| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Receptor Interactions Are Essential for the Presence of Intestinal B Lymphocytes1
,
* Division of Gastroenterology, Departments of
Internal Medicine, and
Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R
is essential for the presence LP B lymphocytes. The LT/LTR-dependent
events relevant for the presence of LP B lymphocytes occur after birth,
do not require the presence of Peyer’s patches, lymph nodes, or the
spleen; and therefore, are distinct and independent from the previously
identified roles of LT/LTR. The LT-dependent signal relevant for the
presence of LP B lymphocytes is optimally supplied by a LT-sufficient B
lymphocyte, and requires a LTR-sufficient radio-resistant, non-bone
marrow-derived cell. Based upon the severity of the deficit of LP B
lymphocytes we observed, these novel LT/LTR-dependent events are of
primary importance in directing the entry and residence of LP B
lymphocytes. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R and TNFRI in the formation of secondary lymphoid
structures and the normal compartmentalization of T and B lymphocytes
in the spleen. LT, a TNF family member, exists in two forms, a
membrane-bound heterotrimer, comprised of two -chains and one
-chain (LT12),
which binds exclusively the LTR, and a soluble homotrimer
(LT3), which has overlapping functions with
TNF, binding both the TNFRI and TNFRII (1, 2). LTR is
expressed on nonlymphoid cells including monocytes. LT
(LT3 and
LT12) is expressed
predominantly on T lymphocytes, B lymphocytes, and NK cells in adult
mice (3). Ligation of the LTR by
LT12 at defined
points during embryogenesis is critical for the formation of organized
lymphoid structures, as evidenced by the lack of lymph node (LN) and
Peyer’s patch (PP) formation in mice deficient in LT, LT, and
LTR (4, 5, 6); and mice in which LTR signaling has been
blocked at specific times during gestation (7). The
relevant
LT12-expressing cell
for the formation of PP and LN during embryogenesis is a non-T, non-B
lymphocyte, as evidenced by the presence of LN in severe combined
immunodeficient mice reconstituted as adults with
LT-/- bone marrow (8).
LT-/- mice have normal lymphatics, and
LT-/- B and T lymphocytes have normal
function and can repopulate LN and PP when placed in an LT-sufficient
environment (4, 9).
In addition to the role of LT in the formation of LN and PP, LT is also
crucial for the normal compartmentalization of T and B lymphocyte areas
of the spleen. Mice in which LTR and/or TNFRI signal
transduction have been blocked fail to develop follicular dendritic
cells (FDC) and germinal centers (GC) (5, 10, 11, 12), and
have diminished expression of secondary lymphoid tissue chemokine
(SLC), B lymphocyte chemoattractant (BLC), and EBV-induced molecule 1
ligand chemokine by splenic stromal cells (13), all of
which contribute to the normal segregation of B and T lymphocytes in
the spleen.
The intestine, as a secondary lymphoid tissue, represents a unique
challenge for the normal segregation of lymphocytes, as the major
effector site, the lamina propria (LP), is distant from intestinal and
nonintestinal inductive sites of the immune response. The relevant
factors for the entry and residence of lymphocytes into intestinal
effector sites are not well-known. Others have demonstrated these
events are at least partially dependent upon the expression of the
47 and
E7 integrins by
lymphocytes, allowing these lymphocytes to home to the LP and
intraepithelial lymphocyte (IEL) compartments (14, 15, 16),
and are dependent upon the expression of thymus-expressed chemokine by
the small intestine epithelia and its corresponding receptor CCR9 by T
lymphocytes (17, 18).
In addition to these observations, recent investigations have
demonstrated a role for NF
-inducing kinase (NIK) in the migration
of peritoneal B-1 B lymphocytes to the intestine (19).
Alymphoplasia (aly) mice have a naturally occurring point
mutation in the gene encoding NIK, and have an absence of LN and PP,
disrupted splenic architecture, immunodeficiency, and lack LP B
lymphocytes (19, 20, 21). Peritoneal B-1 B lymphocytes from
aly/aly mice are unable to populate the intestine of
wild-type mice with IgA producing plasma cells after adoptive transfer.
This is presumed to result from impaired signal transduction downstream
of the receptors for SLC, as evidenced by defective migration of
aly/aly B and T lymphocytes in response to SLC. However,
signal transduction downstream of the LTR has also been shown to
require NIK activity (22). Therefore, deficiencies in
LTR signal transduction in addition to the above defects in signal
transduction downstream of SLC could play a role in the absence of LP B
lymphocytes in the aly/aly mice.
LT is known to play a critical role in the formation of PP and LN, and
the normal segregation of lymphocytes in the spleen. In this study, we
examine the role of LTR-dependent events in directing the entry and
residence of lymphocytes into the intestine. We demonstrate a role for
LT12 interacting with
the LTR in directing B-2 B lymphocytes to the intestinal LP. The
relevant LT12 signal
can be delivered in adulthood, does not require the presence of LN, PP,
or the spleen, and can be blocked by the administration of LTR
antagonists after birth. Bone marrow-derived cell lineages may transmit
the LT-dependent signals relevant for the presence of LP B lymphocytes;
however, these signals are optimally transmitted by LT-sufficient B
lymphocytes, and require a LTR-sufficient, non-bone marrow-derived,
radio-resistant cell population. Our observations define a novel role
for LT/LTR interactions in the intestine in directing the
composition of the intestinal LP. This role is distinct and independent
of the known roles of LT in the formation of PP, LN, and in the
compartmentalization of the spleen. Based upon the severity of the
deficit of LP B lymphocytes, these LTR-dependent events are of
greater hierarchal importance than previously recognized factors
contributing to the entry and residence of LP B lymphocytes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All mice used for this study were housed in a specific
pathogen-free facility and fed routine chow diet. Animal procedures and
protocols were conducted in accordance with the institutional review
board at Washington University School of Medicine (St. Louis,
MO). C57BL/6, TNFRII-deficient, recombination-activating gene
(RAG)-1-deficient, and B cell-deficient JH-/- mice
(23) on the C57BL/6 background were purchased from The
Jackson Laboratory (Bar Harbor, ME). TNFRI-deficient mice (10, 24) on the C57BL/6 background were a gift from Dr. J. J.
