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and Lymphotoxin- Receptor Expression in Mouse Embryos1
* Department of Exploratory Biology, Biogen, Cambridge, MA 02142; and
Department of Dermatology, University of Geneva, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ligand (LT) binds to the
LT receptor (LTR) and provides an essential trigger for lymph
node (LN) development. LTR signaling is also critical for the
emergence of pathological ectopic lymph node-like structures and the
maintenance of an organized splenic white pulp. To better understand
the role of LT in development, the expression patterns of LT and
LTR mRNA were examined by in situ hybridization in the developing
mouse embryo. Images of LT ligand expression in developing
peripheral LN in the E18.5 embryo revealed a relatively early phase
structure and allowed for comparative staging with LN development in
rat and humans. The LTR is expressed from E16.5 onward in
respiratory, salivary, bronchial, and gastric epithelium, which may be
consistent with early communication events between lymphoid elements
and epithelial specialization over emerging mucosal LN. Direct
comparison of mouse fetal and adult tissues by FACS analysis confirmed
the elevated expression of LTBR in some embryonic epithelial layers.
Therefore, surface LTBR expression may be elevated during fetal
development in some epithelial layers. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LT and LT form a heteromeric complex that binds uniquely to the
LTR and deletion of any of these genes leads to loss of lymph node
(LN) development (3, 5, 6). LTR activation is coupled
to NF
B-inducing kinase and IB kinase (IKK) and hence
to NFB activation and indeed NFB-inducing kinase gene
inactivation resembles LTR disruption in terms of LN status
(7, 8). A number of other gene deletions have led to loss
of LN or PP development including RANK and its ligand TNF-related
activation-induced cytokine, CXCR5, CCL13, VLA4, IL-7 and its
receptor, relB, Ikaros, Id2, IKK, and ROR
(9, 10).
The stages of murine LN development have been poorly analyzed due to the difficulty of identifying LN in the mouse embryo. Although there are photographs of developing rat and human LN, an actual microscopic image of a fetal mouse peripheral or mesenteric LN has not been reported (11, 12). In contrast, considerable progress has been made in visualizing PP development by use of in situ whole-mount histological methods (4, 13). Several stages in PP development can be recognized in the mouse from the initial anlage formation and expression of VCAM, to the seeding with the IL-7R+, CD4+, CD3- organizer cell and, finally, filling with mature lymphocytes. In LT-deficient mice, the earliest stages of PP formation cannot be detected (14). Similarly, Kim et al. (9) have postulated five stages of lymphatic and LN development and, in this scheme, LN development commences at stage III shortly after formation of the lymphatics at E12–13. In stage III, mesenchymal connective tissue invaginates into the lumen of the lymphatics, giving rise to a bulbous structure that will form the cortical/paracortical regions of the LN. The invagination is joined to the surrounding tissue by a hilus or stalk that will form the medullary end of the LN. The luminal space is bounded by endothelial cells from the lymphatics and probably capillaries and thin connective bridges link the invagination to the surrounding endothelium (12). Fetal blood flow is present at this point, providing a conduit for lymphocyte trafficking. At stage IV, cellular content increases and the lumen and bridges collapse to form the subcapsular region. In the final stage V, capsule formation occurs and mature lymphocytes begin to populate the structure.
Characterization of the expression patterns of the ligands and
receptors in the LT pathway and the involved cell types is crucial for
a complete understanding of this process. Even though the analysis of
the RNA or protein expression patterns of a particular gene is
technically straightforward, the expression pattern for a new cytokine
system is often one of the last facets to be fully characterized. To
study the question of when and where the LT and LTR genes are
expressed during lymph node organogenesis, we used in situ
hybridization (ISH) methods. This analysis of LT expression led to
visualization of developing LN and thus allowed for a comparative
staging of murine LN formation with that in rat and humans. Surface
LTR expression was elevated in some embryonic mucosal epithelial
layers and expression then decreased in the adult, suggesting that
surface LTR display may be a fetal event in some epithelial
layers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine LT sense and antisense probes were prepared from
pGEM-3Z vector containing a 420-bp fragment of the mouse LT cDNA
covering part of exons 3 and 4 (15). A 700-bp
NotI fragment from the 5' end of the mouse LTR cDNA
(16) was ligated into pcDNA3 (Invitrogen, San Diego, CA),
yielding AN003. LTR sense and antisense probes were prepared from
this vector. Radiolabeled cRNAs were synthesized by in vitro
transcription in the presence of 12.5 µM
35S-labeled UTP (400 Ci/mmol; Amersham).
