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Cutting Edge |
(LT) and Retinoic Acid Receptor-Related Orphan Receptor-
, but the Organization of NALT Is LT Dependent1
*
Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717;
Trudeau Institute, Saranac Lake, NY 12983;
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine and Howard Hughes Medical Institute, New York University School of Medicine, New York, NY 10016;
Department of Immunology, DNAX, Palo Alto, CA 94304; and
¶ Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Furthermore, using mice deficient
in the retinoic acid receptor-related orphan receptor-
, we found
that NALT was formed in the absence of
CD4+CD3- cells, which are thought to be the
embryonic source of LT. However, we also found that NALT of
LT-/- animals was disorganized and lymphopenic,
suggesting that the organization and recruitment of lymphocytes within
NALT remained dependent on LT. Finally, we demonstrated that both
the structure and function of NALT were restored in
LT-/- animals upon reconstitution with normal bone
marrow. These results demonstrate that the organogenesis of NALT occurs
through unique mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is necessary
for the development of Peyer’s patches (PPs) and all peripheral lymph
nodes (LNs) (1, 2). It is now known that several members
of the TNF/TNFR family are involved in lymphoid organogenesis as well
as the maintenance of organized lymphoid tissues (3, 4, 5).
Interestingly, some animals deficient in molecules seemingly unrelated
to the TNF/TNFR family of molecules have phenotypes similar to that of
LT-/- mice. These include retinoic acid
receptor-related orphan receptor-
(ROR)-/- (6),
IL-7R-/- (7), and various
chemokine/chemokine receptor-deficient mice (8). ROR
and IL-7 have been shown to be required for the generation and/or
maintenance of CD4+CD3-
cells in the embryo (6, 7). The
CD4+CD3- cells express
surface LT
(9), which mediates the
differentiation/survival of mesenchymal cells in developing lymphoid
organs and induces the expression of chemokines and adhesion molecules,
which are necessary for the recruitment of hematopoietic cells
(7). Analysis of PP and LN development in mice that are
deficient in one or more of these molecules has resulted in a general
model for lymphoid organ development which includes 1) induction of an
organizing center (anlage), 2) formation of a reticular network, and 3)
recruitment and organization of lymphocytes within the lymphoid organ.
Although LN and PP organogenesis and organization are absolutely
dependent on the expression of LT, the cellular and molecular
interactions involved in the organogenesis of PPs and various subsets
of LNs still differ in their precise details and timing (5, 10, 11). For example, mice deficient in the TNF family member
TNF-related activation-induced cytokine (TRANCE) lack LNs and
retain PPs (4, 10), while LT-/-
mice lack PPs and most LNs, but retain cervical and mesenteric LNs
(5). Thus the structural and functional differences
between these lymphoid organs may be related to the molecular pathways
used during their development. Although much is known about the molecular requirements for LN and PP organogenesis and organization, very little is known about the requirements for development of nasal-associated lymphoid tissue (NALT). NALT is structurally most similar to PPs. Like PPs, NALT is a nonencapsulated mucosal lymphoid organ that does not contain afferent lymphatics, but instead contains M cells that transport Ags across mucosal epithelial surfaces (12). NALT is also functionally similar to PP in that both organs are specifically designed to respond to mucosal Ags. Based on these structural and functional similarities it has been proposed that NALT and PPs are part of a common mucosal immune system and may be developmentally similar. However, despite these structural and functional similarities, we show in this study that NALT and PP are derived through developmentally distinct mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow (BM) chimeras were made by reconstituting irradiated
recipients (900 rad) with 1 x 107 BM cells
from C57BL/6, C57BL/6.Ly5.1, LT-/-, or
TNF-LT-/- animals. When possible,
reconstitution efficiency was determined by measuring the replacement
of Ly5.2-expressing host cells with Ly5.1 donor cells, and in all cases
reconstitution efficiency exceeded 95% replacement. In some
experiments, mice were infected intranasally with 100 egg-infectious
units of PR8 influenza. The MHC class I tetramer
H-2Db containing nuclear
protein366–374 peptide used to identify
influenza-specific T cells was generated by the Trudeau Institute
Molecular Biology Core Facility (Saranac Lake, NY). Viral titers were
measured by plaque assay on Madin-Darby canine kidney cells.