Peschon (Immunex, Seattle, WA). LTR-deficient mice
(6) on the C57BL/6 background were a gift from Dr. K.
Pfeffer (Technical University of Munich, Munich, Germany).
LT-deficient mice (4), a gift from Dr. D.
Chaplin (University of Alabama, Birmingham, AL), were bred onto
the C57BL/6 background for >10 generations before use in experiments.
LT-/- and C57BL/6 mice for flow cytometric
analysis, Table I
, were 7 wk of age.
Fecal IgA levels were measured on feces from
LT-/- and C57BL/6 mice from 9 to 26 wk of
age (Table I). Analysis of resident peritoneal cells, Table II, was performed on 14-wk-old
LT-/-, LTR-/-,
and C57BL/6 mice. TNFRI-/-,
TNFRII-/-, and
LTR-/- mice for flow cytometric analysis,
Table III, were 10–14 wk of age. Timed
pregnant C57BL/6 female mice for use in experiments involving the
injection of LTR-Ig fusion protein, Table IV, were generated by matings with
C57BL/6 male mice. Six to 10-wk-old LT-/-
and LTR-/- mice were used as recipients for
bone marrow transfers (Table V).
|
|
|
|
|
Feces were collected from individual mice, diluted 1/10 wet weight to volume with PBS, vortexed into a uniform suspension, centrifuged at 12,000 rpm for 10 min in a table top microfuge, and supernatants removed. Fecal supernatants or IgA standards (Southern Biotechnology Associates, Birmingham, AL) diluted in PBS containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO) were incubated in 96-well Immunlon 4 plates (Fisher Scientific, Pittsburgh, PA) previously coated with goat anti-mouse Ab (Southern Biotechnology Associates) and blocked with PBS containing 5% BSA and 0.05% Tween 20 at room temperature for 2 h. Plates were washed three times with PBS containing 0.05% Tween 20, then goat anti-mouse IgA alkaline phosphatase-conjugated Ab (Southern Biotechnology Associates) diluted in PBS containing 5% BSA and 0.05% Tween 20 was added to the plate and incubated for 2 h at room temperature. Plates were washed three times with PBS containing 0.05% Tween 20 and p-nitrophenyl phosphate alkaline phosphatase substrate (Sigma-Aldrich) was added. Plates were read at 405 nM using Bio-Tek Instruments Microplate Reader (Bio-Tek Instruments, Winooski, VT). Each sample was measured in duplicate in at least three dilutions. Data are reported as the concentration of IgA in the fecal supernatant prepared as above.
For the determination of LP culture supernatant IgA levels, LP cells, isolated as described below were cultured at a density of 6.25 x 105 cells/ml in RPMI 1640 media (BioWhittaker, Walkersville, MD) containing 10% FCS (HyClone, Logan, UT), 2 mM glutamax I (Life Technologies, Grand Island, NY), 10 mM HEPES (BioWhittaker), 1 mM sodium pyruvate (BioWhittaker), 50 U/ml penicillin-50 mg/ml streptomycin (Life Technologies), and 50 mM 2-ME (Fisher Scientific) for 72 h at 37°C and 5% CO2. After 72 h, the culture supernatants were removed and the concentration of IgA in the supernatant was determined as described above.
Cell isolation; LP mononuclear cells, splenocytes, and resident peritoneal cells
LP mononuclear cells and splenocytes were isolated as previously described (25). Briefly, PP were removed from small intestines, epithelial cells were removed by treatment with HBSS (BioWhittaker) containing 5 mM EDTA (Sigma-Aldrich), and single-cell suspensions were generated from the LP by dispase (Sigma-Aldrich) and collagenase (Sigma-Aldrich) digestion. Cells were counted for viability by trypan blue exclusion. Cellular populations with viability <75% were discarded. Typical yields for LP cell isolation was 1.5 x 107 viable cells/intestine. This population includes both stromal cells and bone marrow-derived cells. Resident peritoneal cells were obtained by flushing the peritoneal cavity with 10 ml of cold PBS. RBCs were lysed in all populations before analysis.
Flow cytometric analysis
Single-cell suspensions obtained as above were resuspended in
PBS with 1% BSA (Fisher Scientific) and 1 mg/ml human IgG (Sandoz
Pharmaceuticals, East Hanover, NJ) at 2 x
107 cells/ml, or for the detection of LT
expression using the LTR-Ig fusion protein, cells were resuspended
in PBS with 1% BSA, 5% normal mouse serum, 5% normal rabbit serum,
and FC block (BD PharMingen, San Diego, CA) at 2 x
107 cells/ml. Cells were stained with the
directly conjugated Abs, biotin-conjugated Abs, the LTR-Ig fusion
protein, or the appropriate isotype control Abs for 30 min on ice.
Cellular suspensions were washed twice in PBS containing 1% BSA, and
where appropriate, stained with directly conjugated secondary Abs,
streptavidin-PE, or steptavidin-FITC for 30 min on ice. Cells were
washed twice as above and fixed with 1% paraformaldehyde in PBS. Flow
cytometric analysis was done on a triple-laser flow cytometer (FACScan;
BD Biosciences, Mountain View, CA) and analysis was performed on a
Macintosh G3 using the CellQuest program (BD Biosciences). Dead cells
were excluded based on forward and side light scatter and 10,000 cells
from the remaining population were analyzed for CD45 expression to
determine the number of bone marrow-derived cells in the LP. For the
analysis of lymphocyte subpopulations, 10,000 cells from the lymphocyte
population (as determined by forward vs side scatter) were analyzed for
the expression of lymphocyte markers. Gates for positive staining were
defined such that 1% of the analyzed population stained positive with
the appropriate isotype control Ab. Therefore, 1% positive staining is
consistent with the absence of a positive population.