35S-Labeled probes were subsequently reduced to
an average size of 50–100 nt by mild alkaline hydrolysis as previously
described (17). For the synthesis of
35S-labeled probes, all of the above reactions
were performed in 10 mM DTT. Total RNA was extracted from 20 selected
tissues of 3-mo-old NMRI mice as described below. Briefly, 0.5 g
of each selected tissue was homogenized with a Polytron in 0.1 M
Tris-HCl (pH 7.4), 0.1 M 2-ME, and 4 M guanidium thiocyanate. After
addition of solid CsCl (0.4 g/ml), the homogenate was layered onto 1 ml
of a 5.7 M CsCl/0.2 M EDTA (pH 7.0) cushion and centrifuged at 35,000
rpm for 20 h at 20°C. The pelleted RNAs were dissolved in 300
µl of 10 mM Tris-HCl (pH 8.1), 5 mM EDTA, and 0.1% SDS, extracted
twice with phenol/chloroform, ethanol precipitated, and resuspended in
water. Five micrograms of total RNA from each tissue was subsequently
denatured with glyoxal, electrophoresed in 1.2% agarose gels and
transferred overnight onto Hybond nylon membranes (Hybond-N; Amersham).
Prehybridizations, hybridizations, and posthybridization washes were
conducted as described previously (18).
In situ hybridizations
Embryos were timed and segments of uteri or, at later stages, whole NMRI embryos were dissected, embedded in Tissue-Tek (Miles, Elkhart, IN), and frozen down in precooled methylbutane as described elsewhere (18). Cryostat tissue sections (5 µm) were mounted on poly-L-lysine (Sigma, St. Louis, MO)-coated microscope slides, fixed in 4% glutaraldehyde in PBS for 1–5 min, rinsed in PBS, and stored in 70% ethanol at 4°C until analyzed. Fixed sections were rinsed in 2x SSC (1x SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0), acetylated with 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine (pH 8.0) at room temperature for 10 min, incubated in 0.1 M Tris-HCl (pH 7.0)/0.1 M glycine at room temperature for 30 min, and prehybridized in 2x SSC/50% formamide at 50°C for 15 min. 35S-Labeled cRNAs (1–3 x 106 cpm) were applied to each section in 20–70 µl of hybridization mixture (50% formamide, 2x SSC, 10 mM DTT, 1 mg/ml BSA (RNase free), 0.15 mg/ml rRNA, and 5% dextran sulfate) for 3 h at 50°C. Slides were subsequently washed for 20 min in 2x SSC, 50% formamide at 50°C, and 10 mM DTT, and in 0.2x SSC and 50% formamide at 50°C for 30 min each. Unhybridized transcripts were digested with 10 µg/ml RNase A at 37°C for 30 min. The slides were washed again in 2x SSC/50% formamide at 50°C for 15 min, four changes of 2x SSC at room temperature, dehydrated in graded ethanol, and air dried. Sections were either directly exposed for 2–3 wk at room temperature to x-ray films (SB5; Eastman Kodak, Rochester, NY) between intensifying screens or immersed in NTB-2 emulsion (Eastman Kodak), diluted 1:1 in deionized water, exposed for 3–4 wk at 4°C, developed in Kodak D-19 developer, fixed in 30% sodium thiosulfate, and counterstained in 1% methylene blue. Controls of specificity included the systematic use of sense cRNA probes in each experiment.
