Interferon-gamma activates EGF receptor and increases TGF-alpha in T84 cells: implications for chloride secretion

Jorge M. Uribe, Declan F. McCole, and Kim E. Barrett

Department of Medicine, University of California San Diego School of Medicine, San Diego, California 92103-8414


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IFN-gamma inhibits intestinal Cl- secretion, in part via downregulation of CFTR and Na+-K+-ATPase activity and expression, but the proximal signaling events were unknown. We have shown that transforming growth factor-alpha (TGF-alpha ) inhibits calcium-activated Cl- secretion, and effects of IFN-gamma in other systems are mediated via EGF family members. We tested whether IFN-gamma inhibits Cl- secretion via EGF receptor (EGFr) activation. IFN-gamma increased tyrosine phosphorylation in T84 cells at 24 h, including the EGFr. IFN-gamma also increased cell-associated pro-TGF-alpha , as well as free TGF-alpha in the bathing media. However, whereas IFN-gamma significantly inhibited carbachol-induced Cl- secretion, neither neutralizing antibodies to TGF-alpha nor an EGFr inhibitor (1 µM tyrphostin AG 1478) were able to reverse this inhibitory effect. AG 1478 also failed to reverse IFN-gamma -induced tyrosine phosphorylation of the EGFr, but receptor phosphorylation was attenuated by both the neutralizing antibody to TGF-alpha and PP2, a Src kinase inhibitor. Moreover, PP2 reversed the inhibitory effect of IFN-gamma on Cl- secretion. In total, our findings suggest an increase in functional TGF-alpha and activation of the EGFr in response to IFN-gamma . The release of TGF-alpha and intracellular Src activation likely combine to mediate EGFr phosphorylation, but only Src appears to contribute to the inhibition of transport. Nevertheless, because TGF-alpha plays a role in restitution and repair of the intestinal epithelium after injury, we speculate that these findings reflect a feedback loop whereby IFN-gamma modulates the extent of cytokine-induced intestinal damage.

chloride secretion; cytokines; inflammation; growth factors; mucosal injury


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHLORIDE SECRETION ACROSS intestinal epithelial cells is regulated by various paracrine, neurocrine, and endocrine factors (4). Such factors bind specific receptors on the epithelial cell surface and thereby stimulate elevations in the levels of various second messengers, such as cyclic nucleotides and calcium. These second messengers activate the transport machinery that comprises the chloride secretory pathway (4). This process is under tight control, and breakdown can lead to overexpression of secretion, resulting in diarrhea. Whereas the mechanisms responsible for initiating the secretory response have been well described, the negative signaling pathways are only now being worked out.

Our laboratory has identified various inhibitory pathways capable of regulating the extent of the secretory response. We have shown that pretreatment of T84 human colonic epithelial cells with muscarinic agonists can render the monolayer refractory to subsequent stimulation by other calcium-dependent secretagogs. The generation of the D-isomer of inositol 3,4,5,6-tetrakisphosphate [D-Ins(3,4,5,6)P4], initiation of the influx of extracellular calcium, and activation of PKC and MAPK have all been implicated in mediating these muscarinic effects (3, 17). We have also demonstrated that epidermal growth factor (EGF) is able to inhibit calcium-activated chloride secretion in T84 cells (31) via modification of potassium channel function secondary to activation of the phosphatidylinositol 3-kinase pathway (32). This finding is important in that EGF and another EGF receptor (EGFr) ligand produced by intestinal epithelial cells, transforming growth factor-alpha (TGF-alpha ), have also been shown to possess various healing functions in the gastrointestinal tract, including control of acid, bicarbonate, and mucus secretion, control of gastrointestinal blood flow, the initiation of epithelial/mucosal restitution, and protection of the mucosa (30). Upregulation of TGF-alpha levels and the EGFr also occurs in the gastrointestinal tract in response to injury and inflammation (3, 7, 23, 28). This may suggest that receptor tyrosine kinase pathways serve an important role in countering the inflammatory response and initiating the healing process.

