Role of STAT6 and Mast Cells in IL-4- and
IL-13-Induced Alterations in Murine
Intestinal Epithelial Cell Function
This information is current as
of June 16, 2017.
Kathleen B. Madden, Lucia Whitman, Carolyn Sullivan,
William C. Gause, Joseph F. Urban, Jr., Ildy M. Katona, Fred
D. Finkelman and Terez Shea-Donohue
J Immunol 2002; 169:4417-4422; ;
doi: 10.4049/jimmunol.169.8.4417
http://www.jimmunol.org/content/169/8/4417
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References
The Journal of Immunology
Role of STAT6 and Mast Cells in IL-4- and IL-13-Induced
Alterations in Murine Intestinal Epithelial Cell Function1
Kathleen B. Madden,* Lucia Whitman,* Carolyn Sullivan,* William C. Gause,†
Joseph F. Urban, Jr.,§ Ildy M. Katona,*‡ Fred D. Finkelman,¶ and Terez Shea-Donohue2‡§
T
he profile of cytokines elicited by an infectious agent orchestrates the host response to the offending pathogen.
Gastrointestinal nematode infections, afflicting nearly 1
billion people worldwide (1, 2), generally invoke a type 2 cytokine
response (3), and are characterized by the production of IL-4, IL-5,
IL-6, IL-9, and IL-13 in the infected host (4 – 6). Up-regulation of
these cytokines results in elevations in serum IgE, eosinophilia,
and hyperplasia of mucosal mast cells (MMC)3 (4) that secrete
soluble mediators, including histamine and PGE2, which affect epithelial cell function (7–11).
IL-4 and IL-13 are of particular interest among the Th2 cytokines elaborated in a type 2 response to nematode infection in that
they share several common biological properties, a functional
overlap that can be explained by the sharing of a common receptor
or receptor component (12). The type 1 IL-4R, which includes
Departments of *Pediatrics, †Microbiology and Immunology, and ‡Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; §Nutritional
Requirements and Function Laboratory, Beltsville Human Nutrition and Research
Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville,
MD 20705; and ¶Division of Immunology, Department of Medicine, University of
Cincinnati, Cincinnati, OH 45267
IL-4R␣-chain and the cytokine receptor common ␥-chain, is expressed predominantly by bone marrow-derived cells and binds
IL-4, but not IL-13. The type 2 IL-4R, containing IL-4R␣-chain
and IL-13R␣-chain, is expressed predominantly by non-bone marrow-derived cells, and binds both IL-4 and IL-13 (13). Binding of
IL-4 (by either receptor) or IL-13 (by the type 2 receptor) initiates
Jak-dependent tyrosine phosphorylation of IL-4R␣-chain and the
transcription factor, STAT6 (14 –16). STAT6 is critical for the
activation or expression of many IL-4-responsive genes, including
class II major histocompatibility molecules, CD23, and the H
chain gene for IgE (17–19).
An integral component of the host response to enteric infection
is to increase the fluid in the intestinal lumen in an effort to facilitate expulsion, limit access to the surface epithelia, and wash
away potential deleterious agents (20). We showed previously that
infection with gastrointestinal nematode Heligmosomoides polygyrus decreased glucose absorption and increased fluid secretion in
response to the mast cell mediators histamine and PGE2, effects
that were mediated by IL-4 (11). In the current studies, we investigated: 1) whether IL-13 has effects on intestinal epithelial cells
similar to those observed with IL-4, and 2) whether the effects of
IL-4 and IL-13 depend on mast cells and/or STAT6 signaling.
Received for publication May 10, 2002. Accepted for publication July 29, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Materials and Methods
Animals
1
This work was supported in part by Uniformed Services University of the Health
Sciences Grant R086CD (to K.B.M.), U.S. Department of Agriculture CRIS 126532000-060 (to J.F.U.), and National Institutes of Health Grants R01AI35987-06 (to
F.D.F.) and R01AI49316-01 (to T.S.-D.). The opinions and assertions contained
herein are the private ones of the authors and are not to be construed as official or
reflecting the views of the Departments of Defense or Agriculture or the Uniformed
Services University of the Health Sciences.
