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*
Immunobiology Vaccine Center, Departments of Microbiology and Oral Biology, University of Alabama, Birmingham, AL 35294;
Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892; and
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Current data support the notion that both B cell activation
(9) and IgA isotype switching (10, 11, 12, 13, 14, 15) can be
stimulated by TGF-ß1. However, interaction with Th2 cells and/or
Th2-type cytokines is necessary to overcome the antiproliferative
effect of TGF-ß1 and to induce B cells to differentiate into
IgA-secreting plasma cells (10, 16, 17, 18, 19, 20, 21, 22, 23, 24). When added to
activated B cell cultures, TGF-ß1 induces a small proportion (
2%)
of B cells to switch to IgA (10, 15, 19, 20). The low
frequency of TGF-ß1-mediated switching could be increased to 10–20%
of B cells, approaching the frequency of IgA+ B
cells in Peyer’s patches, by a combination of TGF-ß1, IL-4, IL-5,
anti-
dextran, and a CD40L-CD8
fusion protein
(11). These results illustrate the importance of multiple
components in the switch to IgA: B cell activation by cross-linking the
B cell Ag receptor, CD40-CD40L interaction to promote switching,
TGF-ß1 to direct the switch to IgA, and Th2-type cytokines for
expansion of postswitch IgA+ B cells and their
differentiation into IgA-secreting plasma cells. The mechanism by which
TGF-ß1 directs switching to IgA appears similar to switching directed
by other cytokines to other Ig classes, the induction of germline
transcription of the targeted constant region gene (12, 21).
In addition to inducing B cells to switch to IgA and to differentiate
into IgA-secreting B cells (15, 20), both of which are
paramount events in mucosal immunity, TGF-ß1 also affects homing of T
cells to mucosal surfaces. In vitro activation of human T cells in the
presence of TGF-ß1 specifically induced expression of the mucosal
homing receptor Eß7
integrin also termed human mucosal lymphocyte-1 (25).
Thus, the lack of TGF-ß1 in TGF-ß1-/- mice
could specifically reduce homing to mucosal compartments by reducing
Eß7 integrin
expression. Enlargement of lymph nodes draining mucosal surfaces in
TGF-ß1-/- mice indicates altered mucosal
lymphocyte trafficking in these mice (1).
The above studies indicate that TGF-ß1 profoundly affects mucosal and systemic immunity and inflammation, whether by inducing B cell switching to IgA or by regulating homing of T lymphocytes into the mucosal compartment. However, most conclusions have been based upon in vitro studies and do not necessarily reflect the effects of TGF-ß1 in vivo. The present study has assessed the role of TGF-ß1 in mucosal and systemic immune compartments in vivo by comparing TGF-ß1-/- and normal mice. Alterations in IgG, IgM, IgE, and IgA levels were observed in serum and external secretions. Those Ig levels were altered by the aberrant production of Igs in both the systemic and mucosal compartments, changes associated with elevated Th2 cytokine production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TGF-ß1-deficient mice were initially produced by disruption of the TGF-ß1 gene in embryonic stem cells by homologous recombination (1). TGF-ß1 gene-deleted (TGF-ß1-/-) mice between 3.5 and 5.5 wk of age were used in this study. Wild-type (+/+) mice, generated by mating +/- mice, were used as age-matched controls. The genotype was analyzed by PCR with tail DNA to assess loss of the TGF-ß1 gene.
Lymphocyte isolation
Mononuclear cells were isolated from the spleen and mediastinal
lymph nodes (MLN)3 by
mechanical disruption, and lymphocytes were enriched by Ficoll-Hypaque
(Lympholyte M; Accurate Chemical, Westbury, NY) density gradient
centrifugation. Lung mononuclear cells were isolated as previously
described (26). Briefly, the lungs were perfused with PBS
to remove the blood and were then mechanically disrupted with a pair of
scissors. Collagenase type IV (Sigma, St. Louis, MO) and DNase I
(Sigma) were then added to the lung tissue and allowed to enzymatically
digest it for 1.0 h at 37°C. The lung suspension was continuously
mixed and pipetted vigorously at 20-min intervals. Undigested lung
tissue was removed by passage over sterile cotton before separation by
Ficoll-Hypaque density gradient centrifugation. This method routinely
produced a 70–80% yield of lymphocytes, as assessed by staining with
eosin and thiazine (Hemocolor; EM Diagnostic Systems, Gibbstown, NJ),
and these lymphocytes were >95% viable, as determined by trypan blue
exclusion.