FACS analysis of LTR expression
The entire intestinal mass was dissected from timed C57BL/6
embryos and any connected mesenteric tissue was removed. The intestines
were gently passed through an 18-gauge syringe needle to crudely
disrupt the gut integrity and then washed in HBSS with 2% FBS and 10
mM HEPES buffer. Gut fragments were suspended in the above HBSS buffer
with 1 mM EDTA (HBSS/EDTA) and rocked for 30 min at 37°C. The
fragments were removed and resuspended in the same buffer for a second
extraction. The cell suspensions were passed through a 70-µm nylon
filter and the resultant mixture of epithelial cells and early
lymphocytes was used for FACS analysis. Adult large or small intestines
were flushed, sliced longitudinally, and the washed fragments subjected
to HBSS/EDTA extraction as defined above. Adult spleen cells were
obtained by subjecting glass slide-mashed spleens to collagenase
digestion for 30 min at room temperature followed by RBC lysis with
Gey’s solution, further EDTA release of cells from residual stroma,
and filtration through a nylon filter. Fetal hepatocytes were obtained
by passage of the liver through a 21-gauge syringe needle. Murine C26
colon carcinoma cells were obtained from D. LePage (Biogen, Cambridge,
MA) and the Lewis lung carcinoma was purchased from the American Type
Culture Collection (Manassas, VA). Embryonic fibroblasts were a gift
from M. Scott (Biogen), grown in DMEM with 10% FBS, and analyzed at
passage 2. Cells were removed with EDTA/PBS. Cells were suspended in
PBS containing 5% FBS, 5% each of normal mouse and rat serum, and 10
µg/ml Fc Block (BD PharMingen, San Diego, CA). Cells were stained
sequentially with 10 µg/ml hamster anti-murine LTBR ACH6
(19) or a control mAb Ha4/8 followed by the
PE-anti-hamster mixture (BD PharMingen). Adult spleen cell
preparations were stained as above for LTBR followed by
FITC-anti-CD11c (HL3), CyChrome-anti-TCR-, and
allophycocyanin-anti-CD11b (Mac-1; all from BD PharMingen). All
fetal or adult epithelial cells were further stained at the end for 5
min with 10 µg/ml 7-amino-actinomycin D and analyzed immediately
without paraformaldehyde fixation. Analysis gates excluded any
7-amino-actinomycin D-positive dead cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and LTR mRNA in mouse
embryos
The macroscopic distribution of LT and LTR mRNA was
determined by ISH of the respective 35S-labeled
cRNA probes to sagittal sections of whole embryos ranging from E5.5 to
E18.5 followed by direct exposure to x-ray films. Using this technique,
LT and LTR RNA were detected in certain macroscopically
identifiable organs from E16.5 onward (Fig. 1
). No signal was detected with the sense
LT or LTR cRNA probes (Fig. 1 and data not shown). As shown in
Fig. 1, and in accordance with previous reported results, embryonic
LT gene expression is fairly restricted, being macroscopically
confined to the developing skin, thymus, gut, and brain
(20). Simultaneous analysis of LTR mRNA revealed a
wider distribution in embryos of the same age (Fig. 1). Significant
levels of LTR mRNA were detected macroscopically in the developing
sinus, submaxillary gland, thymus, muscle, lung, stomach, and gut. The
regional distribution of LT and LTR mRNA at E16.5 was very
similar to that that of the E18.5 embryo (data not shown). A previous
Northern blot analysis of LTR RNA from whole embryos showed
expression from as early as E7 (21). The expression
patterns of LT and LTR are summarized in Table I.
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expression identifies sites of developing LN and PP
The cellular distribution of LT mRNA in embryos ranging from
5.5 to 18.5 days of development was determined by ISH of
35S-labeled cRNA probes to tissue sections
followed by detection with an autoradiographic emulsion. Significant
levels of mRNA were detectable at E18.5 in LN-like structures that
appear above the submaxillary gland, above the thymus, in the abdomen
at the level of the kidneys just anterior to the developing hip joint,
and in the femoral region (Fig. 2).
Tentatively, this labeling is assigned to the developing cervical,
para-aortic (or mediastinal), lumbar/caudal (also sometimes called
para-aortic), and popliteal nodes. Structures shown in Fig. 2 closely
resemble images of rat popliteal LN at E20 (12). One can
see in these images of developing peripheral LN that the primary
LT-positive structure is an invagination into a lumenal space. This
structure closely approximates the stage III described by Kim et al.