Male and female 8- to 12-wk-old BALB/c mice and mast cell-deficient
W/Wv mice and their wild-type (WT) (⫹/⫹) littermates were purchased
from The Jackson Laboratory (Bar Harbor, ME). STAT6-deficient
(STAT6⫺/⫺) mice on a BALB/c background were bred at Uniformed Services University of the Health Sciences (Bethesda, MD), and were age and
sex matched with controls in all experiments.4
2
Address correspondence and reprint requests to Dr. Terez Shea-Donohue, Nutritional Requirements and Function Laboratory, Beltsville Human Nutrition and Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705. E-mail address: [email protected]
3
Abbreviations used in this paper: MMC, mucosal mast cell; 5-HT, 5-hydroxytryptamine; ACH, acetylcholine; Isc, short circuit current; WT, wild type.
Copyright © 2002 by The American Association of Immunologists, Inc.
4
These studies were conducted in accordance with the principles set forth in the
Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, Health and Human Services Publication (National Institutes of Health) 85-23, revised 1996.
0022-1767/02/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Gastrointestinal nematode infections generally invoke a type 2 cytokine response, characterized by the production of IL-4, IL-5,
IL-9, and IL-13. Among these cytokines, IL-4 and IL-13 exhibit a functional overlap that can be explained by the sharing of a
common receptor or receptor component (IL-4R␣). Binding of IL-4 by either the type 1 or 2 IL-4R, or of IL-13 by the type 2
IL-4R, initiates Jak-dependent tyrosine phosphorylation of the IL-4R␣-chain and the transcription factor, STAT6. In the present
study, we investigated: 1) whether IL-13 has effects on intestinal epithelial cells similar to those observed with IL-4, and 2) whether
the effects of IL-4 and IL-13 depend on STAT6 signaling and/or mast cells. BALB/c, STAT6ⴚ/ⴚ, and mast cell-deficient W/Wv mice
or their ⴙ/ⴙ littermates were treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or with IL-13 for seven
days. Segments of jejunum were mounted in Ussing chambers to measure mucosal permeability; chloride secretion in response to
PGE2, histamine, 5-hydroxytryptamine, or acetylcholine; and Naⴙ-linked glucose absorption. IL-4C and IL-13 increased mucosal
permeability, decreased glucose absorption, and decreased chloride secretion in response to 5-hydroxytryptamine. These effects
were dependent on STAT6 signaling. Responses to PGE2 and histamine, which were dependent on mast cells and STAT6, were
enhanced by IL-4C, but not by IL-13. The effects of IL-4 and IL-13 on intestinal epithelial cell function may play a critical role
in host protection against gastrointestinal nematodes. The Journal of Immunology, 2002, 169: 4417– 4422.
4418
STAT6 DEPENDENCE OF CYTOKINE EFFECTS ON INTESTINAL EPITHELIAL FUNCTION EFFECTS
Cytokines
Results
Mice were given vehicle or IL-4, as described previously (21), using a
long-lasting IL-4 formulation (IL-4C), consisting of 10 ␮g IL-4 (PeproTech, Rocky Hill, NJ) mixed with 50 ␮g 11B11, a neutralizing rat IgG1
anti-mouse IL-4 mAb (Verax, Lebanon, NH). Anti-IL-4 mAb in this formulation is saturated with IL-4 to form complexes that contain a single
mAb molecule and two IL-4 molecules. These complexes dissociate in
vivo, releasing free IL-4 with a t1/2 of ⬃1 day. Because these complexes
contain a single IgG molecule, they neither fix complement nor bind more
avidly than uncomplexed, monomeric IgG to Fc␥Rs. Furthermore, because
the mAb in these complexes blocks the binding of IL-4 to its receptors,
complexed IL-4 can only activate its receptor by dissociating from the
complex.
BALB/c or STAT6⫺/⫺ mice were injected i.v. on days 0, 3, and 6 with
IL-4C in 0.1 ml normal saline or with an equal volume of normal saline
only, and were studied 7 days after the initial injection. Additional groups
of mice were injected i.v. with 10 ␮g rIL-13 (Wyeth Research, Cambridge,
MA) in 0.2 ml saline, or an equal volume of normal saline on days 0 – 6,
and were studied 7 days after the initial injection.