IFN-gamma is a 45-kDa dimeric glycosylated protein whose production is upregulated under a variety of pathological circumstances, such as trauma, infection, cancer, and autoimmunity (6). As a cytokine, it is thought to regulate and amplify the immune response, induce tissue injury, and mediate complications of the inflammatory response in the intestine, such as diarrhea and fibrosis (25). However, cytokines also have a critical role in suppressing inflammation and mediating repair and healing (25). IFN-gamma has direct effects on epithelial cells, such as changes in cell morphology, decreased barrier function, increased TNF-alpha receptor and myosin heavy chain II molecule expression, and decreased levels of proteins involved in transport and barrier function (1, 5, 11, 15, 19, 29). IFN-gamma has also been shown to inhibit chloride secretion (11), and downregulation of CFTR, Na+-K+-2Cl- cotransporter (NKCC1), and Na+-K+-ATPase expression has been strongly implicated in this effect (15, 29, 35). However, the signaling pathways that initiate this downregulation have not been examined. Various cytokines, including IFN-gamma , are able to induce the expression of growth factors in various cell types, and it is believed that many of the effects of cytokines may be attributed to these newly expressed growth factors (2, 14, 27). Therefore, the goal of our study was to determine whether IFN-gamma is able to activate the EGFr and/or increase levels of functional TGF-alpha and inhibit chloride secretion in intestinal epithelial cells via these downstream signals.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Carbachol and tyrphostin AG 1478 (Sigma, St. Louis, MO), IFN-gamma (R&D Systems, Minneapolis, MN), TGF-alpha (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti-TGF-alpha (Chemicon, Temecula, CA), monoclonal mouse antiphosphotyrosine and anti-EGFr (Upstate Biotechnology, Lake Placid, NY), PP2, and TGF-alpha ELISA kit (Calbiochem, La Jolla, CA) were obtained from the sources indicated. All other reagents were of at least reagent grade and were obtained commercially.

Cells. Methods for the maintenance of T84 cells for use in transepithelial electrolyte transport studies have been described previously (12). In brief, T84 cells were grown in DMEM/Ham's F-12 (JRH, Lenexa, KS), with the addition of 5% newborn calf serum. Cells were passaged by trypsinization. For the measurement of chloride secretion, 2.5 × 105 cells were seeded onto 12-mm Millicell-HA Transwells (Millipore, Bedford, MA). For experiments involving immunoprecipitation and Western blotting, and for the TGF-alpha ELISA, 106 cells were seeded onto 30-mm Millicell-HA Transwells. All cells were cultured for 7-10 days before use.

Chloride secretion. Chloride secretion was measured as short-circuit current (Isc) across monolayers of T84 cells, mounted in Ussing chambers (0.6-cm2 window area) modified for use with cultured cells (12). Isc (normalized to µA/cm2) was used to quantitate both basal transepithelial chloride secretion and that induced by calcium-dependent secretagogs. T84 cells secrete chloride in response to various calcium-mobilizing agonists, and the resulting changes in Isc are wholly reflective of chloride secretion (13). Isc measurements were carried out in Ringer solution containing (in mM) 140 Na+, 5.2 K+, 1.2 Ca2+, 0.8 Mg2+, 119.8 Cl-, 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 2.4 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 0.4 HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and 10 glucose.

Immunoprecipitation and Western blotting. T84 cells were treated with IFN-gamma on the basolateral surface for the indicated periods of time. On the day of the experiment, cells were washed three times with ice-cold PBS. Ice-cold lysis buffer was then added (consisting of PBS, 1% Nonidet P-40, 1 mM NaVO4, 1 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride), and the cells were incubated at 4°C for 30 min. Cells were then scraped into microcentrifuge tubes and centrifuged at 10,000 rpm for 10 min to remove insoluble material, and an aliquot was removed from each sample to determine protein content (Bio-Rad protein assay). Samples were then adjusted with lysis buffer such that each sample had an identical protein concentration. For the determination of tyrosine-phosphorylated EGFr, 5 µg of monoclonal anti-EGFr was added to each sample and allowed to incubate on ice for 60 min. This was followed by the addition of 50 µl of a 1:1 mixture of protein A-Sepharose and water, and samples were placed on a rotating platform at 4°C for 60 min. Samples were then centrifuged to pellet the protein A-Sepharose-antibody-antigen complex, and the complex was washed three times with cold lysis buffer followed by three more washes with cold PBS. The beads were then resuspended in gel loading buffer (50 mM Tris, pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.2% bromophenol blue, 20% glycerol). For the determination of total tyrosine-phosphorylated proteins and membrane-bound TGF-alpha , the samples were mixed 1:1 with double-strength gel loading buffer. All samples were placed in boiling water for 5 min and then loaded on a 7.5% polyacrylamide gel to resolve proteins (15% gel for TGF-alpha determination). The proteins were transferred from the gel onto a polyvinylidene difluoride membrane (DuPont-New England Nuclear, Boston, MA). The membrane was then blocked with a 1% skim milk solution in PBS for 30 min, followed by further incubation of the membrane with a 1% skim milk solution containing 5 µg monoclonal antiphosphotyrosine or 10 µl polyclonal anti-TGF-alpha for 60 min. This was followed by three 15-min washes with wash buffer (1% skim milk, 0.5% BSA, 0.2% Tween 20, in PBS). After washes, 2 µl of secondary antibody [goat-anti-mouse or goat-anti-rabbit IgG conjugated to alkaline phosphatase (Clontech, Palo Alto, CA)] were added to incubate for an additional 30 min. This was followed by three more washes with wash buffer. The membrane was then treated with a chemiluminescent solution according to the manufacturer's directions (Clontech) and exposed to film. Densitometric analysis of the blot was performed using a digital imaging system.