Peyer’s patches were removed from the small intestine and digested with collagenase type IV, as previously described (27). The remaining intestinal tissue was opened by incision, cut into 1–2 cm segments, and stirred in PBS containing 1 mM EDTA at 37°C for 15 min to remove the epithelium including the intraepithelial lymphocytes. The lamina propria (LP) lymphocytes were subsequently isolated by a collagenase digestion procedure as previously described (27). The mononuclear cells were collected from the interface following centrifugation over a 40-75% discontinuous Percoll gradient (27).
Sample collection
Bronchial lavages were collected as described previously
(28). In brief, a 19-gauge catheter (Intracath; Becton
Dickinson, Sandy, Utah) was inserted into the trachea via a small
incision made without severing the tissue. Bronchial alveolar lavages
(BAL) were obtained by inflating the lung three times with 1 ml sterile
PBS and repeating this step twice. The recovered PBS washes were
pooled, yielding 60–70% fluid recovery, and were stored at
-70°C until analyzed. Nasal and vaginal washes were obtained by
three consecutive 50-µl washes with sterile PBS. The three washes
were pooled and the cellular debris removed by centrifugation. The
samples were stored frozen until analyzed. Fecal pellets were
collected, weighed, and added (100 mg/ml) to sterile PBS containing
0.2% sodium azide. The pellets were homogenized by continuous shaking
for 10 min with a Vortex (Scientific Industries, Bohemia, NY).
Particulate fecal debris was removed by centrifugation for 10 min at
14,000 rpm, and supernatants were collected and stored frozen at
-70°C. Saliva was collected following i.p. injection of 100 µl of
250 µg/ml pilocarpine (Sigma) into anesthetized mice.
Ig ELISA
Ig levels in serum and mucosal samples, including fecal
extracts, vaginal and nasal washes, bronchial alveolar lavages, and
saliva, were determined by an ELISA as previously described
(28). Briefly, levels of IgM, IgG, or IgA were measured by
coating 96-well ELISA plates (Becton Dickinson) with 5 µg/ml of goat
anti-mouse-µ, -
, or - heavy chain-specific Abs (Southern
Biotechnology Associates, Birmingham, AL). The plates were blocked with
1% BSA in PBS-Tween 20 for 1 h at 25°C. Samples were added to
wells and incubated overnight at 4°C, and bound Ig was detected with
HRP-conjugated goat anti-mouse-µ, -, or - heavy
chain-specific Abs (Southern Biotechnology Associates). To determine
IgG subclass levels in biological samples, the plates were coated with
polyclonal anti-1-, -2a-, -2b-, and -3-specific capture
Abs (2.5 µg/ml), and Ig was detected with anti-1, -2a,
-2b, and -3 biotinylated mAbs (PharMingen, San Diego, CA). All
mAbs were used at appropriate concentrations (1:500 for -1 and -3
and 1:1000 for -2a and -2b) for detection. Following incubation
and washing, HRP-conjugated streptavidin was added and color developed
with 2,2'-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium
(ABTS) substrate (Sigma). The absorbance (OD415)
was measured with a Kinetics Reader model EL312 (Biotek Instruments,
Winooski, VT), following a 30-min incubation at 25°C, and the value
was compared with a standard curve from the appropriate Ig isotype or
IgG subclass employing linear regression. Total serum IgE levels were
determined by a sensitive ELISA using IgE-specific polyclonal capture
Ab (2.5 µg/ml) and a biotinylated monoclonal IgE-specific detection
Ab (R35-92) (PharMingen), followed by 1:2000 dilution of
polyHRP80-streptavidin (Research Diagnostics, Flanders, NJ) in
superblock (Pierce, Rockford, IL). The plates were washed extensively
with PBS-Tween 20 between incubation steps, and a final wash with PBS
was performed just before addition of substrate.
