| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Increase Steady State Levels of Polymeric Ig Receptor mRNA in Human Airway and Intestinal Epithelial Cells1
Infectious Diseases Research Laboratory, Veterans Affairs Medical Center, and Department of Internal Medicine, University of Iowa, Iowa City, IA 52242
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Using an RNase protection assay, we
found that IL-4 and IFN- increase steady state levels of pIgR mRNA
in both human intestinal (HT29) and airway (Calu-3) epithelial cells.
Time course studies in HT29 clone 19A cells showed that with each
cytokine alone and with both together: 1) there was a significant lag
before mRNA levels increased; 2) maximal levels were not reached until
48–72 h after the addition of cytokines; 3) mRNA levels remained
elevated in the continued presence of cytokines; and 4) addition of
actinomycin D or removal of cytokines led to decreases in mRNA levels
with a half-life of 
20–28 h. Cytokine-dependent increases in steady
state levels of pIgR mRNA were inhibited by cycloheximide and by
protein tyrosine kinase inhibitors but not by inhibitors of protein
kinase C or cAMP-dependent protein kinase A. Both IFN- and IL-4
increased expression of the inducible transcription factor IFN
regulatory factor-1 (IRF-1), but levels of IRF-1 only weakly correlated
with levels of pIgR mRNA, suggesting that additional transcription
factors are required. These studies provide additional insights into
the mechanisms by which cytokines regulate expression of the pIgR, a
central player in mucosal immunity. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Studies indicate that the pIgR can transport both dimeric and tetrameric IgA, as well as immune complexes that contain pIgA (8). Thus, the receptor may play a dual role: providing mucosal pIgA as a defense against lumenal pathogens, as well as clearing pIgA-containing immune complexes from the submucosal space.
Several immunomodulatory factors increase release of SC (i.e.,
expression of the pIgR) by the human intestinal epithelial cell line
HT29. These factors include TGF-ß (9), TNF-
(10, 11), IFN-
(12, 13, 14), and IL-4 (13, 14). Which of these factors is present at a
given site likely depends upon both the population of cells at that
site and the physiological state (healthy vs diseased) of the mucosal
tissue (15).
We and others demonstrate that IL-4 and IFN- individually increase
cell-associated SC and intact receptor. Furthermore, IFN- increases
receptor expression by increasing steady state levels of pIgR mRNA
(16). This IFN-dependent increase in expression is accompanied by
activation of the transcription factor IFN regulatory factor-1 (IRF-1)
(17). Moreover, in high-affinity pIgA binding studies, IL-4 and IFN-
are synergistic (14). This synergy is due in large part to a
synergistic increase in cell-associated pIgR (13).
Because the pIgR plays a central role in mucosal immunity, our
laboratory is interested in how IL-4 and IFN- regulate pIgR
expression. To address this question, we designed studies to
characterize the effect of these cytokines on pIgR mRNA expression in
human epithelial cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human recombinant IL-4 and IFN- were obtained from R&D
Systems (Minneapolis, MN). Actinomycin D-mannitol was purchased from
Sigma (St. Louis, MO). The kinase inhibitors genistein, KT5720,
staurosporine, and bisindolylmaleimide were obtained from Calbiochem
(La Jolla, CA), and the inhibitor tyrphostin 23 was purchased from
Biomol Research Laboratories (Plymouth Meeting, PA). Abs against IRF-1
and c-Jun were obtained from Santa Cruz Biotechnology (Santa Cruz, CA)
and Upstate Biotechnology (Lake Placid, NY), respectively.
Cell culture
HT29 clone 19A (18, 19), HT29 HTB38 (ATCC HTB38), and Calu-3 (ATCC 55-HTB) cells are human epithelial cell lines isolated from adenocarcinomas of the colon and lung, respectively. All cell lines were cultured in DMEM:Ham’s F12 (1:1) supplemented with 10% FBS, 2 mM glutamine, and 500 U/ml each of penicillin and streptomycin. Cells were subcultured at a 1:10 (HT29) or a 1:4 (Calu-3) dilution. Fresh medium was added every 2–3 days and experiments were done when cultures were 70–90% confluent. Cytokine-dependent expression of the pIgR was reduced >50% if cytokines were added in fresh serum-containing medium or if supplemental serum was added. The basis for this inhibition is currently unknown. Thus, cytokines were routinely added directly to the serum-containing growth medium 1–2 days after it was placed on the cultures.