TGF-alpha ELISA. Enzyme-linked assays (ELISA) for TGF-alpha in basolateral and apical supernatants from T84 cells grown on Nuclepore Transwells were performed using a commercial kit (Calbiochem) according to the manufacturer's instructions. In brief, the kit utilized rabbit polyclonal anti-TGF-alpha bound to microtiter plates as the capturing antibody and biotinylated polyclonal rabbit anti-TGF-alpha as a second-step antibody. This was then followed by the addition of streptavidin-horseradish peroxidase (HRP) as the reporter enzyme. Bound HRP was visualized using O-phenylenediamine and measured colorimetrically at an absorbance of 490 nm using a microtiter reader.

Statistical analysis. ANOVA with Student-Newman-Keuls post hoc test was used to compare mean values where appropriate. P values <0.05 were considered significant. All data are expressed as means ± SE for a series of experiments.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Treatment with IFN-gamma results in phosphorylation of the EGFr. Binding of IFN-gamma to its receptor has been shown to activate a tyrosine kinase cascade in various cell types, leading to the tyrosine phosphorylation of various proteins (18). Our first step therefore was to determine whether IFN-gamma had a similar effect on T84 cells and, specifically, whether it is able to tyrosine-phosphorylate high-molecular-mass transepithelial resistance proteins in the 170-kDa range, the molecular mass of the EGFr (33). Figure 1 shows the time course of effects of IFN-gamma on tyrosine phosphorylation of various proteins in T84 cells. A concentration of 100 ng/ml IFN-gamma was chosen based on prior studies (11, 15, 19, 29) of effects of this cytokine on T84 cells. Under nonstimulated conditions, T84 cells display a low level of tyrosine phosphorylation. On stimulation with IFN-gamma , there is a progressive increase in tyrosine phosphorylation of various proteins, particularly in the 170- to 200-kDa range, with the peak of phosphorylation around the 12- to 24-h time points. To determine whether IFN-gamma stimulation of T84 cells does lead to EGFr tyrosine phosphorylation, IFN-gamma -treated T84 cell lysates were immunoprecipitated with antibodies to the EGFr, and the subsequent Western blots were probed with antiphosphotyrosine. Figure 2 shows that 100 ng/ml IFN-gamma was able to tyrosine-phosphorylate the EGFr in T84 cells in a time-dependent fashion, with the peak of phosphorylation occurring at 24 h. On the basis of these findings, we focused the rest of our studies on the 24-h time point.


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Fig. 1.   Effects of IFN-gamma on overall tyrosine phosphorylation in T84 cells. Monolayers were treated with 100 ng/ml IFN-gamma added basolaterally for 1, 6, 12, and 24 h. Cells were then lysed, separated, transferred onto membranes, and probed with antibodies to phosphotyrosine. Antibody binding was detected through the use of enhanced chemiluminescence methodology as described under MATERIALS AND METHODS. This figure is representative of 3 similar experiments.



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Fig. 2.   Effect of IFN-gamma on tyrosine phosphorylation of the EGF receptor (EGFr) in T84 cells. Monolayers were treated with 100 ng/ml IFN-gamma for 1 min and 1, 6, 12, and 24 h or transforming growth factor-alpha (TGF-alpha ) for 5 min. Cells were lysed and immunoprecipitated with monoclonal antibodies to the EGFr. The immunoprecipitated proteins were then separated, transferred onto membranes, and probed with antibodies to phosphotyrosine. Antibody binding was detected as described in Fig. 1. The arrow denotes the predicted molecular weight of the EGFr. This figure is representative of 3 similar experiments.