The following Abs and secondary staining reagents were used for flow
cytometric studies: FITC-conjugated rat anti-mouse CD45,
PE-conjugated rat anti-mouse CD19, biotin-conjugated rat
anti-mouse CD11b, biotin-conjugated hamster anti-mouse TCR,
PE-conjugated rat anti-mouse CD4, FITC-conjugated rat
anti-mouse CD8, PE-conjugated hamster anti-mouse TCR
,
biotin-conjugated mouse anti-mouse NK1.1, appropriate isotype
control Abs, streptavidin-FITC, streptavidin-PE (all from BD
PharMingen), biotin-conjugated rat anti-mouse CD8 (Caltag
Laboratories, Bulingame, CA), biotin-conjugated rat anti-mouse IgA
(Southern Biotechnology Associates), and PE-conjugated donkey
anti-human Ig (Jackson ImmunoResearch Laboratories, West-Grove,
PA).
ELISPOT assay
96-well multiscreen-HA plates (Millipore, Bedford, MA) were coated with goat anti-mouse Ig (Southern Biotechnology Associates) overnight at room temperature. Plates were washed three times in PBS, blocked with PBS containing 5% newborn calf serum (HyClone) for 1 h at 37°C, washed, and LP cellular suspensions in IMDM (BioWhittaker), 5% FCS (HyClone), 2 mM glutamax I (Life Technologies), 50 U/ml penicillin-50 mg/ml streptomycin (Life Technologies), and 50 mM 2-ME (Fisher Scientific) were added to the plates. Plates were incubated at 37°C 5% CO2 overnight, washed with PBS containing 0.05% Tween 20, and incubated with alkaline phosphatase-conjugated goat anti-mouse IgA Ab (Southern Biotech Associates) overnight at 4°C. Plates were washed with PBS and exposed to 5-bromo-4-chloro-3-indolyl phosphatase/nitro blue tetrazloium substrate (Sigma-Aldrich) and spot-forming cells were counted under a dissecting microscope.
Bone marrow transfers
Six to 10-wk-old LT-/- and
LTR-/- mice (recipients) received 1000 Gy of
irradiation in divided doses over two sequential days. Bone marrow
was harvested from gender-matched adult C57BL/6,
LT-/-, JH-/-, and
RAG-/- donors and treated with anti-Thy1.1
and Thy1.2 Ab (clone AT83; a gift from Dr. O. Kanagawa, Washington
University School of Medicine) and low toxin rabbit complement
(Cedarlane Laboratories Limited, Ontario, Canada) to deplete T
lymphocytes. A total of 1 x 107 T
lymphocyte-depleted bone marrow cells (5 x
106 cells from each donor in experiments
involving multiple donors) from gender-matched donors were injected
i.v. into recipients on the second day of irradiation. All recipient
mice received 280 µg/ml sulfamethoxazole and 58 µg/ml trimethoprim
in drinking water for 3 days before and 5 days following bone marrow
transfer. Mice were allowed 12 wk for reconstitution with donor bone
marrow before use for experiments involving flow cytometric
analysis.
Splenectomy
Laparotomy was performed on 8–10-wk-old anesthetized
LT-/- mice; the splenic artery was ligated
and the spleen removed. Mice received 100 mg/kg ampicillin and 5 mg/kg
gentamicin i.p. daily for 3 days following splenectomy. Two weeks
following splenectomy, mice underwent bone marrow transfers as
described above. The complete absence of the spleen was confirmed by
examination at the time of sacrifice.
LTR-Ig treatment
Soluble LTR-Ig was purified from supernatants generated by a
Chinese hampster ovary cell line producing the LTR-Ig fusion protein
(a gift from Dr. W. Yokoyama, Washington University School of
Medicine). LTR-Ig activity was confirmed by the ability to inhibit
the formation of PP in offspring of pregnant females receiving 100 µg
LTR-Ig i.v. on day 16 postconception (pc).
Treatment groups
Group 1: timed pregnant female C57BL/6 mice were injected with
100 µg LTR-Ig via tail vein on day 16 pc. Group 2: offspring from
timed pregnant female C57BL/6 mice injected with 100 µg LTR-Ig on
day 16 pc received 20 µg LTR-Ig i.p. weekly for 5 wk beginning 7
days after birth. Group 3: offspring from untreated C57BL/6 female mice
received 20 µg LTR-Ig i.p. beginning 3 days after birth and weekly
thereafter for 5 wk. Group 4: mice from group 2 were treated with 20
µg of LTR-Ig i.p. weekly for an additional 10 wk. Group 5: mice
from groups 2 and 3 were followed for 10 wk after the cessation of
LTR-Ig therapy. No differences were noted between groups 2 and 3 at
any time point examined; and therefore, results from these two groups
were combined and reported as group 5. Group 6: 10-wk-old mice were
given 100 µg of LTR-Ig i.p weekly for 3 wk.