RNase protection assay (RPA)
An insert for generating probe was made using an RT-PCR procedure available in the SuperScript II kit (Life Technologies, Gaithersburg, MD). The procedure was performed according to the manufacturer’s instructions, using the following primers: 5'-CCC AGG ACC CTG GCT GAC CTC CAG G-3' and 5'-GGC TAC GTC TCC AGC AAA TAT GCA G-3'. The insert was gel purified and subcloned into the pCRII plasmid (Invitrogen, Carlsbad, CA).
To make biotinylated RNA probe, the plasmid was linearized by treating with BamHI. Transcription was then performed according to the manufacturer’s instructions using the Ambion (Austin, TX) BIOTINscript kit. Briefly, the linearized plasmid was combined with buffer, ribonuclease inhibitor, T7 polymerase, and a nucleotide mixture containing biotin-14-CTP. Samples were then incubated at 37°C for 2 h, and subsequently treated with DNase I. The probe was gel purified by running the final mixture on a 5% polyacrylamide/8 M urea gel (1000 V, 1 h). Biotinylated m.w. markers and probe for 28S rRNA were similarly made using templates purchased from Ambion.
Total cellular RNA was prepared by scraping the cells into TRI REAGENT (Molecular Research Center, Cincinnati, OH) and incubating for 5 min at room temperature. The samples were extracted with chloroform, and the upper aqueous phase was transferred to a fresh tube. The RNA was precipitated by adding isopropanol and centrifuging at 20,000 x g for 30 min at 4°C. The pellet was washed once with 75% ethanol and dissolved in diethylpyrocarbonate-treated water.
The RPA was performed according to the manufacturer’s directions,
using the Ambion HybSpeed RPA kit. Briefly, 20–80 µg of cellular RNA
was combined with 2 ng each of pIgR and 28S rRNA biotinylated probe;
the 28S rRNA probe was diluted 1:30 with nonbiotinylated probe. For
controls, 50 µg of yeast RNA was combined with probe and processed
with and without addition of RNase. Ammonium acetate and ethanol were
added to precipitate the RNA. Samples were centrifuged in a microfuge
at 15,000 x g for 15 min and resuspended in HybSpeed
Hybridization buffer (Ambion). Hybridization with the probe was
accomplished by heating the samples at 95°C for 3 min then at 68°C
for 10 min. Following hybridization, unprotected RNA was digested by
adding HybSpeed Digestion buffer and RNase A/T1 mixture provided by the
kit, and incubating at 37°C for 30 min. For RPAs using >40 µg of
total RNA, twice the recommended concentration of RNase A/T1 was used.
The protected RNA was then precipitated and separated on a 5%
polyacrylamide/8 M urea gel (1200 V, 1.5 h), along with
biotinylated m.w. standards. Samples and controls were electroblotted
onto BrightStar-Plus (Ambion) nylon membrane (200 mA, 1 h) and
UV-cross-linked to the membrane. Biotinylated RNA species were detected
according to the manufacturer’s directions, using
streptavidin-alkaline phosphatase and the Ambion CDP-Star assay kit.
The apparent m.w. of the species were as follows: uncut pIgR, 309; cut
pIgR, 194; uncut 28S rRNA, 176; cut 28S rRNA, 111.