IFN-gamma increases expression of the transmembrane and soluble forms of TGF-alpha . Previous studies (2, 14, 27) have shown that various cytokines, including IFN-gamma , are able to increase the expression of several growth factors. We therefore examined whether IFN-gamma was able to induce the expression and release of a ligand able to activate the EGFr. We focused on TGF-alpha because various studies (3, 23, 28) have demonstrated that this EGFr ligand is upregulated in the intestine in response to inflammation and damage. TGF-alpha is initially expressed as a 20-kDa membrane-bound pro-TGF-alpha precursor protein, which on appropriate stimulation is proteolytically cleaved and released as a 6.0-kDa soluble form (26). As the membrane-bound precursor can itself function as a ligand for the EGFr (20), we first wanted to determine whether IFN-gamma was able to increase the content of this protein in T84 cells. Figure 3 shows the effects of 24-h IFN-gamma on the levels of membrane-bound TGF-alpha . T84 cells display basal levels of a 20-kDa protein that is detected with antibodies to TGF-alpha on Western blot. This is likely pro-TGF-alpha . On stimulation with IFN-gamma , there is a significant increase in the levels of this protein. These findings demonstrate that IFN-gamma is able to increase protein synthesis of this growth factor in T84 cells and in a time frame consistent with EGFr phosphorylation.


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Fig. 3.   Effect of IFN-gamma on the expression of pro-TGF-alpha in T84 cells. Monolayers were treated with 100 ng/ml IFN-gamma or with Ringer's solution for 24 h. Cells were then treated as in Fig. 1 with the use of antibodies to TGF-alpha to probe for pro-TGF-alpha . The arrow denotes the predicted molecular weight of pro-TGF-alpha . This figure is representative of 3 similar experiments.

The membrane-anchored form of TGF-alpha can be cleaved proteolytically by cell-associated proteases that are activated by the appropriate stimulus, including ligand receptor-mediated pathways (24). The released peptide, a 6.0-kDa species, is suggested to be the major TGF-alpha form that activates the EGFr (20). We therefore wanted to determine whether this soluble form was also elevated in response to IFN-gamma . Figure 4 shows the effects of IFN-gamma on the levels of soluble TGF-alpha found in the spent media from T84 cells as determined by ELISA. Under control conditions, detectable levels of TGF-alpha were observed in both apical and basolateral bathing media, although approximately threefold higher concentrations of this growth factor were found in the basolateral compartment. In response to IFN-gamma , basolateral, but not apical, concentrations of TGF-alpha were significantly increased. These data correspond to the known basolateral localization of the EGFr in T84 cells as well as native intestinal epithelial cells (30, 31). Moreover, they imply that IFN-gamma is able not only to increase the synthesis of the membrane-associated precursor of TGF-alpha but also to activate the enzymatic machinery required to cleave the precursor and release the 6.0-kDa species into the basolateral medium, all in a time frame consistent with the increase in pro-TGF-alpha and tyrosine phosphorylation of the EGFr.


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Fig. 4.   Effect of IFN-gamma on the release of soluble TGF-alpha into media bathing T84 cells. Monolayers were treated with 100 ng/ml IFN-gamma or with Ringer's solution for 24 h. An aliquot from apical and basolateral media from treated cells was then sampled, and the concentration of TGF-alpha was determined via ELISA as in MATERIALS AND METHODS. The values are means ± SE for 4 experiments. The asterisk denotes a value that differs significantly from the corresponding value in control cells. *P < 0.05 by Student's t-test.

Activation of the EGFr does not mediate IFN-gamma inhibition of chloride secretion in T84 cells. TGF-alpha has been shown to have many effects on the gastrointestinal system including control of various secretory processes (30). To determine whether the ability of IFN-gamma to release TGF-alpha was involved in the effect of this cytokine on chloride secretion, we conducted control studies to determine whether the effects of exogenously added TGF-alpha could be reversed. Figure 5 shows the effects of a 15-min pretreatment with 50 ng/ml TGF-alpha , added to the basolateral surface of T84 cells, on carbachol-induced chloride secretion. In these studies, carbachol increased Isc by 42.0 ± 8.7 µA/cm2, which was reduced to 14.7 ± 1.7 µA/cm2 in the presence of TGF-alpha (n = 3, P < 0.01). This inhibitory effect was reversed by 24-h coincubation of TGF-alpha with 5 µg of rabbit polycolonal anti-TGF-alpha before its addition to T84 cells. Under these conditions, the response to carbachol returned to 32.7 ± 1.8 µA/cm2. The neutralizing antibody by itself had no significant effect on the response to carbachol. These data suggest that an antibody to TGF-alpha is able to block the effect of a large dose of exogenous TGF-alpha and that the antibody is stable for 24 h.