Statistical analysis
Delta Soft 3 software (BioMetallics, Princeton, NJ) was used to
determine the weighted mean ± the SEM of the fecal IgA
concentration for each sample. Weighted mean =
wi
conci/wi;
conci is the interpolated mean concentration for
dilution i, wi =
1/(SEMi)2,
SEMi is the SE of mean for
conci. Data analysis using an unpaired Student’s
t test was performed using GraphPad Prism (GraphPad, San
Diego, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and LTR is required for the presence of B lymphocytes in
the intestinal LP
The phenotype of mice deficient in the expression of LT
(lacking both LT3 and
LT12) has been well
described (4, 26). These mice lack organized lymphoid
structures, including PP, have disrupted splenic architecture lacking
GC and FDC, have diminished fecal IgA production, and have a diminished
capacity to produce high affinity Ig upon immunization. Importantly,
these mice have normal lymphatics, and studies of B and T lymphocyte
function have not revealed deficits. To assess the role of LT in
directing the composition of the LP lymphocyte populations, we examined
the LP cell populations from C57BL/6 and
LT-/- mice. As shown in Table I
,
LT-/- mice have normal numbers of bone
marrow-derived (CD45+) cells in their intestinal
LP. Further analysis of the lymphocyte subpopulations revealed that
LT-/- mice have an increased proportion of
and TCR+ T lymphocytes, and this
increase is distributed between CD4+ and
CD8+ T lymphocyte subpopulations. We did not
observe CD4, CD8 double positive T lymphocytes in the LP of any mice
examined. However, B lymphocytes are absent from the LP of the
LT-/- mice (Table I and Fig. 1). LT-/- mice
also lack LP lymphocytes expressing surface Ig (data not shown),
confirming the absence of LP B lymphocytes as opposed to a failure of
LT-/- LP B lymphocytes to express an
isolated cell surface marker. The absence of B lymphocytes in the LP of
LT-/- mice cannot be explained by a global
deficiency of B lymphocytes, as an increased proportion of B
lymphocytes were seen in the spleen of LT-/-
mice when compared with controls (Table I and Fig. 1). Notably, the LP
B lymphocytes in C57BL/6 mice are overwhelmingly B-2 B lymphocytes
(CD19+, CD11b-) and,
consistent with previous observations, contain a small population of
CD19+IgA+ cells
(19), suggesting that the deficiency of B lymphocytes in
the LT-/- mice is predominantly a
deficiency in IgA- B-2 B lymphocytes (Table I).
The remainder of the CD45+ population in the LP
of LT-/- mice are also modestly increased,
and include NK cells, granulocytes, and macrophages (our unpublished
observations).
|
-/- and aly/aly mice (5, 19). In aly/aly mice the peritoneal cavity contains
an increased population of B lymphocytes with a disproportionate
expansion of B-1 (CD11b+) B lymphocytes
(19). Analysis of the resident peritoneal cell population
from 14-wk-old LT-/-,
LTR-/-, and C57BL/6 mice (Table II) reveals
an increase in the number of resident peritoneal cells in both the
LT-/- and LTR-/-
mice with a preferential increase in B lymphocytes which are
predominantly CD11b- (B-2 B lymphocytes), the
remaining CD19+ cells are
CD11b+ and therefore, are B-1 B lymphocytes. No
significant change is seen in the proportion of peritoneal B cells
expressing IgA in either the LT-/- and
LTR-/- mice. These findings are consistent
with the inability of IgA- B-2 B lymphocytes to
migrate to appropriate effectors sites (intestinal LP) in the absence
of LTR signaling, resulting in their accumulation in the peritoneal
cavity.
LT-/- mice lack expression of both soluble
LT3 and membrane-bound
LT12; and therefore,
the lack of B lymphocytes in the intestine of the
LT-/- mice could result from a lack of
activation of TNFRI, TNFRII, or the LTR. To determine the relevant
events for the presence of B lymphocytes in the intestinal LP, LP
lymphocyte populations were evaluated from mice deficient in TNFRI,
TNFRII, and LTR. As shown in Table III and Fig. 2a, TNFRI- and
TNFRII-deficient mice have a near normal population of LP B lymphocytes
which is enriched for IgA+ B lymphocytes, while
LTR-deficient mice lack LP B lymphocytes. The deficiency of B
lymphocytes in LTR-deficient mice is not a global phenotype, as B
lymphocytes are present in the spleen of LTR-deficient mice in
proportions equal to or exceeding wild-type mice. Consistent with their
lack of LP B lymphocytes, LTR-/- mice have
diminished fecal IgA production equivalent to that of
LT-/- mice (compare Tables I and III). We
also noted diminished fecal IgA production by TNFRI- and
TNFRII-deficient mice when compared with that of C57BL/6 mice (compare
Tables I and III). Notably, these mice have a population of LP B
lymphocytes which is equivalent to that of C57BL/6 mice and a
comparable or increased number of IgA producing plasma cells when
compared with C57BL/6 mice (Table I and III). However, the ability of
the LP plasma cells in the TNFRI- and TNFRII-deficient mice to produce
IgA is diminished. These observations suggest that TNFRI- and
TNFRII-dependent events play a role in augmenting Ab production by IgA
plasma cells in the LP, and that defects in the epithelial transport of
IgA in these mice strains are unlikely to account for their diminished
fecal IgA production.
|
R-dependent events are essential for the
presence of LP B lymphocytes
The lack of B lymphocytes in the LP of the
LT-/- and LTR-/-
mice demonstrates that a
LT12/LTR-dependent
event is required for the presence of LP B lymphocytes.
LT12/LTR-dependent
events during gestation are essential for the formation of PP and LN,
and
LT12/LTR-signaling
after birth cannot rescue the formation of PP and LN (9).
To assess whether
LT12/LTR-dependent
events, distinct from the embryonic events required for the formation
of PP and LN, are essential for the presence of LP B lymphocytes, we
blocked gestational as well as postgestational LTR signaling with a
LTR-Ig fusion protein (Fig. 3). As
shown in Table IV and Fig. 2b, treatment of pregnant mice on
day 16 gestation, blocking PP formation, had no effect on the
production of fecal IgA or the presence of LP B lymphocytes (group 1).
