Superantigen immune stimulation activates epithelial STAT-1 and PI 3-K: PI 3-K regulation of permeability

Derek M. McKay1,2, Fernando Botelho2, Peter J. M. Ceponis1, and Carl D. Richards2

1 Intestinal Disease Research Programme and 2 Infection and Immunity Programme, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Signal transducers and activators of transcription (STATs) are critical intracellular signaling molecules for many cytokines. We compared the ability of T84 epithelial cells to activate STATs in response to cytokines [interferon-gamma (IFN-gamma ), interleukin (IL)-4, IL-10, and tumor necrosis factor-alpha (10 ng/ml)] and conditioned medium from superantigen [Staphylococcus aureus enterotoxin B (SEB)]-activated peripheral blood mononuclear cells (PBMC) using electrophoretic mobility shift assays (EMSA). Of the cytokines tested, only IFN-gamma caused a STAT-1 response. Exposure to SEB-PBMC-conditioned medium resulted in STAT-1 or STAT-1/3 activation, and inclusion of anti-IFN-gamma antibodies in the conditioned medium abolished the STAT-1 signal. Cells treated with transcription factor decoys, DNA oligonucleotides bearing the STAT-1 recognition motif, and then SEB-PBMC-conditioned medium displayed a reduced STAT-1 signal on EMSA, yet this treatment did not prevent the drop in transepithelial resistance (measured in Ussing chambers) caused by SEB-PBMC-conditioned medium. In contrast, the phosphatidylinositol 3'-kinase (PI 3-K) inhibitor LY-294002 significantly reduced the drop in transepithelial resistance caused by SEB-PBMC-conditioned medium. Thus data are presented showing STAT-1 (±STAT-3) and PI 3-K activation in epithelial cells in response to immune mediators released by superantigen immune activation. Although the involvement of STAT-1/-3 in the control of barrier function remains a possibility, PI-3K has been identified as a regulator of T84 paracellular permeability.

intestine; Staphylococcus aureus enterotoxin B; phosphatidylinositol 3'-kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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CYTOKINE LIGATION OF the appropriate receptor can mobilize one, or more, intracellular signaling pathways, including activation of Janus kinase (JAK)-signal transducers and activators of transcription (STAT), nuclear factor-kappa beta  (NF-kappa beta ) mitogen-activated protein kinases (MAPK), sphingomyelinase-ceramide, and phosphatidylinositol 3'-kinase (PI 3-K), all of which can eventually result in regulation of gene transcription. Thus, although cytokine effects on epithelia (cell lines in vitro and to a lesser extent studies with tissue segments ex vivo) are well documented (26), the intracellular signaling cascades that mediate the responses are less well defined, particularly in terms of the integration of responses to multiple messengers (3).

Recently, the JAK-STAT pathway has been highlighted as an important membrane-to-nucleus signaling pathway for many cytokines (15). Briefly, binding of specific cytokines to their receptors results in JAK phosphorylation followed by the recruitment, tyrosine phosphorylation, and dimerization of cytosolic STAT monomers. The STAT dimers rapidly translocate to the nucleus, where they bind to promoter regions of the DNA and regulate gene transcription (15). Although it is intuitive to accept that exposure of epithelia to cytokines will generate stereotypical JAK-STAT responses, this must nevertheless be tested. Indeed, precise definition of the kinetics of the induction of STAT signaling is crucial if modulation of their function is to be a rational therapy for enteric inflammatory or secretory disease (14). Descriptive data are beginning to emerge on STAT protein activation in a variety of epithelia. For instance, the levels of STAT-1 and STAT-6 in nuclear extracts are increased in human airway epithelium in response to interferon (IFN)-gamma and interleukin (IL)-4, respectively (13). Similarly, IFN-gamma treatment evoked a JAK-2/STAT-1 response in human salivary epithelial cells, and IL-4 stimulation of the human colonic HT-29 epithelial cell line resulted in phosphorylation of JAK-2 (29, 46). A recent report suggests, somewhat unexpectedly, that epidermal growth factor resulted in STAT-2 translocation to the nucleus in the absence of tyrosine phosphorylation in rat IEC-6 cells (17); this does not fit with current dogma (15) and raises the possibility of unique STAT regulatory mechanisms in gut epithelium. The JAK-STAT pathway is central to the mediation of many cytokine and growth factor responses; however, as noted above, alternative signaling pathways are also available. Additionally, a recent study with macrophages presents a model in which the STAT and PI 3-K pathways may be linked (37). PI 3-K activity is involved in vesicle trafficking and cytoskeletal regulation (10) and as such presents itself as a possible candidate molecule involved in the regulation of epithelial tight junction activity (i.e., rate-limiting step in controlling paracellular permeability). Indeed, we have presented data indicating that IL-4-induced decreases in T84 monolayer transepithelial resistance can be elevated significantly by cotreatment with inhibitors of PI 3-K activity (8).