Band intensities for the pIgR mRNA were determined from the
autoradiographs using the AlphaImager 2000 Digital Imaging System
(Alpha Innotech, San Leandro, CA). A calibration film was used to
identify band intensities that were within the linear range of the
x-ray film. Control experiments indicated that the band intensities for
pIgR mRNA were roughly linear within the range of total RNA (20–80
µg) used in our studies (Fig. 1
). The
need to use high amounts of total RNA to detect pIgR mRNA resulted in
internal controls (28S rRNA) that were beyond the linear range. For
this reason, we were unable to determine absolute values expressed as
the ratio of the band intensities for pIgR and 28S rRNA. The 28S rRNA
band intensities were used as a visual confirmation of RNA recovery and
gel loading. To combine data, relative ratios of band intensities for
the pIgR mRNA bands within individual experiments were determined. For
direct comparisons, samples were run in parallel under identical
conditions.
|
To determine mRNA stability, cells were first treated with and without the indicated cytokine(s) for 72 h. For studies on the effect of cytokine removal, the cells were then washed, repleted with medium without cytokine(s), and incubated for increasing times before harvesting the RNA. Conversely, to study turnover of pIgR mRNA in the continued presence of cytokines, actinomycin D (1 µg/ml) was added to inhibit further transcription, and the cells were incubated for increasing times up to 24 h. Actinomycin D was cytotoxic at times >24 h and, hence, longer incubation times were not tested. At the end of the chase period, total RNA was isolated from the cells and pIgR mRNA levels were measured using RPA.
Studies with protein biosynthesis and protein kinase inhibitors
For studies with cycloheximide, the inhibitor (2 mg/ml) was added 1 h before the addition of cytokines, and was removed after 5 h of cytokine treatment to avoid cytotoxic effects (16). For studies with protein kinase inhibitors, the indicated concentration of inhibitor was added for 1 h (24 h for tyrphostin 23) before addition of cytokines, and inhibitor was present throughout the subsequent incubations.
Western blot analysis of transcription factors
At the indicated time after the addition of cytokines, cells were washed with PBS, scraped into PBS, transferred to a microfuge tube, and pelleted by centrifuging at 2500 x g for 3 min. The cells were resuspended in PBS, an aliquot was taken to measure total cellular protein using the micro bicinchoninic acid assay (micro BCA; Pierce, Rockford, IL), and 5x SDS gel sample buffer was added to the remainder. Equal amounts of protein (250 µg) were separated on 7.5% SDS polyacrylamide gels, transferred to nitrocellulose, and subjected to Western blot analysis using the indicated Ab. Briefly, nonspecific binding was blocked by incubating the blots for 1 h at room temperature with nonfat dry milk (NFDM, 5% nonfat dry milk in PBS with 0.05% Tween 20). After each step the blots were washed four to five times with wash buffer (10 mM Tris-HCl (pH 7.3), 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20). The blots were sequentially incubated overnight at 4°C with polyclonal antitranscription factor Ab (1 µg/ml in NFDM) and for 1 h at room temperature with HRP-conjugated donkey anti-rabbit IgG (1:10,000 in NFDM; Amersham, Arlington Heights, IL). Ab binding was visualized using the Pierce SuperSignal reagent and autoradiography.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
on steady state levels of pIgR mRNA in
HT29 cells
Previous studies by our laboratory using HT29 cells demonstrate
that IL-4 and IFN- increase cell-associated pIgR and synergize at
the level of pIgR protein (13). While earlier studies show that IFN-
increases pIgR mRNA levels (16), no similar studies have been reported
for IL-4. To determine whether the observed increases in pIgR protein
are due to increased levels of mRNA, we measured steady state pIgR mRNA
levels in control and cytokine-treated cells using a nonradioactive
RPA. Levels of 28S rRNA were used as a measure of variability in sample
recovery and gel loading.
Fig. 2a shows representative
results using HT29 clone 19A cells treated for 48 h with and
without individual cytokines or with both cytokines together. Basal
mRNA levels (Control) for these cells were consistently below the level
of detection by this assay. Both IL-4 and IFN- individually
increased mRNA levels in these cells and appeared to synergize when
present together. Because of limitations in our ability to determine
absolute values for pIgR mRNA, we compared the responses by arbitrarily
defining the pIgR band intensity from IL-4-treated cells as 1.0.