Effects of exogenous IL-4 or IL-13 on epithelial cell resistance
in STAT6⫺/⫺ and WT mice
Ussing chambers
Solutions and drugs
Krebs’ buffer contained (in mM) 4.74 KCl, 2.54 CaCl2, 18.5 NaCl, 1.19
NaH2PO4, 1.19 MgSO4, and 25.0 NaHCO3 on each side. The tissues were
allowed to equilibrate for 15 min in Krebs’ buffer containing 12 mM glucose on the serosal side and 10 mM mannitol on the mucosal side. All
drugs were obtained from Sigma-Aldrich (St. Louis, MO), unless stated
otherwise. Stock solutions of ACH (1 ␮M) were prepared in ultrapure
water and frozen. PGE2 (1 ␮M) was dissolved in 100% ethanol and stored
at ⫺70°C. On the day of the experiment, 5-HT and histamine were dissolved in water, and appropriate dilutions of ACH, PGE2, 5-HT, histamine,
and glucose were made using distilled water.
Effects of exogenous IL-4 or IL-13 on epithelial cell absorption
in STAT6⫺/⫺ and WT mice
To assess the effect of IL-13 or IL-4C on substrate-linked sodium
absorption, glucose was added to the mucosal (luminal) side of the
tissue. IL-4C and IL-13 significantly decreased Isc responses to
glucose in WT, but not in STAT6⫺/⫺ mice (Fig. 2), indicating the
STAT6 dependence of this effect.
Effects of IL-4 and IL-13 on mast cell numbers
MMC were enumerated in STAT6⫺/⫺ and WT mice after 7 days
of treatment with IL-4C or IL-13. Untreated STAT6⫺/⫺ and WT
mice had similar numbers of MMC (Fig. 3). MMC were significantly elevated in both WT and STAT6⫺/⫺ mice treated with IL4C; however, MMC in IL-4-treated STAT6⫺/⫺ mice were significantly lower than those in IL-4-treated WT mice (Fig. 3). IL-13
had no effect on MMC numbers in either WT or STAT6⫺/⫺ mice
(Fig. 3).
Cytokine, mast cell, and STAT6 dependence of PGE2, and
histamine-induced effects on epithelial cell secretion
In contrast to STAT6-dependent effects of IL-4/IL-13 on intestinal
permeability and glucose absorption, IL-4C, but not IL-13, increased Isc responses to PGE2 and histamine in WT mice (Table I).
Because IL-4, but not IL-13, promotes intestinal mastocytosis (10)
(Fig. 3), we determined whether the IL-4-induced increased responsiveness of intestinal epithelium to PGE2 and histamine is
mast cell dependent. This was accomplished by comparing responses to PGE2 and histamine in WT (⫹/⫹) and mast cell-deficient W/Wv mice after 7 days treatment with saline or IL-4C (Table II). Isc responses to PGE2 and histamine were significantly
enhanced only in the IL-4C-treated ⫹/⫹ mice, suggesting that
these prosecretory effects of IL-4 are mast cell dependent.
Histology
Tissue samples were prepared for visualization of MMC (10). Segments of
midjejunum were excised, slit longitudinally, rolled, and placed immediately in Carnoy’s solution and fixed overnight. Tissues were then transferred to 95% ethanol, embedded in paraffin, and sectioned (5 ␮m). Deparaffinized sections were rehydrated and stained with Alcian blue and
Safranin O (Polysciences, Warrington, PA). The numbers of MMC present
in the lamina propria and mucosa were determined in 50 contiguous highpowered fields (magnification ⫻400) in each section by an investigator
who was unaware of the treatment group.
Data analysis
Statistical analysis was performed using one-way ANOVA to compare
basal Isc and resistance. Cumulative dose responses were compared using
multiple ANOVA with post hoc analysis for multiple comparisons. A value
of p ⬍ 0.05 was considered significant.