(9). Thin connecting bridges can be seen between the
invagination and the enveloping endothelium and these resemble the
bridges described in rat and human embryonic LN (11, 12).
These fetal LN at E18.5 appear to be at a state before stage IV where
the lumen collapses to form the subcapsular sinus. Although these
images are not definitive, in Fig. 2A, one connecting
bridge which does not appear to be the connecting hilus contains
LT+ cells and in Fig. 2C, there is
the suggestion of a LT-positive structure. Interestingly, there was
the appearance of LT+ cells in the lining of
either a capillary or the lymphatic lumen (Fig. 2, A and
C). A whole-mount image of CD4+ cells in the
neonatal mesenteric LN showed scattered colonies of
CD4+ cells with little suggestion of bridges
and/or luminal spaces (9), and images of neonatal
peripheral LN have a fairly mature appearance (22, 23).
Therefore, the E18.5 images shown here appear to be earlier in the
developmental path and the final development from roughly stage III to
stage V must occur relatively quickly within the last 24–36 h of fetal
development.
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mRNA was also observed at the same stage of development in
lymphoid-like aggregates in the intestinal submucosa (Fig. 2
D). This structure strikingly resembles a typical PP
surrounded by well-separated villi. Although we were not able to
precisely differentiate between PP in the small intestine and
potentially submucosal B cell-rich follicles in the large bowel or
perhaps even emerging cryptopatches, the shape of the villi is
consistent with the small bowel. Labeled LN were not observed in the
E16.5 embryo, which may simply reflect a low cellularity and hence an
inability to find them. In general, LT gene expression during murine
embryogenesis appears to be associated with the development of certain
lymphoid organs, as shown here for the fetal thymus, intestinal
lymphoid aggregates, and abdominal LN. Within the 12 sagittal sections
analyzed in this study, there were none sufficiently lateral to include
the fetal spleen.
LT mRNA is also localized in the developing thymus, fetal liver,
and skin
Foci of cells expressing LT were detected in the fetal liver
from E12.5 (Fig. 3) and similar numbers
of foci were found at E14.5 and E16.5, although the colonies were more
diffuse at the later stages. Curiously, there was not a large number of
foci, suggesting that these foci may represent emerging colonies of a
particular cell lineage rather than the massive hemopoiesis that occurs
at this stage. These cells are likely to be hemopoietic since lower
levels of transcript were detectable in the liver at later stages of
development, which would be consistent with an early wave of
hemopoietic cells (Table I). Hybridization of adjacent sections with
the sense cRNA probe only revealed low background levels of signal,
indicating that the observed signal was specific (Fig. 3).
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gene expression were observed
in the thymus, skin, brain, and LN-like structures described above. In
the thymus, thymocytes within both the cortical and medullary regions
were strongly labeled from E16.5 onward (Fig. 3). In contrast, in the
adult thymus, LT expression was only observed in the medulla region
(20). High levels of LT mRNA were also detectable at
E16.5 and E18.5 in the epidermis (Fig. 3). Background levels of
staining are always somewhat higher along the outer edge of the embryo
yet this signal was specific, as no signal was observed in the
epidermis with the sense probe (data not shown). As most cells in the
epithelial layer appear to be LT positive, it is likely that these
cells are developing keratinocytes. Similar skin labeling was also
reported previously (20). The expression of LT mRNA in
the developing brain was noted earlier and its significance remains
unclear (20).
Abundant LTR mRNA is present in the developing epithelial layers
Microscopic analysis of sections hybridized to the
35S-labeled LTR cRNA probe showed detectable
levels of LTR gene expression from E12.5–14.5 and onward with
detectable albeit weak levels of message being seen at the cellular
level in the epithelium of the gut and lung in E14.5 embryos (Table I).
LN-like structures could not be observed using this probe. In E16.5 and
E18.5 embryos, the highest abundance of LTR mRNA was detected in
epithelial cells of the developing gut, and transcripts were present in
epithelial cells of the villi (Fig. 4).