Combined data from ten independent experiments yielded values of
1.6 ± 0.3 and 12 ± 2.7 for cells treated with IFN- and
with both cytokines together, respectively. These data suggest that, in
a side-by-side comparison, IFN- and IL-4 potency are similar and
that the two synergize. These results are consistent with results
measuring pIgR protein (13) and pIgA binding (14). The observed synergy
using maximal concentrations of cytokines suggests that IL-4 and
IFN- increase pIgR expression through different signaling pathways.
|
b), we
found that this cell line appeared to express higher levels of pIgR
mRNA. Basal levels of pIgR mRNA were readily detectable in this cell
line. Additionally, when we compared the pIgR band intensities for
samples from HT29 HTB38 cells treated with IFN- alone and with both
cytokines together with those for cells treated with IL-4 alone (band
intensity defined as 1.0), we found ratios of 1.8 ± 0.37 and
2.1 ± 0.33, respectively (ten independent experiments). These
data suggest that IL-4 and IFN- are somewhat less than additive in
this cell type. Finally, to verify these quantitative differences, we compared results from each cell line treated in parallel under identical conditions. Consistent with our results using individual cell lines, pIgR mRNA levels in clone 19A cells treated with individual cytokines were lower than those in HT29 HTB38 cells using 80 µg of total RNA and were undetectable at RNA concentrations (<40 µg) where signals using HTB38 cells were still observed (data not shown). In contrast, the pIgR mRNA band intensities for cells treated with both cytokines together were similar for the two cell lines in this RNA concentration range (20–80 µg): the ratio of clone 19A to HTB38 was 0.99 ± 0.12 (five independent experiments). This suggests that both cell lines can express similar maximal levels of pIgR mRNA but that individual cytokines are less potent in clone 19A cells. The reason for this difference in potency remains to be determined.
Effect of kinase inhibitors on cytokine-dependent increases in pIgR mRNA
Previous studies by our laboratory demonstrate that
cytokine-dependent increases in cell-associated pIgR protein are
prevented by inhibitors of protein tyrosine kinases (PTKs) but not by
inhibitors of cAMP-dependent protein kinase A (PKA) or protein kinase C
(PKC) (13). Because PTKs are also components of pathways that regulate
gene expression posttranscriptionally (20) and because studies by our
laboratory demonstrate that PTK inhibitors can differentially affect
mRNA and protein levels (21), it was necessary to verify that the
effect of these inhibitors on pIgR protein levels reflected effects on
pIgR mRNA levels. To test this hypothesis, HT29 clone 19A cells were
pretreated with inhibitor as described in Materials and
Methods and then with cytokines in the presence and absence
of inhibitor for an additional 24 h. At the end of the treatment
period, mRNA levels were determined (Fig. 3). The tyrosine kinase inhibitors
genistein and tyrphostin prevented the cytokine-dependent increase in
pIgR mRNA. In contrast, the PKC inhibitor bisindolylmaleimide and the
PKA inhibitor KT5720 had no effect. These data support the hypothesis
that activation of PTKs regulates cytokine-dependent pIgR expression at
the steady state level of pIgR mRNA.
|
We next examined the time dependence of pIgR mRNA induction by
treating cells for increasing times with IL-4, IFN-, or both
together. Because of variability from experiment to experiment, values
were normalized by arbitrarily defining the maximum pIgR band intensity
within each experiment as 100%. Combined data from three independent
experiments (mean ± SEM) for each condition are shown in Fig. 4. Overall, a similar time dependence was
observed, although maximal levels were observed at earlier times with
IFN- alone (48 h) than with IL-4 alone or with both cytokines
together (72 h). As previously reported for IFN- (16), there was a
lag following addition of IL-4 or both cytokines together before mRNA
levels began to rise. In some experiments, bands were barely detectable
at the 12 h time point. Additionally, mRNA levels remained
elevated in the continued presence of cytokines; times greater than
96 h were not tested. This time course mirrors the one that we
observed for pIgR protein levels in these cells (13). A similar lag
before pIgR mRNA levels increased was observed with HT29 HTB38 cells
(data not shown).
