(Received for publication, November 16, 1995; and in revised form, January 19, 1996)
From the
Intestinal epithelia are in intimate contact with subepithelial
and intraepithelial lymphocytes. When stimulated, mucosal lymphocytes
generate cytokines that act locally and influence functional aspects of
many cell types. We have previously defined functional epithelial
receptors for interferon-, interleukin (IL)-4, and a recently
described IL-4-like cytokine IL-13. In this study, we examine the ion
transport properties of T84 cells, a crypt-like epithelial cell line,
following exposure to IL-4 and IL-13. Basolateral exposure of
epithelial monolayers to both IL-4 and IL-13 attenuated epithelial
barrier function and increased paracellular flux of a dextran marker by
greater than 65% in a dose- and time-dependent fashion. Stimulated
Cl
secretion, as measured by epithelial short circuit
current, however, was diminished only by IL-4 and not IL-13,
demonstrating cytokine specificity in this epithelial function.
Decreased Cl
secretion following IL-4 exposure was
associated with diminished Cl
channel activity and
IL-4 pretreatment of epithelia decreased expression of the cystic
fibrosis transmembrane regulator. Finally, stimulated fluid transport
across cultured epithelia was diminished following exposure to IL-4,
but not IL-13. These results indicate that while post-receptor
signaling events induced by IL-13 and IL-4 may be similar, end point
function is cytokine-specific.
Epithelial cells of the intestine are uniquely positioned to
serve as a direct line of communication between the immune system and
the external environment. Intestinal epithelial surfaces are
continuously exposed on the luminal side to foreign antigens and a
myriad of microorganisms, while at the same time, intimately associated
with the immune system via subepithelial lymphoid tissue. In the basal
state, and especially during disease flares, lymphocyte-derived
cytokines are readily detectable in the mucosa(1) . For most
cytokines, target cell function is pleiotropic, and individual
cytokines may act singly, additively, antagonistically, or
synergistically(2) . Previous studies have demonstrated that
intestinal epithelia bear functional receptors for a number of diverse
cytokines, including but not limited to
IFN-(
)(3, 4, 5) ,
IL-2(6) , IL-4(7) , IL-13 (8) , tumor necrosis
factor-
(9) , transforming growth
factor-
(10, 11) , and hepatocyte growth
factor(12) .
Interleukin-13 is a recently described cytokine
produced by activated T lymphocytes(13) . IL-13 and IL-4 share
a number of biological responses when exposed to monocytes,
macrophages, and B cells(14, 15) . We have recently
demonstrated that IL-13, like IL-4, increases paracellular permeability
of cultured intestinal epithelial monolayers(8) . In addition,
human intestinal epithelial cells posess IL-4 receptors(16) ,
and while IL-13 does not bind to the IL-4 receptor, others have
proposed overlapping signal transduction
pathways(14, 15) . The genes for IL-13 and IL-4 are
closely linked in both humans and mice, and these cytokines are
approximately 30% homologous (17) (approximately the same
degree of homology as IL-1 and IL-
(18) ).
In this
study, we demonstrate strict cytokine specificity of IL-4 and IL-13
actions on epithelial ion transport. While both IL-13 and IL-4
specifically diminish epithelial barrier function, only IL-4 attenuates
epithelial electrogenic Cl secretion (the transport
event underlying hydration of mucosal surfaces), indicating cytokine
specificity. Such decreases in Cl
secretion by IL-4
are attributable to diminished Cl
channel activity
and associated with IL-4-mediated down-regulation of CFTR protein
expression. These results suggest that, while the intracellular
signaling events induced by IL-13 and IL-4 reportedly exhibit many
similarities, the resulting end point phenotype of epithelial function
demonstrates clear specificities between these cytokines. Such
observations indicate that differences in IL-13 and IL-4 signal
transduction pathways exist.
T84 epithelial monolayers were exposed to cytokines at indicated concentrations for indicated periods of time. Unless otherwise noted, monolayers were exposed to cytokine on both apical and basolateral surfaces. All incubations included controls consisting of media alone.
Figure 1:
IL-13 and IL-4 attenuate epithelial
barrier function, but only IL-4 modulates chloride secretion. T84
intestinal epithelial monolayers were exposed to human recombinant IL-4 (open circles) or IL-13 (closed circles) at indicated
concentrations for 48 h. Monolayers were exhaustively washed with
Hanks' balanced salt solution and base-line transepithelial
resistance (A) or forskolin-stimulated Cl secretion (short circuit current, Isc, B) were
measured by standard voltage clamp methods. Data are pooled from seven
individual monolayers in each condition, and results are expressed as
the mean ± S.E.