FIGURE 1. Segments of muscle-free intestinal mucosa were mounted
in Ussing chambers to measure changes in tissue resistance (an index of
epithelial permeability) in WT or STAT6⫺/⫺ mice after 7 days of treatment
with IL-4C (A) or IL-13 (B) (n ⫽ 8 –12 mice/group). Values are means ⫾
SE; ⴱ, p ⬍ 0.05 vs WT control.
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Four 1-cm segments of mucosa were stripped of muscle and mounted in
Ussing chambers that exposed 0.126 cm2 to 10 ml Krebs’ buffer. Agar-salt
bridges and electrodes were used to measure potential difference. Every
50 s, the tissues were short circuited at 1 V (World Precision Instruments
DVC 1000 voltage clamp, Sarasota, FL), and the short circuit current (Isc)
was monitored continuously. In addition, every 50 s, the clamp voltage was
adjusted to 1 V for 10 s to allow calculation of tissue resistance using
Ohm’s law.
Following the 15-min equilibrium period, basal Isc, representing the net
ion flux at baseline, and tissue resistance, a measure of tissue permeability,
were determined. After a second 15-min period, concentration-dependent
changes in Isc were determined for the cumulative addition of histamine,
PGE2, 5-hydroxytryptamine (5-HT), or acetylcholine (ACH) to the serosal
side of the stripped mucosae. After the peak response to the final concentration of each secretagogue was recorded, the Krebs’ buffer on each side
of the chamber was replaced, and the tissue was allowed to equilibrate for
30 min. Upon re-equilibration, concentration-dependent changes in Isc
were measured in response to the cumulative addition of glucose to the
mucosal side. Responses from all tissue segments exposed to glucose from
an individual mouse were averaged to yield a mean response per animal.
The effects of exogenous IL-13 or IL-4C on intestinal epithelial
cell resistance were evaluated in STAT6⫺/⫺ and WT mice. Resistance, a measure of tissue permeability, was similar in untreated
WT and STAT6⫺ mice. In contrast, resistance decreased significantly in WT, but not in STAT6⫺/⫺ mice, treated with IL-4C or
IL-13 (Fig. 1), demonstrating the STAT6 dependence of this
response.
The Journal of Immunology
4419
Table I. Changes in epithelial cell secretion in BALB/c (WT) mice
treated with IL-4C or IL-13 for 7 daysa
WT vehicle
WT IL-4C
WT IL-13
PGE2
HIST
52 ⫾ 10
112 ⫾ 8 *
47 ⫾ 14
48 ⫾ 9
85 ⫾ 8*
44 ⫾ 9
a
Isc values are means ⫾ SE expressed as maximum changes in ␮A/cm2; PGE2,
histamine (HIST) ⫽ 1 ␮M; n ⫽ 3– 4 mice/group.
ⴱ, p ⬍ 0.05 vs WT vehicle.
STAT6⫺/⫺ mice, in vivo IL-4C treatment enhanced in vitro mucosal responses to PGE2 and histamine in WT, but not in
STAT6⫺/⫺ mice (Fig. 4, A and B).
IL-4 and IL-13 inhibit secretory responses to 5-HT through a
STAT6-dependent mechanism
Because IL-4 induction of intestinal mastocytosis and mast cell
degranulation are STAT6 independent (22, 23) (Fig. 3), we expected that the mast cell-dependent mechanism by which IL-4 increases the response to histamine and PGE2 would also be STAT6
independent. Surprisingly, although exogenous PGE2 and histamine had similar effects on intestinal epithelial secretory responses
when added to the serosal side of intestine from untreated WT and
Discussion
Increasing the amount of fluid in the lumen is an integral component of the host response to enteric infection that can facilitate
pathogen expulsion, limit pathogen access to the mucosal surface,
and dilute pathogen-produced toxins (20). We showed previously
that IL-4 mediates a decrease in glucose absorption and an increase
in fluid secretion in mice infected with the gastrointestinal nematode parasite H. polygyrus, suggesting that this effect of IL-4 may
contribute to IL-4-dependent worm expulsion (11). In this study,
we compare the effects of in vivo administration of a long-acting
formulation of IL-4 with the effects of in vivo administration of the
related cytokine, IL-13, on intestinal epithelial cell function, and
determine the contribution of STAT6 signaling to these cytokineinduced alterations in intestinal physiology. Because we were concerned about using doses of IL-4 and IL-13 that were biologically
equivalent in vivo, we selected dosing regimens that had equal
ability to induce the expulsion of the gastrointestinal nematode
parasite, Nippostrongylus brasiliensis, from SCID or recombination-activating gene 2-deficient mice (22, 24, 25) (J. Urban, Jr.,
and F. D. Finkelman, unpublished data).