|
Because basal levels of pIgR mRNA were at or below our level of
detection, we were unable to determine whether cytokines alter mRNA
stability relative to unstimulated cells. We could, however, determine
whether there were differences in mRNA stability between IL-4- and
IFN--treated cells and whether increases in mRNA stability might
account for the synergy observed in clone 19A cells. To determine the
effect of cytokines on pIgR mRNA stability, clone 19A cells were
treated with cytokines for 72 h to achieve maximal steady state
levels. Following cytokine treatment, two approaches were used. To
determine the effect of cytokine removal, cells to be chased in the
absence of cytokines were washed once with Hanks’ balanced saline
solution and repleted with complete medium. Conversely, to examine mRNA
stability in the continued presence of cytokines, cells were chased in
the presence of 1 µg/ml actinomycin D. To combine data from multiple
experiments, we defined the band intensity for the pIgR mRNA at time
zero in cytokine-treated cells as 100% and expressed the data at
increasing times as % remaining. Results for combined data from three
independent experiments (mean ± SEM) for each condition are shown
in Fig. 5.
|
Effect of cycloheximide on increases in pIgR mRNA
Previous studies show that protein biosynthesis is required for
IFN-dependent induction of pIgR mRNA (16). To determine whether this is
the case for IL-4, we treated cells with cycloheximide as described in
Materials and Methods, then with cytokines for
24 h. Fig. 6 shows that
cycloheximide inhibits the cytokine-dependent increase in pIgR
expression when either cytokine is present alone and when both are
present together. These data indicate that protein biosynthesis is
required for induction of pIgR mRNA by both IL-4 and IFN-.
Similarly, cytokine-dependent increases in pIgR mRNA were inhibited by
cycloheximide in HT29 HTB38 cells (data not shown). A requirement for
protein biosynthesis is consistent with the lag that is observed in
time course studies.
|
Earlier studies report a lag before IFN- stimulates an increase
in pIgR mRNA and that this increase is inhibited by cycloheximide (16).
Our studies show similar characteristics in cells treated with IL-4
alone and in cells treated with IL-4 plus IFN-. One explanation for
these observations is a requirement for biosynthesis (induction) of
transcription factors. Consistent with this hypothesis are the studies
by Piskurich et al. (17) who find that IFN- increases mRNA levels of
the inducible transcription factor IRF-1 and increases its
transcriptional activity. No studies to date have examined the effects
of IL-4 on IRF-1 in any cell type.
To determine whether IL-4 induces IRF-1 in HT29 cells, whether
induction is different in cells treated with both cytokines together
compared with each cytokine alone, and to characterize the time course
for any observed changes, we treated HT29 clone 19A and HTB38 cells
with cytokine(s) for increasing times and subjected whole cell extracts
to Western blot analysis using polyclonal anti-IRF-1. To facilitate
direct comparisons, samples from each cell type were run in parallel
under identical conditions. Representative results from these
experiments are shown in Fig. 7.
|
a, upper panel), we
observed readily detectable basal levels of IRF-1 (0 h). Moreover,
consistent with previous studies, IFN- caused a marked increase in
IRF-1 levels. This increase was appreciable at 1 h, appeared to be
maximal by 8 h, and remained elevated in the continued presence of
cytokine. Times greater than 48 h were not tested. While less
marked, IL-4 also increased IRF-1 levels in these cells with a time
course similar to that of IFN-. In parallel cultures, results with
both cytokines together mirrored those with IFN- alone (Fig. 7b). This observation suggests that the synergy observed
between IFN- and IL-4 in these cells is not due to a marked change
in the time course nor in the magnitude of IRF-1 expression. If
cytokines were removed following induction of IRF-1, levels of the
protein fell within 4 h of cytokine removal and were reduced to
basal levels by 16 h (data not shown).