We have shown that the mixed mediator milieu produced by immune cells activated by bacterial superantigens can lead to increased epithelial permeability across monolayers of the human colonic T84 epithelial cell line (27). The aim of this study was twofold: 1) to assess STAT protein activation in gut epithelia in response to single recombinant cytokines (principally IFN-gamma ) and the conditioned medium (CM) from immune cells activated by the superantigen, Staphylococcus aureus enterotoxin B (SEB); and 2) given the observation that CM from superantigen-activated immune cells increases T84 monolayer permeability, we wanted to assess the putative involvement of STAT proteins and PI 3-K in the regulation of epithelial paracellular permeability. The findings indicate that immune mediators released from superantigen-activated immune cells elicit a STAT-1 or a STAT-1/3 response and activate PI 3-K in gut epithelium. Although the role of the STAT proteins in the control of epithelial barrier function remains a possibility, our pharmacological studies provide evidence for PI 3-K involvement in the control of epithelial paracellular permeability.


    MATERIALS AND METHODS
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Human Epithelial Cell Culture and Stimulation

Human colonic T84 cells were used primarily throughout this study (some experiments used human Caco-2 or HT-29 epithelial cells). Epithelial cells (3 × 106) were seeded on 6.0-cm-diameter petri dishes and were cultured at 37°C for 7 days as previously described (27). Cells were treated with human recombinant IFN-gamma , IL-4, IL-10, tumor necrosis factor (TNF)-alpha (all at 10 ng/ml; PharMingen, Mississauga, ON, Canada, or Sigma Chemical, St. Louis, MO), or a cell-free SEB-peripheral blood mononuclear cell (PBMC) CM (3-50%, diluted in fresh medium, made by treating 106 PBMC with 1 µg SEB for 24 h). We have previously reported that IFN-gamma in the 50% SEB-PBMC CM is <5 ng/ml (22); however, IFN-gamma levels were measured by sandwich ELISA (paired antibodies from PharMingen) in four randomly selected SEB-PBMC CM samples used in this study. Time-matched control epithelium was grown in culture medium or treated with SEB (1 µg/ml) only. In initial experiments, oncostatin M-stimulated HepG2 cells served as a positive control (36). Subsequently, IFN-gamma -stimulation was used as the positive control for STAT-1 activation in T84 cells (see below). In separate studies, the ability of SEB-PBMC CM preabsorbed against IFN-gamma or TNF-alpha neutralizing antibodies (4 h at 37°C; 10 µg/ml antibody; PharMingen) to activate STAT proteins was examined.

Lamina propria lymphocytes (LPL) were isolated from sections of resected human gut using standard protocols (9) and were exposed to SEB (1 µg/106 LPLs; 24 h). The ability of the SEB-LPL CM to evoke a STAT response in T84 cells was then examined. (Surgical specimens were supplied and used according to the guidelines of the Departments of Surgery and Pathology at McMaster University/Hamilton Health Sciences Cooperation.)