Serum Ab titers were determined by ELISA using plates coated with
purified virus. IgM was detected using HRP-conjugated goat
anti-mouse IgM, while IgG was detected using HRP-conjugated goat
anti-mouse IgG.
Preparation of NALT
The skin, the lower jaw, and the incisors of the upper jaw were removed from the heads of decapitated mice and the remaining tissue was fixed in neutral buffered formalin for 24 h. The skull was decalcified in 7% EDTA in PBS. The decalcified tissue was then embedded in paraffin, sectioned, and stained with H&E. Alternatively, the heads of euthanized animals were prepared as above except that they were fixed in 1% paraformaldehyde in PBS for 24 h then decalcified in 150 mM EDTA in PBS for 5–7 days at 4°C. Upon decalcification, these tissues were embedded in OCT, frozen, and sectioned for immunohistochemical analysis. In some cases NALT was removed from the nasal cavity by cutting along the inside edges of the upper molars with a scalpel and then peeling the tissue away from the roof of the mouth. Such tissues were either frozen in OCT medium and sectioned without decalcification or made into a single cell suspension for FACS analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because NALT is functionally and structurally similar to PP, we
hypothesized that the organogenesis of NALT and PP is dependent on
similar signaling pathways. To test this, we compared NALT from
wild-type (WT) mice with NALT from TNF-/-,
TNFR-1-/-, and LT-/-
mice, which have defects in PP structure (13) or
development (2). NALT in WT mice is a paired lymphoid
organ found in the ventral-lateral corners of the nasal passages that
lie just above the oral cavity (Fig. 1
A). WT NALT contains numerous
lymphocytes that are localized directly subjacent to the nasal
epithelium (Fig. 1B). NALT was present in
TNF-/- and TNFR-1-/-
mice (Fig. 1, C and D) but appeared slightly
smaller than the NALT of WT mice. This result is consistent with the
presence of at least rudimentary PPs in these mice (13).
However, LT-/- and
TNF-LT-/- mice also possessed recognizable
NALT (Fig. 1, E and F), even though these animals
do not develop PPs (2, 14). However, despite the fact that
NALT was present in LT-/- and
TNF-LT-/- mice, it was severely lymphopenic
(Fig. 1, E and F) and consisted primarily of
myeloid cells (see below). These findings suggested that TNF and LT
are unnecessary for the development of the NALT anlage, although these
molecules may be involved in the recruitment or retention of
lymphocytes in the NALT.
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-/-,
and ROR-/- mice because these animals are
deficient in CD4+CD3-
cells, which are thought to be required for LN and/or PP organogenesis
(6, 7, 10). As seen in Fig. 1
G, NALT was
present in TRANCE-/- animals, although it was
aberrantly large and elongated. This is likely due to the abnormal
osteoclast development in the TRANCE-/- mice
(4) and the altered structure of the skull. NALT was also
present in IL-7R-/- animals (data not
shown), although it was very small, most likely due to the paucity of
lymphocytes in these mice (15). Finally, the NALT of
ROR-/- animals was present and of normal
shape, size, and cellularity (Fig. 1H). Together these data
indicate that formation of the NALT anlage does not require the
presence of CD4+CD3- cells
and occurs independently of the TNF, LT, TRANCE, and IL-7 signaling
pathways. Interestingly, NALT was also observed in T cell-deficient
mice (TCR
-/-, Fig. 1I), B
cell-deficient mice (µMT, Fig. 1J), and
lymphocyte-deficient mice (Rag-/-, data not
shown), indicating that NALT anlage development can occur in the
absence of lymphocytes. Mechanism of lymphoid organization in NALT
Although we found that the organogenesis of NALT occurs