B cell enzyme-linked immunospot (ELISPOT)
The ELISPOT assay, which has been described by others
(29), was adapted to quantitate numbers of Ig-isotype
Ab-forming cells (AFC) from the intestinal LP, Peyer’s patches (PP),
spleen, cervical lymph nodes, MLN, and lungs of mice. The isotype of
AFC was determined by ELISPOT assay as described previously
(30). Briefly, 96-well nitrocellulose-based microtiter
plates (Millititer HA; Millipore, Bedford, MA) were coated with 100
µl of anti-, -, -µ (Southern Biotechnology Associates),
-
(PharMingen) (2.5–5.0 µg/ml) diluted in PBS, while control
wells received PBS only. Following washing, the plates were blocked
with complete medium for at least 1 h before addition of
lymphocytes to wells. Ten-fold serial dilutions of cell suspensions
(starting at a density of 1 x 106
cells/well) were added in duplicates and incubated for at least 6
h at 37°C in 5% CO2 in a humidified incubator.
AFC were detected with µ, , or heavy chain-specific Abs
(Southern Biotechnology Associates) conjugated to HRP. For detection of
IgE, a biotinylated anti- mAb was used followed by
anti-biotin-HRP (Vector Laboratories, Burlingame, CA). The AFC were
visualized by addition of the peroxidase substrate
3-amino-9-ethylcarbazole (Moss, Pasadena, CA) for 1 h at 25°C.
The resulting spots were enumerated with the aid of a dissecting
microscope (SZH Zoom Stereo Microscope System; Olympus, Lake
Success, NY).
In vitro stimulation of T lymphocytes
Splenocytes were stimulated in vitro by anti-CD3 mAb
(145-2C11) cross-linking. To this end, 96-well microtiter plates
(Becton Dickinson) were coated with 5 µg/ml 145-2C11 mAb
(PharMingen). Splenic lymphocytes (100 µl/well) were added at a
density of 5 x 106 cells/ml and incubated
at 37°C in 5% CO2 for 48 h. Culture
supernatants were collected and stored at -70°C until analyzed by
cytokine ELISA.
Cytokine ELISA
Falcon Microtest III plates (Becton Dickinson) were coated
overnight at 4°C with 100 µl of mAbs (PharMingen) specific for
IL-2, IL-4, IL-5, IL-6, IL-10, or IFN- diluted in 0.1 M bicarbonate
buffer (pH 8.2) at an optimal concentration (between 2 and 5 µg/ml),
as previously described (31). The plates were blocked for
1 h with PBS Tween 20 (PBS-Tween) (0.05%) and 1% BSA at room
temperature for 1 h. Serial 2-fold dilutions of supernatants were
added to duplicate wells and incubated overnight at 4°C. The wells
were washed with PBS-Tween and incubated with 100 µl of the
appropriate biotinylated cytokine-specific mAb diluted in PBS-Tween
with 1% BSA for at least 4 h. In the case of anti-IL-10 mAb,
the incubation was performed for 45 min. Following three rinses, wells
were incubated with peroxidase-labeled goat-anti-biotin Ab at 0.5
µg/ml (Vector Laboratories) for 1 h at 25°C and developed with
ABTS substrate (Sigma). Standard curves were generated using murine
rIL-2 (PharMingen), rIL-4 (Endogen, Boston, MA), rIL-5, rIL-6, rIL-10,
rIFN- (Genzyme, Cambridge, MA). The sensitivity of the cytokine
ELISA was 15 pg/ml for IFN-, 5 pg/ml for IL-2, IL-4, or IL-5, 100
pg/ml for IL-6, and 200 pg/ml for IL-10.