Pharmacological Inhibitors

The following four additional experiments were performed: 1) T84 cells were pretreated for 1 h with the tyrosine phosphorylation inhibitor genistein (1-10 µM; Sigma Chemical) and then were treated with SEB-PBMC CM for 12 h plus genistein [the inactive analog of genistein, diazein (Sigma Chemical), was included as a control]; 2) T84 cells were exposed to transcription factor decoys (TFDs; 10 µM) for 20 h in serum-free medium. [TFDs are double-stranded DNA oligonucleotides that contain the STAT binding site (see below) and have been phosphorothioated to enhance entry to the cells and render them less susceptible to degradation. In theory, TFDs will capture the STAT dimers and prevent gene regulation (6, 8, 44).] Cells were rinsed (3 times), cultured in serum containing 10% FCS for 1 h, and then exposed to SEB-PBMC CM for 30 min or 24 h; 3) T84 cells were pretreated with LY-294002 [20 µM; Sigma Chemical (10)], a specific inhibitor of PI 3-K activity, for 10 min and then were exposed to SEB-PBMC CM + LY-294002 for 30 min or 24 h; and 4) T84 cells were pretreated with SB-203580 [1 or 10 µM; Sigma Chemical (19)], a specific inhibitor of p38 MAPK activity, for 1 h and then were exposed to SEB-PBMC CM + SB-203580 for 24 h.

Electrophoretic Mobility Shift Assay

After treatment (15, 30, and 60 min and 4, 12, 24, and 48 h), epithelial nuclear extracts were obtained according to Andrews and Faller (2) with the addition of the enzyme inhibitors aprotinin (10 µg/ml), pepstatin A, leupeptin (both at 2 µg/ml), and phenylmethysulfonyl fluoride (20 µg/ml; all from Sigma Chemical). Electrophoretic mobility shift assays (EMSAs) were conducted using a published protocol (35). Briefly, 5-15 µg of nuclear protein extract were reacted in binding buffer with [alpha -32P]CTP (3,000 Ci/mmol; Dupont) end-labeled double-stranded oligonucleotides (2-3 × 105 counts/min) for the high-affinity sis-inducible element (hSIE; sequences: 5'-GTCGACATTTCCCGTAAATC-3' and 5'-TCGACGATTTACGGGAAATG-3'; see Ref. 42) for 20 min at room temperature. Binding buffer contained 250 mM Tris · Cl (pH 7.5), 40 mM NaCl, 10 mM EDTA, 2.5 mM dithiothreitol, 10 mM spermidine, and 25% glycerol plus 2 µg poly(dI-dC) and 1 µg calf thymus DNA (Sigma Chemical). Two microliters of indicator dye [0.25% (wt/vol) bromphenol blue, 5% (vol/vol) glycerol] were added to each sample (total volume 22-25 µl), which was then electrophoresed on a 5% polyacrylamide gel (40:1, acrylamide-bisacrylamide) containing 1.25% glycerol in 0.25× Tris-borate-EDTA (TBE) buffer (10× TBE = 89 mM Tris-borate and 2 mM EDTA). Gels were run for 3.5 h at 95 V and dried, and bands were visualized by autoradiography.

Samples of nuclear extracts were reacted with nonlabeled hSIE (100 ng double-stranded oligonucleotide; 15-20 min on ice) as a cold competitor before exposure to the radiolabeled probe or with a mutant variant of the hSIE sequence that does not bind STAT-1 (42). Supershift EMSAs were performed in which samples were preincubated with antibodies against STAT-1 (2 different polyclonal antibodies were used during this study), tyrosine 701-phosphorylated STAT-1 (monoclonal antibody), or STAT-3 (polyclonal antibody; 2 µg for 20 min on ice; Santa Cruz Biotechnology, Santa Cruz, CA) before reaction with the hSIE probe. An irrelevant IgG-matched antibody (i.e., anti-STAT-6) was used as a specificity control. Finally, EMSAs were also conducted under identical experimental conditions, except that the nuclear extracts were exposed to double-stranded DNA oligonucleotide probes known to bind either STAT-5 or STAT-6 (for sequences see Refs. 20 and 32).