Table II. Changes in epithelial cell secretion in ⫹/⫹ and mast celldeficient (W/Wv) mice treated with IL-4C for 7 daysa
FIGURE 3. Segments of small intestine were taken from WT or
STAT6⫺/⫺ mice after 7 days of treatment with IL-4C or IL-13 (n ⫽ 8 –12
mice/group). The tissues were fixed in Carnoy’s, sectioned, and stained
with Alcian blue and Safranin O. The numbers of MMC present in the
lamina propria and mucosa were determined in 50 contiguous high-powered fields. Values are means ⫾ SE; ⴱ, p ⬍ 0.05 vs WT control; ␾, p ⬍
0.05 vs WT IL-4C.
⫹/⫹ vehicle
⫹/⫹ IL-4C
W/Wv vehicle
W/Wv IL-4C
PGE2
HIST
61 ⫾ 13
122 ⫾ 33*
34 ⫾ 8
49 ⫾ 25
95 ⫾ 14
187 ⫾ 20*
81 ⫾ 14
72 ⫾ 23
a
Isc values are means ⫾ SE expressed as maximum changes in ␮A/cm2; PGE2 ⫽
0.1 ␮M; histamine (HIST) ⫽ 1 ␮M; n ⫽ 3– 4 mice/group.
ⴱ, p ⬍ 0.05 vs ⫹/⫹ vehicle.
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FIGURE 2. Segments of muscle-free intestinal mucosa were mounted
in Ussing chambers to measure concentration-dependent changes in Isc in
response to glucose in WT or STAT6⫺/⫺ mice after 7 days of treatment
with IL-4C (A) or IL-13 (B) (n ⫽ 8 –12 mice/group). Values are means ⫾
SE; ⴱ, p ⬍ 0.05 vs WT control.
To determine whether the effects of IL-4 on intestinal epithelial
cell responses to mediators of physiologic importance are universally prosecretory, we also evaluated the effects of in vivo IL-4C
treatment on in vitro responses to 5-HT and ACH. In vivo treatment with IL-4 significantly inhibited Isc responses to 5-HT in WT,
but not in STAT6⫺/⫺ mice (Fig. 5A). Effects on responsiveness to
5-HT also differed from those observed in response to PGE2 and
histamine, in that they were induced by in vivo treatment with
IL-13 as well as by in vivo treatment with IL-4 (Fig. 5B). In contrast to these positive and negative effects on responsiveness to
PGE2, histamine, and 5-HT, neither IL-4 (79 ⫾ 11 ␮A/cm2) nor
IL-13 (81 ⫾ 21 ␮A/cm2) had marked effects on in vivo responsiveness to ACH when compared with controls (95 ⫾ 8 ␮A/cm2).
4420
STAT6 DEPENDENCE OF CYTOKINE EFFECTS ON INTESTINAL EPITHELIAL FUNCTION EFFECTS
In the current studies, we show that IL-4 and IL-13 induce similar changes in epithelial cell resistance, absorption, and secretion,
and that these changes are STAT6 dependent. However, we also
demonstrate that IL-4, but not IL-13, increases prosecretory responses to PGE2 and histamine, and that these effects are mast cell
dependent. Consistent with this finding, we show that IL-4, but not
IL-13, induces intestinal mastocytosis. The ability of IL-4, but not
IL-13, to induce intestinal mastocytosis in vivo is consistent with
a recent report by Suzuki et al. (26) that IL-4, but not IL-13, promotes in vitro survival and growth of bone marrow-derived mast
cells and that the IL-4 effect requires ligation of the type 1 IL-4R
(IL-4R␣/␥c), which binds IL-4, but not IL-13. Enhancement of the
secretory response to PGE2 may have a similar explanation, because we do not see this response in mast cell-deficient mice. Further evidence that differences between IL-4 and IL-13 effects in our
model are not explainable by lower relative concentrations of
IL-13 than IL-4 comes from our recent observation that IL-13 has
a considerably greater stimulatory effect than IL-4C, at the same
doses that were used in our manuscript, on intestinal smooth muscle contractility (27).