However,
LT12/LTR-dependent
events after birth are essential for the presence of LP B lymphocytes
as evidenced by the lack of LP B lymphocytes in mice receiving the
LTR-Ig fusion protein after birth (groups 2–4). There appeared to
be no additive effect of blocking LTR signaling both during and
postgestation as compared with blocking LTR signaling after birth
alone (compare groups 2 and 3). Discontinuation of the LTR-Ig fusion
protein after 5 wk allowed for the population of the LP with B
lymphocytes which are predominantly CD11b- (B-2
B lymphocytes), while mice continually treated with the LTR-Ig
fusion protein over the same time period continued to lack LP B
lymphocytes (compare groups 4 and 5). Notably, continuous
postgestational treatment with the LTR-Ig fusion protein did not
completely suppress fecal IgA production. In addition, the LP B
lymphocyte compartment could not be modulated by treatment of adult
mice with 100 µg of LTR-Ig weekly for 3 wk (group 6), a dosing
schedule which is sufficient to cause disruption of splenic
architecture and decreased BLC expression in the spleen (Ref.
13 and our unpublished observations). These observations
suggest that the LTR-dependent events required for the presence of
LP B lymphocytes occur after birth and appear to be independent of the
formation of PP and/or LN.
|
As demonstrated above, postgestational LTR events are essential
for the presence of LP B lymphocytes. To determine whether these events
are sufficient for the presence of LP B lymphocytes in the absence of
LN and PP, we performed bone marrow transfers of wild-type
(LT-sufficient) bone marrow into adult
LT-/- recipients. These recipients lack
LT-dependent events until the time of bone marrow transfer, and
transfer of LT-sufficient bone marrow in adulthood cannot rescue LN and
PP formation (Ref. 9 and our unpublished observations). As
shown in Table V and Fig. 2c, transfer of
LT-/- bone marrow to
LT-/- mice does not result in the production
of fecal IgA or the presence of LP B lymphocytes, while the transfer of
C57BL/6 bone marrow to LT-/- recipients
results in the production of normal levels of fecal IgA and the
presence of LP B lymphocytes. LT-sufficient B lymphocytes have been
demonstrated to effect the splenic architecture of
LT-/- mice following bone marrow transfer,
resulting in the formation of GC and the production of FDCs
(11). To assess the contribution of LT-dependent events
in the spleen on the presence of LP B lymphocytes following bone marrow
transfer, bone marrow transfers were performed in splenectomized
recipients. As shown in Table V, splenectomy had no effect upon the
presence of LP B lymphocytes or fecal IgA levels with the exception
that fecal IgA levels in splenectomized recipients showed altered
kinetics, with peak levels occurring 10 wk post bone-marrow transfer as
opposed to 8 wk posttransfer in unmanipulated recipients. These
findings demonstrate that bone marrow-derived cells can deliver the
necessary and sufficient LT signal for the presence of LP B
lymphocytes, independent of the presence of the spleen, PP, and
LN.
LT-sufficient B lymphocytes and radio-resistant, non-bone
marrow-derived, LTR-sufficient cells are required for normal
lymphocyte composition of the intestinal LP
To determine which bone marrow-derived cell lineage delivers the
LT signal needed for the presence of LP B lymphocytes, mixed bone
marrow transfers were performed using RAG-/-
and JH-/- (LT-sufficient) donors (Fig. 2c and Table V). LT-/- mice
receiving a combination of LT-/- and
RAG-/- bone marrow produced fecal IgA which was
significantly greater than LT-/- mice
receiving LT-/- bone marrow alone. Thus,
documenting that LT expression by lymphocytes is not required for the
production of fecal IgA. However, LT-/- mice
receiving mixed LT-/- and
RAG-/- bone marrow had a diminished, but not
absent, population of B lymphocytes when compared with
LT-/- mice receiving wild-type bone marrow,
indicating that LT-expressing lymphocytes are necessary for the optimal
population of the intestinal LP by B lymphocytes.
LT-/- mice receiving mixed
LT-/- and JH-/- bone
marrow had normal production of fecal IgA, and a diminished, but not
absent, population of LP B lymphocytes which was not different from
that seen in LT-/- mice receiving
LT-/- and RAG-/-
bone marrow. These findings indicate that RAG-independent,
LT-expressing, bone marrow-derived cells can transmit the relevant
LT-dependent signal for the production of fecal IgA and can partially
allow the population of the intestinal LP with B-2 B lymphocytes.
However, an LT-expressing B lymphocyte is required for the optimal
population of the intestinal LP with B lymphocytes.
The LTR is expressed by stromal cells and a subpopulation of
monocytes. To identify the LTR-expressing cell population relevant
for the entry of LP B lymphocytes, and to provide direct evidence for a
role for LTR in our bone marrow transfer experiments, we transferred
C57BL/6 bone marrow into irradiated LTR-/-
mice. LTR-/- mice receiving C57BL/6 bone
marrow had diminished fecal IgA production and lacked LP B lymphocytes
(Fig. 2c and Table V). These observations demonstrate a
requirement for LTR-sufficient, radio-resistant, non-bone
marrow-derived cell in directing the entry and residence of LP B
lymphocytes.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Using the LT-/- mice, a well described
induced mutant strain of mice lacking LN and PP, we examined the
requirement of LT-dependent events for the normal composition of
intestinal lymphocytes. We noted no differences in the number or
composition of IEL in the LT-/- mice (our
unpublished observations). These findings are consistent with
observations that the aly/aly mice, which have a spontaneous
mutation in NIK, a signaling molecule necessary for the function of the
LTR (22), have normal composition of IELs
(28). Examination of LT-/- mice
reveals that LT-dependent events, including the development of PP
and LN, are not required for T lymphocytes to enter and reside in the
LP. Consistent with this observation, we noted a normal or
increased population of TCR+
lymphocytes in the LP of TNFRI-/-,
TNFRII-/-, and
LTR-/- mice, as well as
LT-/- mice reconstituted with
LT-/- bone marrow, and mice treated with
LTR-Ig. Flow cytometric analysis of CD69 and CD45RB expression
revealed no differences between LP T lymphocytes from
LT-/- mice and C57BL/6 mice (our unpublished
observations). These findings suggest that LT-dependent events
including events occurring within organized lymphoid structures in
general and PP specifically are not required for T lymphocytes to enter
and reside in the LP. This contrasts with the current understanding
that LP T lymphocytes are predominantly or exclusively comprised from T
lymphocytes undergoing activation in PP. In a broader context, this
observation suggests that events occurring in the PP directing Th cell
development may not influence all T lymphocyte responses in the
LP.
