mice, and acknowledge the assistance of Elena Foulcher (Centenary Institute) for collection and preparation of tissues from the cytokine gene-targeted mice. We thank the Biogen LT project team for the
LT
R-Ig.
Abbreviations used in this paper BCR, B cell receptor; BLC, B lymphocyte chemoattractant; DC, dendritic cell; EBI-1, Epstein Barr virus-induced molecule 1; ELC, EBI-1 ligand chemokine; EF, elongation factor; ES, embryonic stem; FDC, follicular DC; LT, lymphotoxin; MAdCAM, mucosal addressin cell adhesion molecule; MMM, marginal metallophilic macrophage; MZM, marginal zone macrophage; RAG, recombination activating gene; SDF, stromal cell-derived factor; SLC, secondary lymphoid tissue chemokine.
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