We speculated that higher levels of pIgR expression by HT29 HTB38 cells
might reflect higher levels of IRF-1. Unexpectedly, we found that when
run in parallel under identical conditions, clone 19A cells expressed
considerably higher levels of IRF-1 (Fig. 7c) than did HTB38
cells. To detect IRF-1 levels in IL-4-treated HTB38 cells, both higher
protein concentrations and longer exposure times were required (Fig. 7c, lower panel). This weak correlation between
IRF-1 protein levels and pIgR mRNA levels suggests to us that while
IRF-1 may contribute to pIgR expression, other factors may play an
equal or greater role. These data also indicate that the lesser effect
of IL-4 and IFN- on pIgR expression in HT29 clone 19A vs HTB38 cells
is not a general characteristic of the cytokine response in these two
cell lines.
Effect of cytokines on the inducible transcription factor c-Jun
While data support a role for IRF-1 in regulating pIgR expression (17), it is not clear whether IRF-1 induction can fully account for the lag observed before pIgR mRNA levels increase and/or for the effect of cycloheximide. For this reason, we continue to direct studies toward identifying the basis that underlies the requirement for protein biosynthesis and the lag that is observed.
Putative binding sites for the inducible transcription factor AP-1 are
found in the 5' flanking sequences of the pIgR gene (22). Whether these
binding sites contribute to cytokine-dependent regulation of pIgR
expression remains to be determined. Because one of the members of the
AP-1 family, c-Jun, shows a delayed pattern of induction in some cell
types (23), we hypothesized that c-Jun induction might contribute to
the kinetics of the pIgR response and to the requirement for protein
biosynthesis. To test this hypothesis, we reprobed our IRF-1 Western
blots with anti-c-Jun Abs (Fig. 7a, lower
panel). Detectable levels of c-Jun were observed in unstimulated
cells (0 h). In contrast to IRF-1, however, neither cytokine alone nor
both together (data not shown) increased levels of c-Jun protein over
the time period of our study. These data suggest that IL-4 and IFN-
do not influence expression of c-Jun in HT29 cells over these time
periods. However, the data do not rule out a role for AP-1 in
regulation of pIgR expression since phosphorylation of AP-1 family
members that are already present could activate AP-1 transcriptional
activity (23).
IL-4 and IFN- increase pIgR mRNA levels in human airway
epithelial cells
Studies of cytokine-dependent regulation of pIgR expression have
focused primarily on intestinal epithelial cells. Since the pIgR likely
plays an important role in mucosal immunity in the airway, we wished to
examine expression of the pIgR in airway epithelial cells. To do this,
we determined the effect of cytokines on expression of pIgR mRNA using
the human airway epithelial cell line Calu-3 (Fig. 8). Previous studies show that IFN-
increases transcytosis of dimeric IgA by these cells (24). Results with
Calu-3 cells were similar to those with HT29 HTB38 cells in that basal
expression was easily detectable and band intensities were similar when
samples from each cell type were run in parallel under identical
conditions. Moreover, like in HT29 cells, cycloheximide blocked the
cytokine-dependent increase in pIgR mRNA in Calu-3 cells (data not
shown). Finally, IRF-1 protein levels in Calu-3 cells were increased by
cytokines and, when run in parallel, were intermediate between HT29
clone 19A and HTB38 cell lines (data not shown). These data suggest
that cytokine-dependent regulation is similar in airway and intestinal
epithelial cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
increase pIgR mRNA expression in Calu-3 cells.
Moreover, a close correlation between pIgR mRNA and protein levels
under all of our experimental conditions suggests that the major
mechanism for regulating pIgR expression is at the level of
transcription and/or mRNA stability.
In addition, our results using HT29 cells indicate that regulation of
pIgR expression by each cytokine alone and by both together share
common features. First, similar time courses of induction were
observed, including a lag before mRNA levels increase. This lag is
consistent with the requirement for protein biosynthesis in each case.