Figure 2:
Only IL-4 diminishes cAMP and
Ca-mediated electrogenic chloride secretion. T84
intestinal epithelial monolayers were exposed to media alone (CTL,
open squares), human recombinant IL-4 (open circles), or
human recombinant IL-13 (closed circles) at 5 ng/ml for 48 h.
Shown here are dose-response curves for epithelial stimulation of
Cl
secretion (measured as short circuit current, Isc) by the cAMP-mediated agonist forskolin (top
panel) or the Ca
-mediated agonist carbachol (bottom panel). Transepithelial resistance and I
were measured by standard methods (see ``Materials and
Methods''). Data are pooled from six to nine individual monolayers
in each condition, and results are expressed as the mean ± S.E.
peak I
.
This IL-4-induced decrease in Cl secretion cannot be competed by excess IL-13. Experiments were
performed in which 10- and 100-fold excess concentrations of IL-13 (50
and 500 ng/ml, respectively, 48 h) were added to IL-4 treated (5 ng/ml,
48 h) monolayers and assayed for forskolin-stimulated (1 µM final concentration) Cl
secretion. Indeed
compared with no cytokine control, addition of IL-4 alone decreased
Cl
secretion by 65 ± 10% (I
of 58
± 8.6 and 20 ± 6.2 for control and IL-4 treated,
respectively, p < 0.01), while no competitive effect was
observed with addition of 50 ng/ml IL-13 (I
of 22 ±
7.1, p = not significant compared with IL-4 alone) or
500 ng/ml IL-13 (I
of 25 ± 6.6, p =
not significant compared with IL-4 alone).
Figure 3:
IL-4 diminishes apical chloride channel
activity. T84 intestinal epithelial monolayers were exposed to media
alone (open circles), human recombinant IL-4 (closed
squares, 5 ng/ml), IL-13 (closed circles, 5 ng/ml), or
IFN- (open squares, 1000 units/ml) for 48 h. Shown here
are sequential 1-min rate constants of
I efflux from T84
cells, as calculated by Venglarik et al.(21) . After
two 1-min washout periods, forskolin (5 µM final
concentration) was added, and
I efflux was assayed in
sequential 1-min periods. In some monolayers (no agonist, closed
triangles) no forskolin was added. Data are pooled from four to
six monolayers each, and results are expressed as the mean ±
S.E.
Figure 4:
IL-4
diminishes immunoprecipitable CFTR protein levels. T84 epithelial
monolayers were exposed to media alone, human recombinant IL-4 (10
ng/ml, 48 h), IL-13 (10 ng/ml), or IFN- (1000 units/ml, 48 h).
Phosphorylated immunoprecipitates were derived from equivalent numbers
of T84 cells using anti-CFTR monoclonal antibody. Protein samples were
separated under reducing conditions on a 7% polyacrylamide SDS gel. A shows resulting blots from immunoprecipitation. B depicts densitometry tracings from immunoprecipitated bands shown
in A. One of two experiments are
represented.
Figure 5:
IL-4 diminishes fluid transport across
cultured T84 epithelial cells. T84 epithelial monolayers were exposed
to media alone, human recombinant IL-4 (10 ng/ml, 48 h), IL-13 (10
ng/ml), or IFN- (1000 units/ml, 48 h). After 24 h, cells were
stimulated (+cAMP, open bars) or not stimulated
(-cAMP, closed bars) with the cAMP agonist forskolin (50
µM final concentration) in the presence of the
phophodiesterase inhibitor IBMX (100 µM final
concentration). The dashed line indicates the initial volume
of fluid on the mucosal surface. Values represent the recovered fluid
volume after 24 h. Data are pooled from six to eight monolayers each,
and results are expressed as the mean ±
S.E..
Soluble factors produced by lymphocytes adjacent to the
epithelium modulate epithelial function. We and others have
demonstrated that a diverse array of cytokines regulate the function of
model intestinal
epithelia(3, 5, 26, 27) . Here we
use polarized model intestinal epithelia, which demonstrate readily
detectable biophysical responses as a means to probe potential
differences in IL-4- and IL-13-mediated signal transduction pathways.
Our results indicate that both IL-4 and IL-13, which share many
biological properties in a variety of systems (14, 15) , attenuate epithelial barrier function; IL-4
selectively down-regulates electrogenic Cl secretion,
indicating distinct cytokine specificity. In addition, these studies
reveal that IL-4 attenuation of Cl
secretion, which
we have observed previously(7) , occurs through regulation of
expression of the apical Cl
efflux channel and
results in diminished epithelial fluid secretion.
IL-13 is a
recently described cytokine that is secreted by activated T lymphocytes
and shares many properties with IL-4(14, 15) . To
date, functional IL-13 receptors have been demonstrated on
monocyte/macrophages, B cells, NK cells, mast cells, fibroblasts, renal
epithelia, and intestinal
epithelia(8, 14, 15, 28, 29) .