independently of the TNF/LT signaling pathways, it was possible that
the structure of NALT and the organization of lymphocytes within it
require expression of one or more TNF/TNFR family members. To determine
the lymphoid architecture in NALT from animals lacking various TNF/TNFR
family members, sections of NALT were stained with Abs to identify B
cells, T cells, dendritic cells (DCs), follicular DCs (FDCs), and the
vascular addressin, peripheral lymph node addressin (PNAd). These
results are summarized in Table I, and
examples of the immunohistochemical analysis are shown in Fig. 2. In WT NALT, B cells were located in a
large central follicle, while T cells were clustered to the sides of
the follicle and were scattered underneath the epithelium (Fig. 2A). CD11c+ DCs were clustered in the
T cell areas to the sides of the follicle and were highly concentrated
directly under the dome epithelium (Fig. 2B), while
CD21+CD35+ FDCs were found
in the center of the follicle (Fig. 2C). WT NALT also had
numerous high endothelial venules (HEV) that stained strongly for PNAd
(data not shown). In contrast to the highly organized lymphoid
structures seen in WT NALT, the NALT of
TNF-LT-/- mice was disorganized and
lymphopenic. The few B and T lymphocytes present in the NALT of
TNF-LT-/- animals were not segregated (Fig. 2D), and DCs filled most of the available space (Fig. 2E). Furthermore, FDCs were completely absent (Fig. 2F), and the HEVs had reduced expression of PNAd (data not
shown). These defects were also observed in the
LT-/- mice (Table I), indicating that LT
is required for the recruitment and organization of lymphocytes within
the NALT. Interestingly, the NALT of ROR-/-
mice appeared nearly normal (Fig. 2, G–I).
Although there were relatively few T cells in these mice, due to
defects in thymocyte survival (6), those present in NALT
were found around the edge of the B cell follicle (Fig. 2G)
and were colocalized with the DCs, which were found at edge of the
follicle and underneath the dome epithelium (Fig. 2H). FDCs
were prominently visible in the center of the follicle (Fig. 2I) and HEVs were present and expressed PNAd at normal
levels (data not shown). Thus, ROR expression is not required for
either NALT organogenesis or organization of the lymphocytes
within NALT.
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-/-
mice by reconstitution with WT cells. Therefore,
TNF-LT-/- mice were lethally irradiated and
reconstituted with BM from WT mice. As controls, WT mice were also
reconstituted with either TNF-LT-/- or WT
BM. After 9 wk, the NALT of reconstituted mice was examined by
histochemistry. As expected, WT mice reconstituted with WT BM possessed
a normal-sized NALT (Fig. 3A)
that was appropriately organized (data not shown). Interestingly, the
NALT of TNF-LT-/- animals was returned to
normal size after reconstitution with WT BM (Fig. 3B), even
though neither PPs nor LNs were ever reconstituted in these animals
(data not shown and Refs. 11 and 16).
Additionally, the NALT of TNF-LT-/- mice
reconstituted with WT BM was organized with B cells forming a
follicle under the epithelium and T cells clustering off to the edges
of the follicle (Fig. 3D). Furthermore, FDCs were found in
the B cell area and CD11c+ DCs were found
primarily in the T cell area (Fig. 3E). In contrast, the
NALT of WT mice that had been reconstituted with
TNF-LT-/- BM (Fig. 3C) was
smaller than WT NALT (Fig. 3A), B and T cells were
intermingled and disorganized (Fig. 3F), and FDCs could not
be found (data not shown). Together, these data show that LT is not
required for the formation of the NALT anlage but is needed to
facilitate the recruitment and organization of lymphocytes in the
NALT.