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Fig. 5.   Effect of an antibody to TGF-alpha on the ability of TGF-alpha to inhibit carbachol (Carb)-induced chloride secretion in T84 cells. Monolayers were mounted in Ussing chambers as described in MATERIALS AND METHODS and pretreated with 50 ng/ml TGF-alpha to the basolateral surface 15 min before stimulation with carbachol (100 µM). In those experiments with antibodies to TGF-alpha , 5 µl of polyclonal antibodies to TGF-alpha were coincubated with either TGF-alpha or BSA for 24 h at 37°C and then added to monolayers as described in RESULTS. The data are means ± SE for 3 experiments and are expressed as the peak increase in short-circuit current (Delta Isc) induced by the addition of carbachol. Asterisks denote responses that differ significantly from those induced by carbachol alone. **P < 0.01 by ANOVA with Student-Newman-Keuls post hoc test.

We then focused on whether the neutralizing antibody to TGF-alpha was capable of reversing the ability of IFN-gamma to inhibit chloride secretion. Figure 6 shows the effects of 24-h IFN-gamma on carbachol-induced chloride secretion. Pretreatment with IFN-gamma decreased chloride secretion from 45.0 ± 5.7 to 20.0 ± 2.6 µA/cm2 (n = 3-4, P < 0.05). However, unlike its effects on TGF-alpha , coincubation with the neutralizing antibody to TGF-alpha had no effect on the ability of IFN-gamma to inhibit chloride secretion. Because we have shown that the dose of antibody used in our studies is able to inhibit effects of TGF-alpha at concentrations far greater than those found in media from IFN-gamma -treated T84 cells, these data suggest that soluble TGF-alpha is unlikely to mediate the inhibitory effect of IFN-gamma on chloride secretion.


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Fig. 6.   Effect of antibodies to TGF-alpha on the inhibitory effect of IFN-gamma on carbachol-induced chloride secretion in T84 cells. Monolayers were mounted in Ussing chambers as described in MATERIALS AND METHODS and treated with 100 ng/ml IFN-gamma on the basolateral surface 24 h before stimulation with carbachol (100 µM). In those experiments with antibodies to TGF-alpha , 5 µl of polyclonal antibodies to TGF-alpha were additionally added to the apical and basolateral aspect of T84 cell monolayers and incubated for 24 h. Data are means ± SE for 3-4 experiments and are expressed as Delta Isc induced by the addition of carbachol. Asterisks denote responses that differ significantly from those induced by carbachol alone. *P < 0.05 by ANOVA with Student-Newman-Keuls post hoc test.

In addition to TGF-alpha , IFN-gamma has also been shown to induce the expression of other members of the EGF family of peptides from epithelial cells (2). To determine whether these peptides were involved in activating the EGFr, subsequent experiments used a specific inhibitor of the EGFr kinase, tyrphostin AG 1478 (22). Inhibition of the receptor's intrinsic kinase activity should block activation of the receptor mediated by cognate ligand binding. Figure 7 shows the effects of tyrphostin AG 1478 on the ability of IFN-gamma to inhibit calcium-activated chloride secretion. Pretreatment with IFN-gamma for 24 h decreased carbachol-stimulated chloride secretion from 38 ± 1.3 to 9.0 ± 2.0 µA/cm2 (n = 3-4, P < 0.001). However, similar to the lack of effect of the neutralizing antibody to TGF-alpha , 24-h coincubation with tyrphostin AG 1478 failed to reverse the ability of IFN-gamma to inhibit chloride secretion. These findings suggest that extracellular activation of the EGFr, whether from soluble or membrane-bound ligand, is probably not involved in mediating the inhibitory effect of IFN-gamma on chloride secretion. To examine further whether extracellular ligand is activating the receptor, we examined the level of EGFr tyrosine phosphorylation in the presence and absence of tyrphostin AG 1478. Figure 8 shows the densitometric analysis of these results. A 24-h incubation with 1 µM tyrphostin AG 1478 inhibited EGFr phosphorylation occurring in response to 5-min treatment of 50 ng/ml TGF-alpha as expected. However, 24-h coincubation of tyrphostin AG 1478 with IFN-gamma had no effect on IFN-gamma 's ability to phosphorylate the EGFr.


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Fig. 7.   Effect of tyrphostin AG 1478 (Tyr) on the inhibitory effect of IFN-gamma on carbachol-induced chloride secretion in T84 cells. Monolayers were mounted in Ussing chambers as in MATERIALS AND METHODS and treated with 100 ng/ml IFN-gamma on the basolateral surface 24 h before stimulation with carbachol (100 µM). In those experiments with tyrphostin AG 1478, 1 µM of the inhibitor and 100 ng/ml IFN-gamma were coincubated with T84 cell monolayers for 24 h before stimulation with carbachol. The data are means ± SE for 3-4 experiments and are expressed as Delta Isc induced by the addition of carbachol. Asterisks denote responses that differ significantly from those induced by carbachol alone. ***P < 0.001 by ANOVA with Student-Newman-Keuls post hoc test.