In contrast to the finding of a normal composition of LP T lymphocytes,
we observed an absence of LP (CD19+) B
lymphocytes in the LT-/- mice. CD19, the
earliest B lymphocyte lineage marker, is first expressed as early pro-B
cells arise from stem cells in the bone marrow and is continually
expressed by B lymphocytes until their differentiation into plasma
cells. CD19 is a more specific marker for B lymphocytes when compared
with B220 (29, 30, 31). We observed a small population of LP
cells which are B220+,
CD19- in LT-/- and
RAG-/- mice, suggesting that
B220+ is not an accurate marker for the
evaluation of intestinal B lymphocyte populations. Analysis of C57BL/6
mice revealed that the LP B lymphocyte population is overwhelmingly
comprised of B-2 B lymphocytes, and thus, the
LT-/- mice are predominantly deficient in LP
B-2 B lymphocytes.
Examination of the resident peritoneal cell population from the
LT-/- and LTR-/-
mice revealed an increase in cell number with a disproportionate
expansion of B lymphocytes which were predominantly
CD11b- (B-2 B lymphocytes). Consistent with this
finding, we also observed an increase in the splenic B lymphocyte
population in LT-/- and
LTR-/- mice. Together these observations
suggest that the lack of LT/LTR-dependent signals result in the
inability of intestinal B lymphocytes to migrate appropriately to their
effector site(s).
We were able to further demonstrate that the relevant event(s) for B
lymphocytes to enter and reside in the LP are not dependent upon TNFRI
and/or TNFRII binding soluble LT3, but are
dependent upon LTR, which binds membrane-bound
LT12. Consistent with
the absence of B lymphocytes in the LP in the
LT-/- and
LTR-/- mice, we saw an increase in the
splenic B lymphocyte populations in these mice, suggesting an inability
of B lymphocytes to migrate to their appropriate effector sites. In
contrast to our observations in the knockout mice, we have not
observed an increase in resident peritoneal cell number or a
shift in the B-1 or B-2 B lymphocyte populations in mice treated with
the LTR-Ig fusion protein or in LT-/- and
LTR-/- bone marrow recipients.
We also noted diminished production of fecal IgA by both TNFRI-
and TNFRII-deficient mice; however, the levels were significantly
higher than that seen in the LT-/- and
LTR-/- mice. TNFRI-deficient mice have a
normal population of B lymphocytes in their LP; however, they lack GC
(10) and have hypoplastic PP which may contribute to their
diminished fecal IgA production. Fecal IgA production by
TNFRII-deficient mice was significantly lower than that seen for
TNFRI-deficient mice. TNFRII-deficient mice are not known to have a
defect in the formation of GC and FDC, are felt to have normal PP and
LN formation, and by our observations have a normal population of LP B
lymphocytes. In addition, we observed a normal or increased number of
IgA producing cells in the LP of TNFRII-/- and
TNFRI-/- mice. However, despite the normal or
increased number of IgA producing plasma cells in these mice, the
production of IgA by these plasma cells was significantly decreased in
comparison with wild-type mice. These observations suggest that TNFRI-
and TNFRII-dependent events may be necessary for the optimal
production of IgA by LP plasma cells, and is consistent with the
diminished Ig production in response to T lymphocyte-dependent Ags seen
in TNF-deficient mice (32).
LTR-dependent events occurring during embryogenesis are essential
for the development of PP and LN. Using a LTR-Ig fusion protein, we
demonstrate that blocking LTR-dependent events during gestation
alone, which have been shown to be essential for the formation of PP,
does not affect the production of fecal IgA or the population of the LP
by B lymphocytes. However, blocking LTR-dependent events after birth
results in the absence of LP B lymphocytes and reduced, but not absent,
fecal IgA production. The effects of LTR blockade were reversible
with the discontinuation of LTR-Ig treatment in adulthood,
demonstrating that the relevant LT-dependent signal for the entry of LP
B lymphocytes can be delivered in adulthood. In contrast to the rapidly
reversible effects of LTR signaling on the normal segregation of
lymphocytes in the spleen, we were unable to modulate the LP B
lymphocyte compartment of adult mice with short-term LTR-Ig
treatment. Together these findings document a role for postgestational
LT/LTR interactions important for the presence of LP B
lymphocytes.
To assess the requirement of PP and LN in the entry and residence of LP
B lymphocytes, we performed adoptive transfers of wild-type bone marrow
into adult LT-/- mice. These mice lack LN
and PP, and do not receive LT-dependent signals until after bone marrow
transfer. LT-/- mice reconstituted with
wild-type bone marrow have normal fecal IgA and increased LP B
lymphocytes when compared with LT-/- mice
reconstituted with LT-/- bone marrow.
Previous observations have suggested postgestational LTR-dependent
events may play a role in the formation of PP. Transgenic mice
expressing concentrations of the LTR-Ig fusion protein sufficient to
block LTR signal transduction on day 3 of life have decreased size
and number of PP (33). However, we feel that the formation
of PP is not the relevant LTR-dependent event for the presence of LP
B lymphocytes, as LP B lymphocytes are present in mice treated with the
LTR-Ig fusion protein at day 16 in utero, and in
LT-/- mice receiving C57BL/6 bone marrow,
both of which lack PP. LT expressing B lymphocytes have been shown to
direct normal compartmentalization of the spleen of LT mice after
adoptive bone marrow transfer (9, 34). To rule out a role
for events in the spleen contributing to the presence of LP B
lymphocytes, we performed bone marrow transfers on splenectomized
LT-/- recipients. Splenectomized recipients
had normal production of fecal IgA and normal population of the LP with
B lymphocytes. Together these findings suggest that postgestational
LT/LTR interactions, independent of the formation of LN and PP, and
the independent of events occurring in the spleen, are both essential
and sufficient for the entry and residence of LP B lymphocytes.