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To test whether NALT from LT-/- animals
could be made immunologically competent, we produced chimeric (CH) mice
by reconstituting irradiated LT-/- animals
with WT BM and then compared the immune response to influenza infection
of CH mice with that of WT and LT-/-
animals. Within 10 days of infection, the NALT of WT mice contained
activated influenza-specific CD8 T cells (Fig. 4A, WT) and germinal center B
cells (Fig. 4B, WT). Furthermore, the NALT of CH mice
also contained flu-specific CD8 T cells and germinal center B cells at
frequencies similar to those in WT NALT (Fig. 4, A and
B, CH). In contrast, the NALT of
LT-/- mice did not generate activated
flu-specific CD8 T cells or germinal center B cells by day 10 post-flu
infection (Fig. 4, A and B, KO). Consistent with
the appearance of germinal center B cells in WT and CH NALT but not
LT-/- NALT, both WT and CH mice had elevated
levels of flu-specific IgM (Fig. 4C) and IgG (Fig. 4D) by day 10 postinfection, while no increase in
flu-specific IgM or IgG was observed in the
LT-/- mice (Fig. 4, C and
D). Furthermore, although all groups of mice were infected
with similar levels of influenza at day 6 (Fig. 4E), the WT
and CH mice were able to clear detectable virus from their lungs by day
10, while LT-/- mice retained high levels of
virus (Fig. 4F). This is consistent with the appearance of
activated flu-specific T cells in WT and CH mice but not in
LT-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+
cells, 2) the formation of a reticular network, and 3) the recruitment
and organization of lymphocytes. Step one of PP organogenesis is
blocked in ROR-/- and
IL-7R-/- mice due to the lack of
CD4+CD3- cells
(6) and is also blocked in
LT-/- mice due to failure of
CD4+CD3- cells to express
surface LT. LT is also necessary for steps two and three of PP
development, as LT is required for proper FDC development
(17) and appropriate chemokine expression
(18) within the PP and LN. We now demonstrate that,
despite the functional and structural similarities between PP and NALT,
the general model of PP development cannot be applied to NALT
development. ROR-/- mice that completely
lack CD4+CD3- cells retain
NALT of normal size and structure. Although this might suggest that the
CD4+CD3--mediated LT
signal is provided by another LT-expressing cell, we also show that
LT expression is unnecessary for step one of NALT development. Thus,
CD4+CD3- cells are not
required for NALT formation because LT is not needed for the
development of the NALT anlage. Although the development of NALT anlage
proceeds in the absence of LT, the recruitment, organization, and
function of lymphocytes (step three) is impaired in
LT-/- NALT. These structural and functional
defects can be easily corrected by transferring
LT+/+ BM cells to
LT-/- mice. Importantly, neither PPs nor LNs
can be reconstituted in these chimeric animals (11, 16).
Thus, the basic scaffold of the NALT (but not that of the PP or LN) is
still formed in the absence of LT, demonstrating that this important
step in the organogenesis of NALT must occur by a novel mechanism.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Troy D. Randall, Trudeau Institute, 100 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: trandall{at}trudeauinstitute.org
3 Abbreviations used in this paper: LT, lymphotoxin; ROR, retinoic acid receptor-related orphan receptor-; NALT, nasal-associated lymphoid tissue; DC, dendritic cell; TRANCE, TNF-related activation-induced cytokine; FDC, follicular DC; WT, wild type; BM, bone marrow; HEV, high endothelial venule; PNAd, peripheral lymph node addressin; CH, chimeric.
Received for publication October 2, 2001. Accepted for publication November 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient mice: effects on secondary lymphoid organ development and humoral immune responsiveness. J. Immunol. 155:1685.[Abstract]
receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9:59.[Medline]
and revealed in lymphotoxin -deficient mice. Immunity 6:491.[Medline]
in thymocyte survival and lymphoid organ development. Science 288:2369.+ cells that can differentiate to APC, NK cells, and follicular cells, but not T or B cells. Immunity 7:493.[Medline]
/ complex is required for the development of peripheral lymphoid organs. J. Exp. Med. 184:1999. and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue. Eur. J. Immunol. 27:2600.[Medline]
(LT) supports development of splenic follicular structure that is required for IgG responses. J. Exp. Med. 185:2111./ and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J. Exp. Med. 189:403.This article has been cited by other articles:
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