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Fig. 8.   Effect of 24-h tyrphostin AG 1478 treatment of T84 cells on the ability of IFN-gamma and TGF-alpha to cause tyrosine phosphorylation of the EGFr. T84 cell monolayers were either coincubated with 1 µM tyrphostin AG 1478 and 100 ng/ml IFN-gamma for 24 h or incubated with 1 µM tyrphostin AG 1478 for 24 h followed by treatment with 50 ng/ml TGF-alpha for 5 min. Cells were then treated as in Fig. 2. Data were obtained by digital image analysis of the respective blots and are normalized to the control band in the absence of tyrphostin AG 1478, set at a value of 100% on the same blot. Data represent duplicate determinations for both conditions.

It remained possible that signaling events evoked by IFN-gamma , secondary to the release of TGF-alpha , might be mediated by mechanisms that were insensitive to tyrphostin AG 1478. We first examined whether the neutralizing antibody to TGF-alpha used previously had any effect on EGFr phosphorylation evoked by IFN-gamma . In fact, when cells were coincubated with IFN-gamma (100 ng/ml) and the neutralizing antibody (5 µg/ml), tyrosine phosphorylation of the EGFr was significantly attenuated (Fig. 9). Similarly, the ability of IFN-gamma to evoke EGFr phosphorylation was blocked by PP2, an inhibitor of the soluble tyrosine kinase, Src (Fig. 9). These data indicate that IFN-gamma may recruit intracellular signaling events and soluble tyrosine kinase(s) in evoking EGFr phosphorylation. Moreover, extracellular TGF-alpha released after IFN-gamma treatment, as opposed to exogenous addition of the growth factor, may be capable of stimulating EGFr phosphorylation that is independent of the kinase activity of the receptor.


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Fig. 9.   IFN-gamma induces EGFr phosphorylation by a mechanism involving activation of Src kinase and release of TGF-alpha . T84 cell monolayers were treated with IFN-gamma (100 ng/ml) for 24 h in the presence or absence of the Src kinase inhibitor PP2 (20 µM) added bilaterally, or an anti-TGF-alpha antibody (5 µg/ml) added basolaterally. Cells were lysed and immunoprecipitated with monoclonal antibodies to the EGFr. The immunoprecipitated proteins were then separated by gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and probed with anti-phosphotyrosine antibodies. A: representative blot of EGFr phosphorylation after 24-h treatment of T84 cells with IFN-gamma  ± inhibitors. B: densitometric analysis of phosphorylated EGFr blots expressed in arbitrary units (a.u.) is shown as means ± SE for six experiments. Asterisks denote phosphorylation levels that are significantly lower than those observed in cells treated with IFN-gamma alone. *P < 0.05 by Student's t-test.

Because PP2 was able to reverse the ability of IFN-gamma to cause EGFr phosphorylation, we performed a final experiment to test whether this drug could also block the effect of the cytokine on chloride secretion. As shown in Fig. 10, IFN-gamma reduced carbachol-stimulated chloride secretion, as expected. PP2 alone significantly potentiated chloride secretion evoked by carbachol alone, consistent with our prior reports (16). Moreover, in the presence of PP2, the inhibitory effect of IFN-gamma on carbachol-stimulated chloride secretion was reversed. These data imply that Src family kinases mediate the effect of IFN-gamma on chloride secretion, perhaps by mediating intracellular activation of the EGFr.