No significant background expression was observed in adjacent sections
hybridized to the sense LTR cRNA probe (Fig. 4). Likewise,
significant levels of LTR mRNA were detected from E16.5 onward in
the epithelium lining of the stomach (Fig. 4), in respiratory
epithelial cells of the lung and sinuses (Fig. 4), and in acinar
glandular cells of the developing submaxillary gland (data not shown).
In the developing thymus, LTR transcripts were detected in both
cortical and medullary thymic regions, but at comparatively lower
levels than observed for LT (Table I). Taken together, during late
embryogenesis, LTR gene expression appears to predominate in several
epithelial layers (Table I).
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).
Adult large and small intestinal epithelial cells expressed very low
levels of LTBR. At birth, LTBR expression in the intestinal epithelium
had already begun to drop down to the low levels seen in the adult.
Several other cells were assessed for comparison. Two cultured
epithelial tumor lines, the C26 colon and Lewis lung carcinomas, showed
the highest level of surface expression (Fig. 5) and the B16-F10
melanoma line was similar (data not shown). As noted previously,
splenic B cells (Fig. 5) were receptor negative as were T cells (data
not shown). CD11b+ splenic monocytes were
receptor positive (Fig. 5) as were
CD11b+CD11c+CD11b-CD11c+
and CD11c+CD8a+ splenic
dendritic cells (data not shown). The ISH analysis did not reveal
general expression in the embryo that one would perhaps expect for
global fibroblast expression, yet early passage murine embryonic
fibroblasts were receptor positive. Since we find that most cultured
primary nonlymphoid cells from either murine or human sources are LTBR
positive, receptor expression may be simply up-regulated upon transfer
into tissue culture. This analysis shows that the high level of RNA
expression revealed by ISH was paralleled by higher surface protein
expression as assessed by FACS analysis of uncultured primary cells.
Thus, surface LTBR display appears to be down-regulated upon maturation
of the intestinal epithelial layer and this event may occur in several
mucosal epithelial layers.
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R gene expression contrasts with fairly restricted LT
gene expression in adult mouse tissues
LT and LTR gene expression was assessed in 20 adult mouse
tissues using Northern blot analysis. As shown in Fig. 6, expression of LT is fairly
restricted in adult tissues. Strong levels of expression are observed
in the thymus and spleen and lower levels are detectable in the large
intestine, small intestine, and lung. Several bands are detectable in
the bone, but only a fraction appears to migrate at the expected size.
This may be due to some RNA degradation in this sample. This pattern of
LT expression in adult tissues is in accordance with that observed
by others who have detected high levels of LT mRNA by RT-PCR and
Northern blot analyses in the thymus and spleen (15, 20).
Therefore, it appears that both during development and in the adult
mouse, LT gene expression occurs predominately in the lymphoid
tissues.
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R gene expression revealed a distinct
pattern of expression compared with that of LT, as most of the
analyzed tissues contain detectable amounts of LTR transcripts (Fig. 6). Expression was observed in the lung, kidney, stomach, small
intestine, large intestine, liver, and adrenal gland. Receptor
expression was noted to occur in liver, lung, and kidney in previous
studies (16, 21). Moderate to low levels of LTR mRNA
were detected in all other tissues analyzed except for the pancreas.
Therefore, the distribution of the receptor is relatively ubiquitous,
although beyond the myeloid and follicular dendritic lineages, the
types of cells that express the receptor remain ill-defined. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression was observed using ISH at sites of both developing
peripheral LN and PP. Although the appearance of LT-positive cells
in these embryonic LN was not surprising, the ISH signal allowed one to
find the early LN within the embryos and visualize their structure.
These images of developing E18.5 peripheral murine LN resemble closely
those from the rat E20 and human embryos (11, 12). Given
that E18.5 in murine development is close to birth, these early stage
peripheral murine LN appear to be emerging late relative to the rat. It
is possible that LT expression within the LN anlagen occurs earlier
than E18.5 and we were simply unable to find the LN. Alternatively,
given the rather rudimentary structure observed at E18.5, rapid LN
development and filling may occur only after E16.5 and for this reason
the rudimentary LN are effectively invisible.