These findings expand those of our previous report (11) in two
significant ways. First, our observation that IL-4 and IL-13 affect
the intestinal epithelial secretory response to 5-HT is novel, in that
it was not investigated in mice treated with anti-IL-4R␣ mAb.
Second, and more importantly, our studies in the STAT6-deficient
mice demonstrate that this IL-4R-induced pathway is critical for a
number of the effects of IL-4 and/or IL-13 on intestinal mucosal
physiology (Fig. 6). This latter observation was not an obvious
consequence of the IL-4R dependence of these effects, given that
we had shown previously that some important effects of IL-4R
signaling, such as the induction of mucosal mastocytosis and mast
cell degranulation, were STAT6 independent (22).
Our observations confirm and extend some previous reports
(e.g., stimulation of mast cell responses by IL-4, but not IL-13)
(10) (Fig. 3) and appear to conflict, in part, with others (e.g., that
1) IL-4 treatment of the human intestinal cell line T84 in vitro
inhibits Cl⫺ secretion (28, 29), and 2) IL-4/IL-13 induce a STAT6independent, phosphatidylinositol 3-kinase pathway-dependent,
increased transepithelial permeability in vitro in the human T84
intestinal cell line (30)). This apparent conflict may be explained
by the in vivo administration of the cytokines in our study, as well
as by inherent differences in the function of homogeneous intestinal epithelial cell lines vs excised intestinal mucosae with its
intact neural circuitry. However, it is of interest to note that two
FIGURE 6. Schematic depiction of the roles of STAT6, IL-4, and/or
IL-13 in murine intestinal epithelial cell function and MMC hyperplasia.
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FIGURE 4. Segments of muscle-free intestinal mucosa were mounted
in Ussing chambers to measure concentration-dependent changes in Isc in
response to PGE2 (A) or histamine (B) in WT or STAT6⫺/⫺ mice after 7
days of treatment with IL-4C (n ⫽ 8 –12 mice/group). Values are means ⫾
SE; ⴱ, p ⬍ 0.05 vs WT control; ␾, p ⬍ 0.05 vs STAT6⫺/⫺ control.
FIGURE 5. Segments of muscle-free intestinal mucosa were mounted
in Ussing chambers to measure concentration-dependent changes in Isc in
response to 5-HT in WT or STAT6⫺/⫺ mice after 7 days of treatment with
IL-4C (A) or IL-13 (B) (n ⫽ 8 –12 mice/group). Values are means ⫾ SE;
ⴱ, p ⬍ 0.05 vs WT control.
The Journal of Immunology
well as the effects of specific mediators on this process. The role of
inhibitory effects of IL-4/IL-13 on intestinal epithelial function,
and the mechanisms by which inhibitory and stimulatory effects
interact during parasite infection, remain to be determined.
Acknowledgments
We thank Dr. Debra Donaldson (Respiratory Disease, Wyeth Research) for
the generous gift of IL-13.
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1993. IL-13 induces proliferation and differentiation of human B cells activated
by the CD40 ligand. Int. Immunol. 5:657.
19. Punnonen, J., G. Aversa, B. G. Cocks, A. N. McKenzie, S. Menon, G. Zurawski,
R. de Waal Malefyt, and J. de Vries. 1993. Interleukin 13 induces interleukin
4-independent IgG4 and IgE synthesis and CD23 expression by human B cells.
Proc. Natl. Acad. Sci. USA 90:3730.
20. Cooke, H. J. 1998. Enteric tears: chloride secretion and its neural regulation.
News Physiol. Sci. 13:269.