Epithelial Paracellular Permeability

T84 monolayers grown on filter supports (27) were cultured with 25 or 50% SEB-PBMC CM (placed in the basal compartment of the culture well) for 24 h with or without pretreatment with TFDs (10 µM), genistein (5 µM), LY-294002 (20 µM), or SB-203580 (1 or 10 µM). T84 monolayers were then mounted in Ussing chambers and clamped at 0 V, and permeability (transepithelial ion resistance measured by the differential pulse method) was assessed [IFN-gamma only-treated cells were not used in this portion of the study, since it takes 48-72 h to observe changes in T84 function with low-dose IFN-gamma (10 ng/ml)].

Data Presentation and Analysis

EMSA analyses were conducted on at least three separate epithelial preparations for each experimental condition. Data from the epithelial physiology studies are presented as means ± SE, where n is the number of experiments (with 2 monolayers per condition/experiment). Data were normalized based on control responses within each experiment and were analyzed by one-way ANOVA followed by post hoc Newman-Keuls comparisons (27). P < 0.05 was accepted as a level of statistically significant difference.


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IFN-gamma Evokes Epithelial STAT-1 Activation

T84, HT-29, and Caco-2 epithelial cells displayed no or negligible constitutive nuclear STAT-1 and no STAT-3 identifiable by EMSA using the hSIE probe (n = 2-7). STAT-1 activation was apparent in T84 cells 15-240 min post-IFN-gamma treatment (10 ng/ml; n = 2-5). The signal was most prominent after 30 min (Fig. 1A), and, although the signal was significantly diminished, it was still detectable in T84 nuclear extracts 24 h post-IFN-gamma (n = 3; data not shown). STAT protein DNA binding activity on EMSA was not apparent 48 h post-IFN-gamma treatment (n = 3). Inclusion of the cold competitor, but not a mutated cold competitor, blocked detection of the STAT-1 complex (Fig. 1B). Similarly, inclusion of polyclonal anti-STAT-1 antibodies in the reaction mixture either blocked detection of the STAT-1 band (Fig. 1B) or supershifted the band (Fig. 2A). This discrepancy is most likely due to differences in batches of antibodies, and indeed others have shown that STAT-specific antibodies can elicit supershifts or prevent detection of the suspect band (33, 44). The isotype-matched anti-STAT-6 antibody did not interfere with EMSA detection of STAT-1 (Fig. 1B). Also, use of a monoclonal antibody directed against the tyrosine (residue 701)-phosphorylated motif of STAT-1 did not cause a supershift on EMSA but significantly reduced the intensity of the detected STAT-1 band (n = 3; data not shown). T84 cells treated with IL-4, IL-10, or TNF-alpha (Fig. 1C) for 30 min, 240 min, or 24 h (data not shown) did not result in a detectable STAT-1 response. In comparison with T84 cells, nuclear extracts from HT-29 or Caco-2 cells exposed to IFN-gamma or 50% SEB-PBMC CM for 30 min had a readily detectable STAT-1 signal that was abrogated by inclusion of the appropriate cold competitor (n = 3).


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Fig. 1.   Electrophoretic mobility shift assay (EMSA) autoradiographs showing signal transducers and activators of transcription (STAT) in epithelial nuclear extracts. A: STAT-1 activity in T84 cells exposed to interferon (IFN)-gamma (10 ng/ml) or Staphylococcus aureus enterotoxin B (SEB)-peripheral blood mononuclear cell (PBMC)-conditioned medium (CM; 50%) for 30 min or 4 h (positive control is oncostatin M-treated HepG2 cells; see Ref. 36 for appropriate controls for this activity and resolution into discernable STAT-1 and STAT-3 bands; n = 3-7). B: specificity of the T84 STAT-1 signal is confirmed by loss of the band by inclusion of the anti-STAT-1 antibody (also see Fig. 2A) or a cold competitor but not an anti-STAT-6 antibody or mutant cold competitor (representative of 3 experiments). NS, nonspecific band. C: STAT-1 is not activated in T84 cells treated with interleukin (IL)-4, IL-10, or tumor necrosis factor (TNF)-alpha (all at 10 ng/ml; n = 2-3). fp, Free probe; arrowhead, STAT-1 band.