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Fig. 10.   Effect of PP2 on the inhibitory effect of IFN-gamma on carbachol-induced chloride secretion in T84 cells. Monolayers were mounted in Ussing chambers and treated with 100 ng/ml IFN-gamma on the basolateral surface 24 h before stimulation with carbachol (100 µM). In parallel, T84 cell monolayers were coincubated with PP2 (20 µM apically and basolaterally) and IFN-gamma (100 ng/ml) for 24 h or with PP2 alone before stimulation with carbachol. The data are means ± SE for 4 experiments and are expressed as a percentage of Delta Isc induced by carbachol alone. Asterisks denote responses to carbachol that differ significantly from those in cells pretreated only with IFN-gamma . **P < 0.01; ***P < 0.001 by ANOVA with Student-Newman-Keuls post hoc test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we investigated the mechanism by which IFN-gamma is able to inhibit calcium-activated chloride secretion in intestinal epithelial cells. We focused on the possible role of growth factors as mediators of the inhibitory effect, because the literature supports such a hypothesis: 1) in the setting of gastrointestinal inflammation, various growth factors, including TGF-alpha , have been shown to be upregulated (3, 23, 28); 2) we have previously demonstrated that EGF is rapidly able to inhibit calcium-activated chloride secretion in T84 cells (31); 3) Asano et al. (2) have shown that IFN-gamma is able to induce expression of TGF-alpha in human bronchial epithelial cells; and 4) Prenzel et al. (24) initially showed that, in response to G protein-dependent ligands, at least the membrane-bound precursor to heparin-binding EGF, a member of the EGF family of peptides, is cleaved, releasing soluble EGFr ligand into the media and subsequently activating the EGFr. More recent studies (21) from our own laboratory have likewise indicated that carbachol can evoke TGF-alpha release from T84 cells. Thus we hypothesized that the inhibitory mechanism evoked by IFN-gamma involved activation of the EGFr via increased expression and/or release of TGF-alpha and that this ligand might then mediate the inhibitory effect of IFN-gamma on chloride secretion via an autocrine TGF-alpha /EGFr loop.

We demonstrated that IFN-gamma is able to increase both the expression and release of TGF-alpha from T84 cells over a time frame consistent with its ability to inhibit chloride secretion. However, the concentration of TGF-alpha found in the spent media from T84 cells treated with IFN-gamma is well below the IC50 for TGF-alpha to inhibit chloride secretion (3.7 nM) (31). Therefore, whereas TGF-alpha levels are elevated, it is unlikely that soluble TGF-alpha alone would activate the EGFr sufficiently to inhibit chloride secretion. However, we also demonstrated a significant increase in the amount of membrane-bound pro-TGF-alpha present on T84 cells in response to treatment with IFN-gamma . This is significant in that the membrane-bound precursor has been shown to possess biological activity (20) and thus can serve as an additional source of ligand. Furthermore, it has been suggested that the membrane-bound precursor may function as a means to concentrate the growth factor within a defined area, thereby decreasing the effective IC50 (20). Despite this, we were unable to reverse the IFN-gamma -mediated inhibition of secretion using either neutralizing antibodies to TGF-alpha or a specific inhibitor to the EGFr, tyrphostin AG 1478, at maximal concentrations, although the neutralizing antibodies at least could partially reduce (although not abolish) IFN-gamma -stimulated EGFr phosphorylation. These findings suggest that IFN-gamma mediates its inhibition of chloride secretion utilizing pathways that are, at least in part, independent of extracellular ligand activation of the EGFr.

Currently, it is believed that the inhibitory effect of IFN-gamma on chloride secretion is secondary to downregulation of the activity and expression of transport proteins, including CFTR. However, this is speculative at present because no studies have been performed to determine whether the decrease in CFTR expression is directly responsible for the inhibitory effect. Furthermore, Fish et al. (15), utilizing the T84 cell model, have shown that decreases in CFTR protein do not parallel changes in chloride secretion induced by IFN-gamma . Similarly, Sugi et al. (29) failed to see an effect of IFN-gamma on CFTR expression, reporting instead a reduction in Na+-K+-ATPase and the Na+-K+-2Cl- cotransporter NKCC1. Whereas they do not dismiss the possibility of CFTR downregulation as an important factor in the inhibition of secretion by IFN-gamma , our findings and those of others suggest that other pathways are likely to be also involved. In our studies, the EGFr remained phosphorylated in response to IFN-gamma despite the use of tyrphostin AG 1478, suggesting that the signaling events for inhibition of secretion due to EGFr activation may be still active. On the other hand, IFN-gamma -induced EGFr phosphorylation could be blocked by an antibody to TGF-alpha or by the Src inhibitor PP2. Our data therefore imply at least a partial role for the EGFr in mediating the effect of IFN-gamma on secretion, perhaps acting in concert with Src. We have shown that potassium channel modification via PKCepsilon is required for EGFr-mediated inhibition of secretion (10). Therefore, future studies should emphasize examination of the functional properties of the potassium channel to better understand the possible role of the EGFr in the inhibitory effect of IFN-gamma . It is also possible that Src and/or other signaling events linked to EGFr activity could negatively regulate Na+-K+-ATPase activity, thereby accounting for the effect reported by Sugi et al. (29).