21. Finkelman, F. D., K. B. Madden, S. C. Morris, J. M. Holmes, N. Boiani,
I. M. Katona, and C. R. Maliszewski. 1993. Anti-cytokine antibodies as carrier
proteins: prolongation of in vivo effects of exogenous cytokines by injection of
cytokine-anti-cytokine antibody complexes. J. Immunol. 151:1235.
22. Urban, J. F., Jr., N. Noben-Trauth, D. D. Donaldson, K. B. Madden, S. C. Morris,
M. Collins, and F. D. Finkelman. 1998. IL-13, IL-4R␣, and Stat6 are required for
the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis. Immunity 8:255.
23. Urban, J. F., Jr., L. Schopf, S. C. Morris, T. Orekhova, K. B. Madden, C. J. Betts,
H. R. Gamble, C. Byrd, D. Donaldson, K. Else, and F. D. Finkelman. 2000. Stat6
signaling promotes protective immunity against Trichinella spiralis through a
mast cell- and T cell-dependent mechanism. J. Immunol. 164:2046.
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other recent reports have shown 1) a dose-dependent decrease in
the resistance of rat glomerular visceral epithelial cells in vitro in
response to IL-4 or IL-13 (31), and 2) decreased Na⫹ absorption
and increased Cl⫺ secretion in vitro in response to IL-4 treatment
of human bronchial epithelial cells (32). Results of these studies
suggest that the effects of IL-4 and IL-13 on epithelial cell function
may not be limited to the gastrointestinal tract, but rather may
represent a broader mechanism of immunoregulation at epithelial
cell surfaces.
More importantly, our observations indicate that the Th2 cytokines, particularly IL-4 and IL-13, change intestinal epithelial
function through multiple effects that additively or synergistically
interact to shift the balance of ion and fluid flow toward the gut
lumen, creating the increase in luminal fluid that may protect the
host against pathogens. The complexity of these interactions is
illustrated by the IL-4 effects on intestinal responsiveness to PGE2
and histamine, which must have at least two components. The mast
cell dependence of this effect of IL-4 and its failure to be induced
by IL-13 (which does not stimulate mast cells) suggest that it requires IL-4 stimulation of mastocytosis. However, IL-4 induction
of mast cell hyperplasia and mast cell degranulation (as measured
by an increase in serum levels of mouse mast cell protease) (22,
23) (Fig. 3) is STAT6 independent, while IL-4 enhancement of the
prosecretory effects of PGE2 and histamine is STAT6 dependent.
It remains to be determined whether STAT6 signaling is required
to induce mast cells to release specific mediators that promote
increased responsiveness to PGE2 and histamine, or whether there
is a separate, STAT6-dependent effect of IL-4 on intestinal epithelial cells that acts with a STAT6-independent mast cell effect to
increase intestinal epithelial responsiveness. In support of the latter
possibility, IL-4 has been shown to act through a STAT6-dependent mechanism to: 1) increase responsiveness to platelet-activating factor, histamine, 5-HT, and leukotriene C4 in an anaphylaxis
model (33, 34); 2) induce increased expression of a receptor for
cysteinyl leukotrienes (35); and 3) promote mast cell-dependent
expulsion of Trichinella spiralis by infected mice through an effect
on non-bone marrow-derived cells (24).
This difference in the effects of IL-4 and IL-13 on mast cells and
mast cell-dependent epithelial function probably has consequences
for host responses to intestinal worm infection and may explain
differences in the relative importance of IL-4 and IL-13 in host
protection against different parasites. Mice infected with N. brasiliensis do not require mast cells for parasite expulsion and exhibit
a stronger dependence on IL-13 than IL-4 for worm expulsion
(22). This greater dependence on IL-13 probably reflects either
greater production of IL-13 than IL-4 by infected mice or increased potency of IL-13 vs IL-4 in the induction of a host-protective effect, because treatment of N. brasiliensis-infected mice
with IL-4 induces worm expulsion in the absence of IL-13. In
contrast, the mast cell-dependent expulsion of T. spiralis is more
dependent on IL-4 than on IL-13, particularly during a second
infection with this parasite (23). Thus, the secretion of both IL-4
and IL-13 during worm infections and the multiple mechanisms by
which these cytokines promote changes in intestinal epithelial cell
function appear to extend the ability of the Th2 cytokine response
to protect against a spectrum of intestinal nematode parasites.