To determine the LT expressing bone marrow-derived cell lineage
relevant for the presence of LP B lymphocytes, we performed mixed bone
marrow transfers into LT-/- recipients. Mice
receiving combinations of LT-/- with
RAG-/- or JH-/- bone
marrow had similar populations of LP B lymphocytes that were
significantly increased over those seen in
LT-/- recipients receiving
LT-/- bone marrow and significantly less
than those seen in LT-/- recipients
receiving wild-type bone marrow. These observations suggest that while
LT expressing nonlymphocyte bone marrow cell lineages may provide the
LT-dependent signal necessary for the production of fecal IgA and the
entry of B lymphocytes into the intestinal LP, optimal population of
the intestinal LP by B lymphocytes requires an LT expressing B
lymphocyte. In vitro studies demonstrated up-regulation of LT
expression upon lymphocyte activation (3, 35); however,
the kinetics of LT expression in vivo is less well understood. Using
flow cytometric analysis we observed 5–10% of
CD19+ PP and mesenteric LN cells and <5% of
CD19+ LP cells express LT in normal mice (our
unpublished observations).
The LTR is expressed by non-bone marrow-derived stromal cells and a
subset of monocytes (3). To determine the LTR
expressing cell type relevant for the presence of LP B lymphocytes, and
to provide direct evidence for the role of LTR in our bone marrow
transfer experiments, we transferred wild-type bone marrow to
LTR-/- recipients. In contrast to
LT-/- recipients receiving C57BL/6 bone
marrow, LTR-/- recipients of C57BL/6 bone
marrow lacked LP B lymphocytes. These findings document a requirement
for a LTR-expressing, radio-resistant, non-bone marrow-derived cell
for the presence of LP B lymphocytes.
The events leading to the production of fecal IgA are complex, and
involve the maturation and migration of B-1 and B-2 B lymphocytes, both
of which contribute to the pool of fecal IgA (19, 36, 37, 38).
Multiple studies have investigated the role of LT in the development of
IgA producing plasma cells and the generation of fecal IgA. It is
currently not clear where the final development of B lymphocytes into
IgA producing plasma cells occurs and it is also unclear as to what
proportion of the LP B lymphocytes contribute to the pool of these
plasma cells. Recent descriptions suggest that B lymphocyte
differentiation into plasma cells may take place after B lymphocytes
arrive in the LP (39). Given these observations, we feel
it is important to make a distinction between our findings and the
findings of previous investigations examining the roles LT plays in
promoting the production of Ig. LT has been shown to play a role in the
development of PP and LN which are essential to the production of high
affinity Ab from B-2 B lymphocyte precursors (4, 5, 6, 7, 40, 41). In this study, we demonstrate a role of LT in directing the
composition of B lymphocytes in the intestinal LP. Our observations
suggest that the roles LT plays in the production of fecal IgA and in
directing the composition of LP B lymphocytes may be independent as
evidenced by the normal fecal IgA levels in mice with absent or
diminished LP B lymphocytes (mice receiving continuous LTR-Ig and
mice receiving mixed bone marrow transfers, respectively).
Our observations parallel the role LT plays in directing the compartmentalization of the spleen. LT expressing B lymphocytes have been demonstrated to promote the development of splenic stromal cells which produce chemokines facilitating the normal segregation of splenic T and B lymphocytes (2, 11, 13, 34, 42). Similar to our findings, the LT-dependent signal important for the normal segregation of splenic lymphocytes can be transmitted by an LT-expressing B lymphocyte in adulthood. These observations suggest that an intestinal stromal cell with a function similar to the previously described splenic stromal cell may require LT for its development, and would expand the recently described roles of intestinal stromal cells in the intestinal immune response (39, 43).
Diminished BLC expression in the intestine is an attractive explanation
for our observations, as BLC is a B lymphocyte selective chemokine, and
its expression by splenic stromal cells is diminished in the absence of
LTR signal transduction (13). However, mice deficient
in the Burkitt’s lymphoma receptor 1, the BLCR, have a normal number
of IgA-producing cells in the LP (44), and BLC expression
is not detected in non-PP bearing intestine (Ref. 19 and
our unpublished observations), suggesting that the loss of
LTR-induced intestinal BLC expression is unlikely to explain our
findings.
Recently, a primitive mechanism of intestinal IgA production
independent of the presence of T lymphocytes and requiring lymphoid
structures has been described (38). These observations
were based upon the lack of IgA production in the
LT-/- and aly/aly mice (both
lacking PP and LN). In addition, recent investigations have described a
pathway of intestinal IgA production independent of the expression of
the µ- or -chain (45). This pathway of IgA production
is felt to require PP or LN based upon the inability of bone marrow
from µ-chain-deficient mice to reconstitute IgA production in
aly/aly mice. Based upon our observations, these pathways of
IgA production may not be dependent upon the presence of organized
lymphoid structures, but rather require LT/LTR-mediated events,
independent of the formation of PP and LN, necessary for the normal
entry of B lymphocytes into the intestinal LP.