Next, a similar mRNA turnover rate was observed for each cytokine alone
and for both together. These data suggest that neither cytokine has a
unique effect on mRNA stability and that synergy occurs at the level of
transcription rather than at the level of mRNA stability. Third,
increases in pIgR mRNA in response to each cytokine can be blocked by
PTK inhibitors, suggesting a role for PTKs in regulating pIgR
transcription and/or mRNA stability. Finally, each cytokine can
increase IRF-1 protein levels: IRF-1 is implicated in IFN-dependent
regulation of pIgR expression (17). These common characteristics,
however, should not obscure the fact that synergy/additivity suggests
that there are differences between the mechanisms by which IL-4 and
IFN- regulate pIgR expression in these cells. Other evidence for
mechanistic differences are the observations that butyrate (26) and
vitamin A (27) differentially affect the response to each cytokine.
It seems likely that biosynthesis of transcription factors accounts, at
least in part, for the observed lag that precedes the increase in pIgR
mRNA levels and for inhibition by cycloheximide. However, our results
suggest that cytokine-dependent changes in IRF-1 expression may not
fully account for these observations. This includes the observation
that cycloheximide inhibits cytokine-dependent increases in pIgR mRNA
in HT29 HTB38 cells, despite the relatively low levels of IRF-1.
Similarly, induction of the AP-1 family member c-Jun cannot account for
the lag, for the requirement for protein biosynthesis, nor for synergy,
since we saw no change in the expression of c-Jun under the conditions
of our study. Whether induction of other members of the AP-1 family
accounts for the observed lag remains to be determined. In addition to
induction of transcription factors, alternative mechanisms to
explain the lag and the effect of cycloheximide must also be
considered. Potential mechanisms include biosynthesis of secreted
factors that regulate pIgR expression, increased expression of the
receptors for IL-4 and/or IFN-, and biosynthesis of proteins that
regulate transcription, mRNA stability, and/or translation of the pIgR.
PTK inhibitors prevent cytokine-dependent increases in pIgR mRNA. There
are at least two general signaling pathways that could explain this
effect. First, both IL-4 and IFN- activate signaling pathways that
include members of the Janus family of tyrosine kinases (JAKs) (28, 29). JAKs, in turn, phosphorylate and activate members of the STAT
family of transcription factors (30). The JAK-STAT pathway regulates
the expression of numerous immunomodulatory factors (31). While
cytokines can activate overlapping groups of JAK proteins, each
cytokine activates specific STATs. It seems likely that the JAK-STAT
pathway contributes to cytokine-dependent IRF-1 expression (32) and,
thus, indirectly to pIgR expression. Additionally, while there are no
reported STAT binding sites in the pIgR promoter (22), STATs may bind
to sites in the promoter that were not identified based on known
consensus sequences, may bind to other transcription factors that
regulate pIgR expression, and/or may regulate pIgR transcription
indirectly by modulating expression of other regulatory proteins.
A second major group of cellular signaling pathways are the
mitogen-activated protein kinase (MAPK) pathways (33). There are three
distinct MAPK pathways, those for extracellular signal-regulated kinase
(ERK), c-Jun terminal kinase (JNK), and for p38; PTKs are early
intermediates in each of these pathways. These pathways, in turn, have
been shown to activate transcription factors for which there are
putative binding sites in the pIgR promoter, specifically the
transcription factors NF-
B (20) and AP-1 (34). Since transcription
factors, in addition to IRF-1, are likely required for maximal
expression of the pIgR, it seems reasonable to explore a possible role
for NF-B and AP-1 and for signaling pathways that regulate their
activity.
With respect to the physiological relevance of our studies, it should
be noted that there are several reported subpopulations of T cells in
the intestine and that each of these subpopulations exhibits a
different pattern of cytokine expression; intraepithelial lymphocytes
secrete more IFN- than IL-4, while the opposite is true for lamina
propria lymphocytes (15). Additionally, different types of challenge
(bacterial, antigenic, viral, parasitic) can stimulate a different
pattern of cytokine secretion. Finally, there are differences between
the intestine and the airway in T cell subpopulations and responses
(35). Thus, regulation of pIgR expression in vivo likely involves the
coordinate action of numerous immunomodulatory factors, among them IL-4
and IFN-. The relative contribution of specific immunomodulatory
factors to pIgR expression is likely determined by the mucosal tissue
of interest, by the population of resident immune cells at a particular
site, and by the physiological state (healthy vs diseased) of the
tissue.