Immunofluorescence of tissue sections
The jejunum and ileum were obtained from
TGF-ß1-/- mice and normal littermates for
quantitation of Ig-containing cells as previously described
(27). The small intestine (jejunum and ileum) was opened
longitudinally, and 1-cm sections were mounted on cards (2.5 x
1.0 cm). The tissue was washed thoroughly with cold PBS (4°C) over a
2-h period to remove interstitial tissue-associated Ig. The tissue was
then immediately fixed in 5% glacial acetic acid in 95% ethanol at
-20°C, embedded in paraffin, and cut into 4-µm thick sections. The
tissues were mounted on slides and stained for IgM with FITC-conjugated
goat F(ab')2 anti-mouse µ (Southern
Biotechnology Associates), for IgA with biotinylated
F(ab')2 anti-mouse (Southern
Biotechnology Associates), followed by
avidin-7-amino-4-methyl-coumarin-3-acetic acid (Jackson ImmunoResearch,
West Grove, PA), and for IgG with RITC-labeled goat
F(ab')2 anti-mouse (Southern
Biotechnology Associates). Fluorescent images were visualized with a
Leica/Leitz DMRB microscope equipped with appropriate filter cubes
(Chromtechnology, Battleboro, VT), as previously described
(32). Images were collected with a C5810 series digital
color camera (Hamamatsu Photonic System, Bridgewater, NJ) and processed
with Adobe Photo Shop and IP LAB Spectrum software (Signal Analytics
Software, Vienna, VA).
Statistics
The results are expressed as the mean ± 1 SE. Statistical
significance (p 
0.05) was determined by
Student’s t test, employing the computer program Statview.
For statistical analysis, cytokine levels below the detection limit
were recorded as one-half the detection limit (e.g., IL-2 = 2.5
pg/ml).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the effects of TGF-ß1 on the mucosal immune system, in
vivo analyses were done with TGF-ß1-/- and
TGF-ß1+/+ mice between 3.5 and 5.5 wks of age
due to their abbreviated life span. Higher levels of serum IgM and IgG
were noted in TGF-ß1-/- mice than in
wild-type TGF-ß1+/+ mice. The serum Ig levels
in TGF-ß1-/- mice increased 3.0-fold and
1.4-fold for IgG and IgM, respectively, while IgA levels decreased
by 50% (Fig. 1
A). The IgG
subclasses, which reflect important immune parameters, such as Th1 and
Th2, and the ability to activate complement, were assessed in these two
groups of mice. The IgG1 levels were the least affected, with only a
2.3-fold increase in TGF-ß1-/- mice, while
the IgG2b subclass was elevated 11-fold (Fig. 1B). Serum
IgG2a and IgG3 levels increased 7- to 8-fold on average; however,
these ratios varied between different groups of
TGF-ß1-/- mice, as opposed to IgG1 and IgG2b
subclasses, where ratios remained constant. All IgG subclasses were
significantly elevated in TGF-ß1-/- mice
(p values varied between 0.03 and 0.0005). In
TGF-ß1-/- mice younger than 3.5 wks
(n = 4), Ig levels were comparable to wild-type
controls, suggesting abberrant shifts in the immune system at 3.5
wks of age (data not shown).
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The effects of TGF-ß1 on mucosal immunity were assessed in fecal
extracts, saliva, nasal, and vaginal washes, and BAL. The IgA levels in
saliva were 3.1-fold lower, while IgM and IgG levels increased 5.3- and
2.0-fold, respectively (Fig. 2). All
mucosal secretions of TGF-ß1-/- mice,
including nasal washes and BAL, displayed higher levels of IgM and IgG
and lower levels of IgA (Fig. 2). In our studies, rather dramatic
differences in Ig isotypes were noted in the gastrointestinal (GI)
tract. For example, fecal extracts of
TGF-ß1-/- mice contained 9.0-fold less IgA
and 6.9-fold more IgG than did those of control mice (Fig. 3A). The decreased production
of IgA in the intestinal tract was further confirmed by
immunohistochemical staining of the ileum (Fig. 4, A and B) and PP
(Fig. 4, C and D) with anti-, -, and
-µ heavy chain-specific Abs, where a dramatic decrease in
IgA-positive B cells and increases in IgG and IgM-positive B cells were
observed.