The mucosal immune system is a network of physically distinct lymphoid
compartments with the capacity to transmit Ag-specific immune responses
between distant surfaces. Essential to the generation of these
coordinated responses is the appropriate entry and residence of
lymphocytes in these lymphoid compartments. Recent investigations have
defined roles for the 7 integrins and the
CCR9/thymus-expressed chemokine receptor/chemokine pair in directing
the compartmentalization of the intestinal immune system. We have
demonstrated a role for the cytokine
LT12 and its receptor
LTR in directing the specific compartmentalization of B lymphocytes
to the intestinal LP. Surprisingly, these
LT12/LTR-dependent
events are distinct and independent of the role
LT12/LTR plays in
the formation of PP, LN, and the segregation of lymphocytes in the
spleen. Based on the severity of the deficit of LP B lymphocytes when
LTR signal transduction is blocked, we believe these events are of
primary importance in directing the entry and residence of LP B
lymphocytes.
|
Acknowledgments |
|---|
R-Ig; E. Newberry,
S. Amadeus, and the members of the St. Louis Institute of
Mucosal Immunology (St. Louis, MO) for assistance and advice
with manuscript preparation. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Robin G. Lorenz, Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8118, St. Louis, MO 63110. E-mail address: lorenz{at}pathbox.wustl.edu
3 Abbreviations used in this paper: LT, lymphotoxin; PP, Peyer’s patch; FDC, follicular dendritic cell; GC, germinal center; SLC, secondary lymphoid tissue chemokine; BLC, B lymphocyte chemoattractant; LP, lamina propria; IEL, intraepithelial lymphocyte; NIK, NF-inducing kinase; LN, lymph node; RAG, recombination-activating gene; pc, postconception.
Received for publication November 1, 2001. Accepted for publication March 14, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
complexes on the surface of mouse lymphocytes. J. Immunol. 159:3288.[Abstract]
-deficient mice. Proc. Natl. Acad. Sci. USA 94:9302. receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9:59.[Medline]
/ complex is required for the development of peripheral lymphoid organs. J. Exp. Med. 184:1999.-deficient mice: effects on secondary lymphoid organ development and humoral immune responsiveness. J. Immunol. 155:1685.[Abstract]
(LT)-deficient mice is due to abnormal organ development, not defective lymphocyte migration. J. Inflamm. 45:72.[Medline]
and the type I tumor necrosis factor (TNF) receptor in the establishment of follicular dendritic cells from non-bone marrow-derived cells. J. Exp. Med. 186:1997./ 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. 189:403.4 integrins during fetal and adult hematopoiesis. Cell 85:997.[Medline]
7 integrins in formation of the gut-associated lymphoid tissue. Nature 382:366.[Medline]
E (CD103)-deficient mice. J. Immunol. 162:6641.B-inducing kinase (NIK) causes defects in secondary lymphoid tissue chemokine receptor signaling and homing of peritoneal cells to the gut-associated lymphatic tissue system. J. Exp. Med. 191:1477. B-inducing kinase. J. Immunol. 165:804. receptor-induced NF-B transcriptional activity in NIK-deficient mice. Science 291:2162.-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers. Immunol. Rev. 156:137.[Medline]
receptor-IgG1 fusion protein. Proc. Natl. Acad. Sci. USA 93:13102.-dependent fashion. J. Exp. Med. 187:1009.- deficient mice. Nature 382:462.[Medline]
chain expression in developing B cells. Nat. Immunol. 2:625.[Medline]This article has been cited by other articles:
|
|
 |
|
  P. Velazquez, B. Wei, M. McPherson, L. M. A. Mendoza, S. L. Nguyen, O. Turovskaya, M. Kronenberg, T. T. Huang, M. Schrage, L. N. Lobato, et al. Villous B Cells of the Small Intestine Are Specialized for Invariant NK T Cell Dependence J. Immunol., April 1, 2008; 180(7): 4629 - 4638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. T. Taylor, S. R. Patel, E. Lin, B. R. Butler, J. G. Lake, R. D. Newberry, and I. R. Williams Lymphotoxin-Independent Expression of TNF-Related Activation-Induced Cytokine by Stromal Cells in Cryptopatches, Isolated Lymphoid Follicles, and Peyer's Patches J. Immunol., May 1, 2007; 178(9): 5659 - 5667. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. G. McDonald, J. S. McDonough, C. Wang, T. Kucharzik, I. R. Williams, and R. D. Newberry CC Chemokine Receptor 6 Expression by B Lymphocytes Is Essential for the Development of Isolated Lymphoid Follicles Am. J. Pathol., April 1, 2007; 170(4): 1229 - 1240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Wang, K. G. McDonald, J. S. McDonough, and R. D. Newberry Murine isolated lymphoid follicles contain follicular B lymphocytes with a mucosal phenotype Am J Physiol Gastrointest Liver Physiol, October 1, 2006; 291(4): G595 - G604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. W. Spahn, H.-P. Eugster, A. Fontana, W. Domschke, and T. Kucharzik Role of Lymphotoxin in Experimental Models of Infectious Diseases: Potential Benefits and Risks of a Therapeutic Inhibition of the Lymphotoxin-{beta} Receptor Pathway Infect. Immun., November 1, 2005; 73(11): 7077 - 7088. [Full Text] [PDF]
|
|
|
|
|
|
K. G. McDonald, J. S. McDonough, and R. D. Newberry Adaptive Immune Responses Are Dispensable for Isolated Lymphoid Follicle Formation: Antigen-Naive, Lymphotoxin-Sufficient B Lymphocytes Drive the Formation of Mature Isolated Lymphoid Follicles J. Immunol., May 1, 2005; 174(9): 5720 - 5728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T W Spahn and T Kucharzik Modulating the intestinal immune system: the role of lymphotoxin and GALT organs Gut, March 1, 2004; 53(3): 456 - 465. [Full Text] [PDF]
|
|
|
|
|
|
R. G. Lorenz, D. D. Chaplin, K. G. McDonald, J. S. McDonough, and R. D. Newberry Isolated Lymphoid Follicle Formation Is Inducible and Dependent Upon Lymphotoxin-Sufficient B Lymphocytes, Lymphotoxin {beta} Receptor, and TNF Receptor I Function J. Immunol., June 1, 2003; 170(11): 5475 - 5482. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Time of conception, birth, and date of analysis.
, Time points of LT