In contrast to the relatively late appearance of LT-positive
structures, it has been shown using maternal transfer to deliver in
utero an LT inhibitor that LT:LTR communication was critical
between E12 and E15 for the development of the LN anlagen after stage
II (24). Studies on PP development have led to a model
whereby a hemopoietic "inducer" cell displays surface LT in
response to IL-7 signaling. The LT-positive cell triggers
LTR-positive mesenchymal cells to become a
VCAM+/ICAM+ "organizer"
(4, 13, 25). The genetic lack of LT blocks PP development
at the earliest detectable stage, i.e., formation of the
VCAM/ICAM+ anlage at E16; however, it has not
been formally proven that a hemopoietic cell delivers this LT signal at
the early stages. Clearly, the Ikaros mouse reveals the need for
hemopoietic cells in general and Ikaros null mice lack the critical
IL-7R+CD4+/-CD3-LT+
cell that populates the rudimentary gut and mesenteric LN (22, 25, 26). It is certainly reasonable that the LT signal is
delivered by a hemopoietic cell since expression in the adult appears
to be limited to lymphoid cells and specifically to activated T, B, and
NK cells and a subset of resting B cells (10, 19, 27, 28, 29).
Nonetheless, the possibility remains that stromal elements may express
LT and LN development may be more comparable to other organs, e.g., the
liver or the pancreas, where a dialogue occurs between endothelial and
endodermal tissues early in the process (30). There may be
expression of LT along the lining of the lumen surrounding the
rudimentary LN. This expression could be simply an artifact and
certainly this study is not definitive. Alternatively, the luminal
lining LTR-positive cells may be hemopoietic cells in transit into
the LN via the hilus or perhaps LT expression by stromal/endothelial
cells.
LT was found to be present on the surface of a unique
IL-7R+CD4+CD3-
oligolineage progenitor cell and this cell is a likely source of
LT during the population of rudimentary mesenteric LN and PP by
cells of the hemopoietic lineage (22, 26). Abundant
numbers of the
CD4+CD3-LT+
cells are easily observed by FACS in the rudimentary mesenteric LN and
by whole-mount histological methods in PP. Thus, LT expression by
this cell is likely to be a dominant, if not sole, contributor to the
LT signal observed at E18.5 in this study. Whether this cell
provides the critical ontological LT input both early and later during
filling of the rudimentary LN has not been unequivocally established.
Only low numbers of these cells are detectable at E14.5 in the blood
and spleen, yet none are found in the fetal liver (26).
Attempts to identify surface LT-positive cells in the E11.5–15.5
fetal liver by FACS were unsuccessful and therefore the nature of the
LT-positive cell foci reported here is not clear (J. L.
Browning, unpublished observations). It is possible that LT mRNA is
expressed in the absence of LT and in some settings LT expression
appears to be constitutive (31). LT RNA expression was
not examined in this study and even in the adult spleen where LT
expression is readily detected, LT expression is relatively low
(15, 32). RT-PCR analysis of LT expression using RNA
from various embryo parts showed LT expression from E11 to E13 in
placenta, head, liver, and yolk sac/blood; however, yolk sac expression
ceased by E12–13 (33).
Expression of LT was seen in the other immune organs. The
E16.5–18.5 thymus was LT positive, roughly correlating with the
first wave of thymocyte processing. Since the fetal liver does not
contain the CD4+CD3- cell,
this LT-positive cell must be derived directly within the thymus or
perhaps the gut cryptopatch. LTR is also expressed in the thymus at
this time even though a role for the LT system in thymic development or
function has not been revealed in genetically deleted mice
(34). The embryonic expression of LT in the skin is
intriguing given reports of nonlymphoid expression of LT RNA or
protein in inflamed epithelial layers such as in lichenoid skin, in
keratinocytes from the middle ear with the hyperproliferating disease
cholesteatoma, in skin following hair regression, and in melanocytes
(35, 36, 37, 38). The expression of LT or LT in
nonlymphoid/myeloid cells at sites of inflammation has not been
extensively studied. Since LTR signals through IKK and deletion
of IKK results in an epidermal defect, the skin expression of LT
is potentially interesting (39). However, such a skin
defect was not observed in LT- or LTR-deficient mice and
therefore the epidermal defect is not linked to the LT pathway. LT
expression was observed in the developing brain and LT proteins were
found in astrocytes and oligodendrocytes in the human brain
(28). Since some aspects of ontogeny can be recapitulated
in inflammatory processes, these observations may be linked and hence
LT ligand expression may not be solely limited to the T, B, and NK
lineages.