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Significantly elevated serum IgE levels (p
= 0.004) were observed in 5-wk-old TGF-ß1-/-
mice, i.e., a 31-fold increase over age-matched littermates (Fig. 5). In addition, elevated numbers of IgE
AFC were observed in the spleen, lungs, and MLN in 5.5-wk-old
TGF-ß1-/- mice. The highest numbers of IgE
AFC were detected in the MLN and the draining lymph nodes of the lungs.
Elevated IgE levels are normally associated with high IL-4 levels,
which are, in turn, associated with B cell switches to IgE.
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The observations of altered Ig levels were corroborated by
enumerating Ig-secreting cells in various tissues, including spleen,
MLN, lungs, cervical lymph nodes (Fig. 6), PP, and intestinal LP (Fig. 3
, B and C). Numbers of IgM and IgG AFC were higher
in all tissues analyzed from TGF-ß1-/- mice
than those of their normal littermates, with the exception of PP, for
which IgM AFC were lower in TGF-ß1-/- mice,
based on ELISPOT assay. On the other hand, all tissues analyzed
displayed lower numbers of IgA AFC in
TGF-ß1-/- than in
TGF-ß1+/+ mice, confirming the notion that
TGF-ß1 plays an important role in IgA responses in vivo.
Approximately 5-fold fewer IgA AFC were observed in spleen- and
lung-derived lymphocytes in TGF-ß1-/- mice.
The increases in IgG and IgM AFC were most pronounced in MLN, which
showed 100- and 49-fold increases, respectively, and were lowest in
spleen, with 1.8- and 1.7-fold increases, respectively (Fig. 6). Thus,
the draining lymph nodes of the lung, an organ severely inflamed in the
absence of TGF-ß1, were characterized by high IgM and IgG AFC and
much lower IgA AFC.
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B), a pattern
noted in other lymphoid tissues as well. No PP were detected in
TGF-ß1-/- mice that were <3.5 wks of age
(data not shown). T cell cytokine production
T lymphocytes derived from TGF-ß1-/-
mice produced more Th2-type cytokines after activation by CD3
cross-linking than did those derived from littermate controls (Fig. 7). IL-4, IL-5, IL-6, and IL-10 levels
were elevated 13-, 16-, 5-, and 2-fold, respectively, over those
observed in TGF-ß1+/+ mice. Comparable levels
of the Th1-type cytokine IFN- (<2-fold increase) and 2-fold
decreases in IL-2 were noted in anti-CD3-stimulated T cells from
TGF-ß1-/- mice. Due to variations in absolute
cytokine levels between different experiments, only IL-6
(p = 0.005) and IL-4 (p
= 0.05) levels were significantly elevated in anti-CD3-stimulated
TGF-ß1-/- lymphocyte cultures. Though
elevated, the level of the Th1 cytokine IFN- in
TGF-ß1-/- mice did not differ significantly
from that observed in TGF-ß1+/+ mice. It is
noteworthy that IL-2 is the only cytokine that actually decreased in
cultures of TGF-ß1-/- mice. Such data
indicate a preferential increase in TGF-ß1-/-
mice of Th2 cytokines, which are associated with humoral immune
responses and are consistent with elevated Ig production in vivo in
TGF-ß1-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TGF-ß1-/- mice had fewer PP than did control mice, indicating impaired development of this mucosal inductive tissue. Variables such as gut microflora and enteral nutrition affect the gut-associated lymphoreticular tissue (GALT) as well as the numbers of PP present in the GI tract (34, 35). It could be hypothesized that these TGF-ß1-/- mice eat considerably less due to oral and intestinal inflammation. Interestingly, enteral feeding also enhanced immunity in the respiratory tract (36), indicating that a lack of GALT development also influences the immune responses observed in the respiratory tract.