Finally, our observations demonstrate that not all effects of IL-4
and IL-13 on intestinal epithelial cells are prosecretory. Although
5-HT normally increases intestinal epithelial cell secretion, IL-4
and IL-13 inhibit this effect through a STAT6-dependent process.
Furthermore, treatment of STAT6⫺/⫺ and WT mice with IL-4C or
IL-13 in vivo had no effect on the secretory response to ACH.
Thus, exposure to IL-4 and IL-13 shifts the relative importance
of different mediators in regulating intestinal epithelial ion flow as
4421
4422
STAT6 DEPENDENCE OF CYTOKINE EFFECTS ON INTESTINAL EPITHELIAL FUNCTION EFFECTS
24. Urban, J. F., Jr., N. Noben-Trauth, L. Schopf, K. B. Madden, and
F. D. Finkelman. 2001. Cutting edge: IL-4 receptor expression by non-bonemarrow-derived cells is required to expel gastrointestinal nematode parasites.
J. Immunol. 167:6078.
25. Urban, J. F., Jr., C. R. Maliszewski, K. B. Madden, I. M. Katona, and
F. D. Finkelman. 1995. IL-4 treatment can cure established gastrointestinal nematode infections in immunocompetent and immunodeficient mice. J. Immunol.
154:4675.
26. Suzuki, K., H. Nakajima, N. Wantanbe, S. Kagami, A. Suto, Y. Saito, and
I. Iwamoto. 2000. Role of common receptor ␥ chain (␥c)- and Jak3-dependent
signaling in the proliferation and survival of murine mast cells. Blood 96:2172.
27. Zhao, A., D. P. Mulloy, J. F. Urban, Jr., W. C. Gause, and T. Shea-Donohue.
2001. Role of Stat6 in hypercontractility of murine small intestinal smooth muscle induced by nematode infection. Gastroenterology 120:A534.
28. Colgan, S. P., M. B. Resnick, C. A. Parkos, C. Delp-Archer, D. McGuirk,
A. E. Bacarra, P. F. Weller, and J. D. Madara. 1994. IL-4 directly modulates
function of a model human intestinal epithelium. J. Immunol. 153:2122.
29. Zund, G., J. L. Madara, A. L. Dzus, C. S. Awtrey, and S. P. Colgan. 1996.
Interleukin-4 and interleukin-13 differentially regulate epithelial chloride secretion. J. Biol. Chem. 271:7460.
30. Ceponis, P. J. M., F. Botelho, C. D. Richards, and D. McKay. 2000. Interleukins 4 and 13 increase intestinal epithelial permeability by a phosphatidylinositol 3-kinase pathway: lack of evidence for Stat6 involvement. J. Biol.
Chem. 275:29132.
31. Van den Berg, J. G., J. Aten, M. A. Chand, N. Classen, L. Dijikink, J. Wijdenes,
F. G. Lakkis, and J. J. Weening. 2000. Interleukin-4 and interleukin-13 act on
glomerular visceral epithelial cells. J. Am. Soc. Nephrol. 11:413.
32. Galietta, L. J. V., P. Pagesy, C. Folli, E. Caci, L. Romio, B. Costes, E. Nicolis,
G. Cabrini, M. Goossens, R. Ravazzolo, and O. Zegarra-Moran. 2002. IL-4 is a
potent modulator of ion transport in the human bronchial epithelium in vitro.
J. Immunol. 168:839.
33. Malaviya, R., and F. M. Uckun. 2002. Role of Stat6 in IgE/Fc⑀RI-mediated late
phase allergic responses of mast cells. J. Immunol. 168:421.
34. Strait, R. T., S. C. Morris, M. Yang, X. W. Qu, and F. D. Finkelman. 2002.
Pathways of anaphylaxis in the mouse. J. Allergy Clin. Immunol. 109:658.
35. Hsieh, F. H., B. K. Lam, J. F. Penrose, K. F. Austen, and J. A. Boyce. 2001. T
helper type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound
induction of leukotriene C4 synthase expression by interleukin 4. J. Exp. Med.
193:123.
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