Staining for LTR using FACS methods showed a low level of expression
in various monocytic and follicular dendritic populations (Fig. 5)
(19, 40, 41). In this light, the inability to detect
LTR RNA in the developing LN is possibly not surprising. Lacking a
strong signal or the presence of only a few LTR-positive cells, the
LN cannot be visualized. Therefore, even a signal of intermediate
intensity may be difficult to score and the inability to detect LTBR
expression that must exist in the fetal LN represents a limitation of
the approach.
LTR is expressed from E16 onward in mucosal epithelium lining cells,
especially in the intestine, bronchi, and potentially in several ductal
epithelial systems, e.g., salivary gland. Expression on the thymic
epithelium may account for the receptor expression observed in the
thymus. Likewise, in the liver, LTR is not expressed on the
hepatocytes as ascertained by FACS analysis, suggesting that either
stromal expression accounts for the ISH results or the surface
appearance is modulated. Why this receptor is expressed in these
various developing epithelial cell types in the apparent absence of
ligand is not clear. The ISH method may limit the ability to detect low
levels of the LT ligand or other ligands such as LIGHT that bind
LTR may be critical. It is enticing to speculate that the receptor
serves in the differentiation process whereby these cells specialize to
form either lymphoid anlagen or the layer covering mucosal lymphoid
organs in both the gut and the bronchus. During PP development, a
VCAM/ICAM-positive mesenchymal cell in the emerging anlage is believed
to be a critical source of chemokine expression and this cell
presumably expresses LTR (13). In this study, we have
shown that the bulk of the fetal intestinal epithelial cells at E16 are
LTBR positive. At this stage the PP anlage is just beginning to develop
and the numbers of VCAM/ICAM-positive cells are very few. Therefore, it
is likely that if this VCAM/ICAM-negative LTR-positive cell is
recruited into anlage formation, a conversion must occur that would
resemble an inflammatory process. Mature intestinal epithelial cells
are known to be capable of initiating an inflammatory program and
therefore this model has some precedent (42). Inhibition
of LTR signaling in an adult mouse decreases the number of M cells
in the follicle-associated epithelium, suggesting that lymphoid cells
communicate with the epithelium via this system (43). It
is reasonable to assume that during development the LTR may play a
similar regulatory role. Direct comparison by FACS of fetal and adult
intestines confirmed that LTR expression is higher in the fetus and
begins to decrease at birth. This observation is interesting since many
human breast and colorectal carcinomas are LTR bright by
immunohistochemical staining while much of the surrounding
nontransformed stromal tissue is receptor dull (J. Browning, V. Bailly,
and S. Violette, unpublished data) and a similar observation was
reported for lung carcinomas (44). Therefore, in some
epithelial layers, surface LTR is displayed during development and
perhaps some carcinomas recapitulate ontology by reinstating the higher
fetal level of surface expression.
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Acknowledgments |
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R expression in breast carcinomas and
Tom Crowell, Sheila Violette, Bill Yang, Veronique Bailly, and Linda
Griffith for further immunohistological assessment. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeffrey L. Browning, Biogen, 12 Cambridge Center, Cambridge, MA 02142. E-mail address: jeff_browning{at}biogen.com
3 Abbreviations used in this paper: LT, lymphotoxin; LN lymph node, PP, Peyer’s patch; ISH, in situ hybridization; IKK, IB kinase.
Received for publication November 28, 2001. Accepted for publication March 8, 2002.
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