It is also of interest that, despite higher Ag exposure in the GI tract, the function of the lungs was more affected by the absence of TGF-ß1 (1). The lungs, like the GI tract, are protected by secretory-IgA Abs. Reasons why IgA deficiency might have a more pronounced effect on the lung include: 1) the oxygen exchange is much more critical than food uptake and even a brief disruption of this function due to inflammation would be detrimental; and 2) the intestinal tract of the TGF-ß1-/- mice may also be protected by maternal IgA and other antimicrobial and antiinflammatory components present in breast milk. The GI tract would benefit more than other systems, such as the lungs, from such protection because of the presence of breast milk in the intestinal lumen (37, 38). It has been well documented that TGF-ß1-/--deficient mice born to heterozygous females contain TGF-ß1 in many of their tissues (38). Placental transfer of TGF-ß1 has been shown to occur before birth as well as transfer through breast milk into the GI tract after birth (38).
B cells from TGF-ß1-/- mice secrete large
amounts of IgG and IgM, and these Igs are markedly increased in serum
and external secretions. In addition, elevated numbers of IgG and IgM
AFC occur in spleen and mucosa-associated lymph nodes, confirming the
notion of elevated Ig production. One explanation for this increased
IgM and IgG production would be enhanced B cell proliferation in the
absence of TGF-ß1, as indicated by in vitro studies (9).
TGF-ß1 inhibits B cell proliferation by halting
G1 to S transition, seemingly without affecting B
cell activation (9). Thus, the absence of TGF-ß1 could
allow for extended proliferation of B cells after activation and
consequently would lead to higher levels of Ab and Ab-secreting B
cells, as we observed in TGF-ß1-/- mice. It
is interesting that IgM and IgG, including all four IgG subclasses, and
IgE were elevated in TGF-ß1-/- mice. In this
regard, it has been suggested that IgA deficiency in children
predisposes them to inflammatory myopathy and intestinal abnormalities,
such as blunted villi and interstitial inflammation (39),
the same symptoms reported in TGF-ß1-/- mice.
In humans, IgA functions to: 1) down-regulate the respiratory burst in
monocytes and granulocytes (40); 2) inhibit production of
inflammatory cytokines, such as IL-1ß, IL-6, and TNF-
(41); and 3) induce IL-1 receptor antagonist
(41), presumably by signaling through the Fc receptor.
In addition, IgA deficiency may shift humoral immune responses to food
proteins or bacteria-derived Ags to the IgG isotype. Although the
increased IgG and IgM Ab levels may prevent bacterial invasion and aid
in mucosal defense, they can also contribute to local inflammation and
tissue injury (42). Following elevation of
complement-activating IgG subclasses in
TGF-ß1-/- mice, pronounced inflammation and
pathology would be expected. The fact that IgA-deficient patients
display mucosal inflammation with locally elevated IgG production
supports this notion (43).
Previous work has shown that TGF-ß1 is a switch factor for murine IgG2b, which enhances IgG2b secretion in LPS-activated splenic B cells (19). Serum IgG2b levels in TGF-ß1-/- mice were consistently elevated above other serum IgG subclasses when compared with wild-type littermates. Although appearing contradictory, there may be multiple additional regulatory components operative in the TGF-ß1-/- mice that could influence IgG2b secretion when compared with in vitro studies.
The high serum IgE levels detected in
TGF-ß1-/- mice also indicate a preferred Th2
cytokine environment. In addition to the somewhat elevated IL-4
production in the absence of TGF-ß1, higher IgE levels noted in serum
of TGF-ß1-/- could also be explained by the
ability of TGF-ß1 to inhibit germline RNA expression, as reported
in the 1.29µ B cell line and human B cells (44, 45).
Thus, the increase in IgE may be a direct effect of increased germline
expression and would explain the low increase of serum IgG1 relative to
other IgG subclasses.
Given the elevated number of AFCs in the lymphoid organs and effector sites, elevated Ig levels observed in serum and mucosal secretions would seem to be the consequence of increased production by B cells rather than of decreased catabolism of Igs as reported in ß2-microglobulin knockout mice (46). Because Th2-type cytokines are associated with humoral immunity (11), it could be hypothesized that elevated Th2 cytokine levels may decrease the requirement of TGF-ß1 needed for IgA Ab production in vivo in TGF-ß1-/--deficient mice and would result in detectable IgA levels in serum and mucosal secretions. These Th2-type cytokines may operate in conjunction with the TGF-ß2 or -ß3 isoform to enable B cell switching to IgA in TGF-ß1-/- mice, although no formal proof exists for such a mechanism.