In general, mucosal lymphocytes are subdivided into two populations,
intraepithelial lymphocytes and lamina propria lymphocytes, each
defined by their spatial proximity to the epithelia and each with
characteristic surface markers and cytokine production patterns. The
primary source of IL-13 and IL-4 appears to be the lamina propria
lymphocyte population of CD4, Th-0, Th-1, or Th-2
T-cells, although other cell types may also produce these
cytokines(14, 15) . The present study demonstrates
that in a time- and dose-dependent manner, exogenous exposure of
intestinal epithelial monolayers to IL-13 diminishes barrier function
(measured as transepithelial resistance and paracellular flux), an
essential and primary role of mucosal epithelial cells(30) .
The functional IL-13 receptor is localized to the basolateral surface
on T84 cells. This feature of diminished barrier following ligation of
the IL-13 receptor on T84 cells resembles that of IL-4 (7) and
IFN-
(4, 5) . At present, a commonality between
these cytokines and modulation of epithelial barrier has not been
identified.
The structure of the IL-13 receptor has remained
elusive. While IL-13 does not bind to the IL-4 receptor, it has been
shown that IL-13 can inhibit the binding of labeled IL-4 to cells that
are responsive to both cytokines(31) , and thus it has been
proposed that the IL-13 receptor shares a component of the IL-4
receptor important in signal transduction. Of note on this accord,
Reinecker and Podolsky (16) recently demonstrated that
intestinal epithelia possess transcripts for and signal transduction
capacity (tyrosine phosphorylation) for a number of cytokines that
share the IL-2 receptor c chain, including the IL-4 receptor.
Alternatively, the IL-4 and IL-13 receptors may share redundant signal
transduction pathways. We demonstrate that addition of IL-13 in
combination with IL-4 at approximate ED
does not result in
additive or synergistic effects on diminished transepithelial
resistance. Moreover, addition of even 100-fold excess exogenous IL-13
failed to inhibit IL-4-elicited diminution of Cl
secretion (see ``Results''), indicating that it is
unlikely IL-4 and IL-13 bind to the same receptor, or if this is the
case, receptor affinities favor binding of IL-4 to a great extent.
Finally, it is worthwhile to note that we have not examined whether
IL-4 and/or IL-13 might liberate epithelial-derived cytokines that
could act through autocrine mechanisms to regulate such functions as
barrier and ion transport. Further work on IL-13 receptor binding
should reveal important information regarding the mechanism by which
this cytokine regulates epithelial function.
While a number of
studies have demonstrated that IL-4 and IL-13 share many biological
properties, the present studies clearly demonstrate differences between
IL-4 and IL-13 at the level of epithelial electrogenic Cl secretion, the transport event responsible for mucosal
hydration(23) . Here we show that IL-4 selectively
down-regulates Cl
transport. Such down-regulation was
evident in response to both cAMP (forskolin) and Ca
(carbachol)-stimulated Cl
secretion, was
observed at the level of apical Cl
channel activity,
and resulted in decreased transmonolayer fluid movement. Additionally,
diminished levels of the CFTR were associated with IL-4 pre-exposure.
Whether such decreases in CFTR (
65%) result in our observed
decrease in Cl
secretion are not known at the present
time. Another possible explanation for diminished Cl
secretion in response to IL-4 is attenuation of basal
K
efflux pathways. Current methods available to study
K
channel activity lack the sensitivity necessary to
detect decrements that might be induced by IL-4, and thus, we have not
directly examined basal K
channel activity in T84
cells.
Such findings with IL-4 are remarkably similar to previous
investigations using IFN-, including diminished epithelial barrier (3, 4, 5) , decreased electrogenic
Cl
secretion in response to a variety of
agonists(4) , decreased Cl
channel
activity(4) , and decreased CFTR protein(25) . We have
defined such IFN-
-elicited changes as a ``cell surface
phenotype switch,'' in which epithelia exposed to such stimuli
lose many epithelial characteristics and assume other more immune
accessory cell-like features (increased major histocompatibility class
I and II expression, regulated neutrophil trafficking,
etc.)(4) . Such results suggest that a T-cell-derived cytokines
specifically modulate the extent of electrolyte and water loss across
the intestinal barrier, a hypothesis that has been proposed previously
for IFN-
(26) .
In summary, these data show that a human intestinal crypt epithelial model has diverse functional characteristics modulated by IL-4 and IL-13 exposure to the basolateral surface. Furthermore, end point functions elicited by IL-4 and IL-13 on intestinal epithelial biology are distinct. Such observations strengthen the hypothesis that local mucosal cytokine profiles will contribute significantly to the regulation of epithelial barrier function and ion transport characteristics.