Understanding the complex regulation of this critically important
protein will require additional studies both in vivo and in vitro. By
examining the molecular mechanisms by which IL-4 and IFN- regulate
pIgR expression using human airway and intestinal epithelial cell
lines, our studies contribute toward our general understanding of
pIg-mediated mucosal immunity.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Gerene M. Denning, Infectious Diseases Research Laboratory, Building 3, Room 139, Veterans Affairs Medical Center, Highway 6 West, Iowa City, IA 52246. E-mail address:
3 Abbreviations used in this paper: pIgA, polymeric IgA; IRF-1, IFN regulatory factor-1; SC, secretory component; PKA, cAMP-dependent protein kinase A; PKC, protein kinase C; PTK, protein tyrosine kinase; RPA, RNase protection assay.
Received for publication June 24, 1998. Accepted for publication February 8, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
up-regulates expression of secretory component, the epithelial receptor for polymeric Ig. J. Immunol. 140:3086.[Abstract]
enhances expression of secretory component, the epithelial receptor for polymeric immunoglobulins. J. Immunol. 138:4303.[Abstract]
synergistically increase total polymeric IgA receptor levels in human intestinal epithelial cells. J. Immunol. 156:4807.[Abstract]
on the expression of polymeric Ig receptor (secretory component) and IgA binding by human epithelial cells. J. Immunol. 145:1740.[Abstract]
induces polymeric immunoglobulin receptor mRNA in human intestinal epithelial cells by a protein synthesis dependent mechanism. Mol. Immunol. 30:413.[Medline]
. Mol. Immunol. 34:75.[Medline]
B activation by MAP kinase cascades. Immunobiology 198:35.[Medline]
. Am. J. Physiol. 272:L951. in a human intestinal epithelial cell line. J. Nutr. 128:1063. chain, and IL-4 induces the phosphorylation of Jak2 tyrosine kinase in human colon carcinoma cells. J. Biol. Chem. 270:30829.This article has been cited by other articles:
|
|
 |
|
  K. Pal, C. S. Kaetzel, K. Brundage, C. A. Cunningham, and C. F. Cuff Regulation of polymeric immunoglobulin receptor expression by reovirus J. Gen. Virol., August 1, 2005; 86(8): 2347 - 2357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Lesur, M. Brisebois, A. Thibodeau, F. Chagnon, D. Lane, and T. Fullop Role of IFN-{gamma} and IL-2 in rat lung epithelial cell migration and apoptosis after oxidant injury Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L4 - L14. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Schjerven, P. Brandtzaeg, and F.-E. Johansen Hepatocyte NF-1 and STAT6 Cooperate with Additional DNA-Binding Factors to Activate Transcription of the Human Polymeric Ig Receptor Gene in Response to IL-4 J. Immunol., June 15, 2003; 170(12): 6048 - 6056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Simmons, N. S. Goncalves, M. Ghaem-Maghami, M. Bajaj-Elliott, S. Clare, B. Neves, G. Frankel, G. Dougan, and T. T. MacDonald Impaired Resistance and Enhanced Pathology During Infection with a Noninvasive, Attaching-Effacing Enteric Bacterial Pathogen, Citrobacter rodentium, in Mice Lacking IL-12 or IFN-{gamma} J. Immunol., February 15, 2002; 168(4): 1804 - 1812. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. V. Galietta, P. Pagesy, C. Folli, E. Caci, L. Romio, B. Costes, E. Nicolis, G. Cabrini, M. Goossens, R. Ravazzolo, et al. IL-4 Is a Potent Modulator of Ion Transport in the Human Bronchial Epithelium In Vitro J. Immunol., January 15, 2002; 168(2): 839 - 845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
H. Schjerven, P. Brandtzaeg, and F.-E. Johansen Mechanism of IL-4-Mediated Up-Regulation of the Polymeric Ig Receptor: Role of STAT6 in Cell Type-Specific Delayed Transcriptional Response J. Immunol., October 1, 2000; 165(7): 3898 - 3906. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