Upon anti-CD3 mAb stimulation of T cells, both Th1- and Th2-type
cytokines were produced, although only the Th2 cytokines were elevated
over those observed with wild-type littermates. These findings are
consistent with those of others who have demonstrated that in vitro
stimulation of CD4+ T cell precursors in the
presence of IL-2 and TGF-ß induced Th0 cells to develop into the
Th1-type (25). This response was characterized by
induction of IFN- expression and lack of IL-4 and IL-5 synthesis.
Thus, the presence of TGF-ß1 favors Th1 cell induction and indicates
that the absence of TGF-ß1 would generate a Th2-type of immune
response. Indeed this was the prevalent cytokine profile detected in
anti-CD3 mAb-stimulated,
TGF-ß1-/--derived, splenic T cells.
Furthermore, others have noted that TGF-ß1 inhibits Th2 cell
development of human or murine CD4+ T cells
(25, 47, 48). Treatment with anti-TGF-ß1 supported
Th2 cell induction (47), and this is consistent with a
preferential Th2-type environment in the absence of TGF-ß, as
reported in our study. In contrast, others have reported that TGF-ß1
down-regulates Th1-type responses in vitro (49, 50).
Others have reported down-regulation of both IL-4 and IFN-
production in the presence of TGF-ß1 and have suggested IL-2/IL-2
receptor signaling as a possible target for TGF-ß1-mediated
inhibition of human T cells (51). Consistent with a role
of IL-2 in T cell regulation in TGF-ß1-/-
mice is the decreased secretion of IL-2 by anti-CD3 mAb-activated T
cells in our study, or by Con A-stimulated T cells in a previous study
(52).
In summary, TGF-ß1-deficient mice display highly altered B cell immunity when compared with littermate controls. This change was characterized by a shift from IgA to IgG, IgM, and IgE production in both the systemic and mucosal immune compartments and reflected uncontrolled B cell activation. The lack of mucosal immunity in the GI tract, as manifested by decreased IgA AFC at mucosal surfaces, low IgA Ab levels in mucosal secretions, and lack of GALT development, could contribute to an inflammatory mucosal environment that ultimately contributes to the premature death of these mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frederik W. van Ginkel, Department of Microbiology, Immunobiology Vaccine Center, University of Alabama, BBRB Room 775, 845 19th Street South, Birmingham, AL 35294-2170. E-mail address:
3 Abbreviations used in this paper: MLN, mediastinal lymph nodes; LP, lamina propria; PP, Peyer’s patches; GALT, gut-associated lymphoreticular tissue; AFC, Ab-forming cell; BAL, bronchial alveolar lavage; GI, gastrointestinal; ELISPOT, enzyme-linked immunospot; ABTS, 2,2'-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium.
Received for publication February 24, 1999. Accepted for publication June 8, 1999.