The role of elevated TGF-alpha in response to IFN-gamma in T84 cells is currently unknown. TGF-alpha has been shown to disrupt tight junctions in mammary epithelial cells as evidenced by a reduction in monolayer transepithelial electrical resistance, an increase in paracellular transport, and a redistribution of the ZO-1 tight junctional protein (8). These findings are in keeping with the fact that Sugi et al. (29) reported recently that IFN-gamma decreases ZO-1 protein expression in T84 cells. Similar responses have been previously ascribed to IFN-gamma on functional grounds (19). It is therefore possible that TGF-alpha may be mediating these effects of IFN-gamma . In our study, there was little, if any, change in transepithelial resistance in response to IFN-gamma after 24 h (data not shown). These findings are consistent with the findings of previous studies (1, 29). As peak changes in transepithelial resistance occur at 48-72 h of IFN-gamma treatment, examination of this potential effect will require a longer time course than that used in this study. TGF-alpha has also been shown to posses mitogenic and restitutive properties in intestinal epithelial cells and has been implicated in the healing process in the gastrointestinal tract in response to inflammation (30). Various studies have shown a significant increase in the levels of TGF-alpha in response to inflammation and injury to the gastrointestinal tract (3, 23, 28). One can therefore speculate that cytokines increase TGF-alpha expression to limit the extent of inflammation-induced intestinal damage and/or excessive secretion.

Another significant finding of our study was that tyrosine phosphorylation of the EGFr occurred in response to IFN-gamma treatment. This finding is consistent with other studies that show transactivation of the EGFr in other cell types. Both intra- and extracellular pathways for EGFr transactivation have been studied and include release of soluble ligand, the activation of G protein-coupled receptors, and the activation of cytokine receptors (17, 24, 34). We show that levels of TGF-alpha are increased in response to IFN-gamma , and our group (17) has previously shown that T84 cells can undergo activation of the EGFr via a G protein-coupled receptor pathway. However, the inability of a specific inhibitor of the EGFr kinase to reverse the tyrosine phosphorylation of the receptor suggests that other mechanisms are involved in the response to IFN-gamma , as these latter pathways, at least, are sensitive to tyrphostin AG 1478 (17). On the other hand, Yamauchi et al. (34) have shown that growth hormone can tyrosine-phosphorylate the EGFr via pathways independent of the receptor's intrinsic kinase activity. They demonstrated a requirement for the cytosolic tyrosine kinase, janus kinase 2 (Jak2), for tyrosine phosphorylation of the EGFr at the Grb2 binding site, thus making the EGFr kinase activity dispensable. Such a pathway in T84 cells would predictably be insensitive to tyrphostin AG 1478. Because the IFN-gamma receptor is similar to the growth hormone receptor in that they are both in the class of cytokine receptors, neither possesses intrinsic tyrosine kinase activity, and both are able to activate Jak2 (16), it is quite likely that activation of the EGFr occurs via this pathway. Indeed, we show that the ability of IFN-gamma to induce EGFr phosphorylation is blocked by an inhibitor of the soluble tyrosine kinase Src. However, it should also be noted that we have recently shown that growth hormone at least activates EGFr phosphorylation in T84 cells in a manner that is, in fact, sensitive to tyrphostin AG 1478 (9). This underscores the fact that the details of complex signaling pathways may depend on the cell type studied, particularly when cross talk among various signaling cascades is involved.

In conclusion, the present study demonstrates an increase in the expression and release of TGF-alpha and the phosphorylation of the EGFr in response to IFN-gamma treatment of T84 cells. Although we were unable to reverse the inhibitory effect of IFN-gamma on chloride secretion with antibodies to TGF-alpha or the tyrosine kinase inhibitor tyrphostin AG 1478, we were able to block inhibition by antagonizing Src activity. Moreover, at the very least, our findings do expand on the various roles of IFN-gamma in the gastrointestinal tract. We speculate that the increase in functional growth factor and activation of the EGFr may serve as a mechanism to restrict the extent of cytokine-induced intestinal damage.


    ACKNOWLEDGEMENTS

We thank Glenda Wheeler for administrative support.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28305 (to K. E. Barrett). D. F. McCole is the recipient of a Research Fellowship Award from the Crohn's and Colitis Foundation of America. J. M. Uribe was the recipient of an American Digestive Health Foundation student research fellowship award while a medical student in the University of California San Diego School of Medicine.

Present address of J. M. Uribe: Dept. of Anesthesiology and Critical Care Medicine, Blalock 1415, The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21287-4965.

Address for reprint requests and other correspondence: K. E. Barrett, Univ. of California San Diego Medical Center, 8414, 200 West Arbor Drive, San Diego, CA 92103-8414 (E-mail: kbarrett{at}ucsd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 11, 2002;10.1152/ajpgi.00237.2002

Received 25 February 2002; accepted in final form 26 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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