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  G.-Y. Seo, J. Youn, and P.-H. Kim IL-21 ensures TGF-{beta}1-induced IgA isotype expression in mouse Peyer's patches J. Leukoc. Biol., May 1, 2009; 85(5): 744 - 750. [Abstract] [Full Text] [PDF]
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P. Bergqvist, E. Gardby, A. Stensson, M. Bemark, and N. Y. Lycke Gut IgA Class Switch Recombination in the Absence of CD40 Does Not Occur in the Lamina Propria and Is Independent of Germinal Centers J. Immunol., December 1, 2006; 177(11): 7772 - 7783. [Abstract] [Full Text] [PDF]
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D. W. Smith and C. Nagler-Anderson Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa J. Immunol., April 1, 2005; 174(7): 3851 - 3857. [Abstract] [Full Text] [PDF]
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S. Borsutzky, B. B. Cazac, J. Roes, and C. A. Guzman TGF-{beta} Receptor Signaling Is Critical for Mucosal IgA Responses J. Immunol., September 1, 2004; 173(5): 3305 - 3309. [Abstract] [Full Text] [PDF]
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 Y. Nambu, M. Sugai, H. Gonda, C.-G. Lee, T. Katakai, Y. Agata, Y. Yokota, and A. Shimizu Transcription-Coupled Events Associating with Immunoglobulin Switch Region Chromatin Science, December 19, 2003; 302(5653): 2137 - 2140. [Abstract] [Full Text] [PDF]
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 I R Korponay-Szabo, I Dahlbom, K Laurila, S Koskinen, N Woolley, J Partanen, J B Kovacs, M Maki, and T Hansson Elevation of IgG antibodies against tissue transglutaminase as a diagnostic tool for coeliac disease in selective IgA deficiency Gut, November 1, 2003; 52(11): 1567 - 1571. [Abstract] [Full Text] [PDF]
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 M. Y. Sangster, J. M. Riberdy, M. Gonzalez, D. J. Topham, N. Baumgarth, and P. C. Doherty An Early CD4+ T Cell-dependent Immunoglobulin A Response to Influenza Infection in the Absence of Key Cognate T-B Interactions J. Exp. Med., October 6, 2003; 198(7): 1011 - 1021. [Abstract] [Full Text] [PDF]
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A. Sato, M. Hashiguchi, E. Toda, A. Iwasaki, S. Hachimura, and S. Kaminogawa CD11b+ Peyer's Patch Dendritic Cells Secrete IL-6 and Induce IgA Secretion from Naive B Cells J. Immunol., October 1, 2003; 171(7): 3684 - 3690. [Abstract] [Full Text] [PDF]
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 C. Schramm, M. Protschka, H. H. Kohler, J. Podlech, M. J. Reddehase, P. Schirmacher, P. R. Galle, A. W. Lohse, and M. Blessing Impairment of TGF-beta signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice Am J Physiol Gastrointest Liver Physiol, March 1, 2003; 284(3): G525 - G535. [Abstract] [Full Text] [PDF]
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S. Dennler, M.-J. Goumans, and P. ten Dijke Transforming growth factor {beta} signal transduction J. Leukoc. Biol., May 1, 2002; 71(5): 731 - 740. [Abstract] [Full Text] [PDF]
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M. Shimoda, T. Nakamura, Y. Takahashi, H. Asanuma, S.-i. Tamura, T. Kurata, T. Mizuochi, N. Azuma, C. Kanno, and T. Takemori Isotype-specific Selection of High Affinity Memory B Cells in Nasal-associated Lymphoid Tissue J. Exp. Med., December 3, 2001; 194(11): 1597 - 1608. [Abstract] [Full Text] [PDF]
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 C. Pilette, Y. Ouadrhiri, V. Godding, J-P. Vaerman, and Y. Sibille Lung mucosal immunity: immunoglobulin-A revisited Eur. Respir. J., September 1, 2001; 18(3): 571 - 588. [Abstract] [Full Text] [PDF]
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 M.-J. Shi, S.-R. Park, P.-H. Kim, and J. Stavnezer Roles of Ets proteins, NF-{{kappa}}B and nocodazole in regulating induction of transcription of mouse germline Ig {{alpha}} RNA by transforming growth factor-{beta}1 Int. Immunol., June 1, 2001; 13(6): 733 - 746. [Abstract] [Full Text] [PDF]
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F. W. van Ginkel, R. J. Jackson, Y. Yuki, and J. R. McGhee Cutting Edge: The Mucosal Adjuvant Cholera Toxin Redirects Vaccine Proteins into Olfactory Tissues J. Immunol., November 1, 2000; 165(9): 4778 - 4782. [Abstract] [Full Text] [PDF]
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 S. Kodama, S. Suenaga, T. Hirano, M. Suzuki, and G. Mogi Induction of Specific Immunoglobulin A and Th2 Immune Responses to P6 Outer Membrane Protein of Nontypeable Haemophilus influenzae in Middle Ear Mucosa by Intranasal Immunization Infect. Immun., April 1, 2000; 68(4): 2294 - 2300. [Abstract] [Full Text] [PDF]
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