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Fig. 2.   EMSA autoradiographs showing T84 STAT activation in response to 50% SEB-PBMC CM. A: exposure to SEB-PBMC from 4 different donors activates STAT-1 (arrow) and a weaker STAT-3 signal (large arrowhead). Small arrowhead, supershifted STAT-1; ab, antibody. B: STAT-1 homodimers (arrowhead on bottom), STAT-1/-3 heterodimers (arrowhead in middle), and STAT-3 homodimers (arrowhead on top) in T84 nuclear extracts from cells treated with SEB-PBMC CM for 30 min from this particular blood donor. The identify of STAT-3 in the EMSA banding pattern was confirmed by supershifts with anti-STAT-3 antibodies (small arrow). Large arrow, position of STAT-1 (n = 7).

SEB-PBMC CM Elicits Epithelial STAT-1 and STAT-3 Signals

Exposure of T84 cells to SEB only (or the related superantigen, Staphylococcus aureus enterotoxin A) did not elicit a detectable STAT response. This would be expected since T84 cells have very low constitutive expression of major histocompatibility class (MHC) II, the ligand for superantigens. Treatment with SEB-PBMC CM resulted in a time- and dose-dependent induction of STAT activation. STAT-1 activation occurred within 5 min of treatment (data not shown); like IFN-gamma stimulation, was most prominent 30-60 min posttreatment (Fig. 1A); and was still readily detectable on EMSAs 12-14 h after treatment. A weak STAT band was detectable at 24 h, but not 48 h, after exposure to SEB-PBMC CM (n = 3; data not shown). SEB-PBMC CM from all blood donors consistently gave a strong T84 STAT-1 signal (Fig. 2A shows CM from 4 different donors), the specificity of which was confirmed by cold competitor and antibody experiments (Fig. 1B). STAT-1 activation was detectable with as little as 3% and increased dose dependently to 25-50% SEB-PBMC CM, which elicited similar STAT responses as detectable by EMSA. Additionally, exposure (30 min) to SEB-PBMC CM from some, but not all, PBMC preparations resulted in detectable STAT-3 homodimers and STAT-1/3 heterodimers in T84 nuclear extracts (Fig. 2B and see Figs. 5A and 6A). The EMSA band corresponding to STAT-3 [supershifted by anti-STAT-3 antibodies (Fig. 2B)] was consistently less prominent than that for STAT-1. EMSA analysis with STAT-5- or STAT-6-specific probes revealed no positivity in nuclear extracts from SEB-PBMC CM-treated cells [n = 2-4; data not shown; with the use of the above conditions STAT-6 was activated in IL-4-treated T84 cells (8)].

The level of IFN-gamma in the 50% SEB-PBMC CM was approximately fourfold less than that used in the recombinant cytokine studies (2.78 ± 0.53 ng/ml; n = 4). Neutralization of IFN-gamma via anti-IFN-gamma antibodies significantly reduced the intensity of the STAT-1 signal observed after 30 min of exposure to 50% and abolished the signal elicited in response to 25% SEB-PBMC CM (Fig. 3). Inclusion of the control isotype-matched anti-TNF-alpha antibody did not interfere with the T84 STAT response to SEB-PBMC CM (data not shown).


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Fig. 3.   STAT-1 activation in T84 epithelial cells in response to 30 min exposure to SEB-PBMC CM at concentrations of 50 and 25% is partially or wholly inhibited by addition of anti-IFN-gamma antibodies to the CM (IFN-gamma at 10 ng/ml; anti-IFN-gamma at 10 µg/ml). cc, Cold competitor; arrowhead, position of STAT-1. (representative of 3 experiments).

When examining enteric epithelial responses to immune mediators, it is appropriate to conduct studies with gut-derived lymphocytes whenever they are available. Thus, as shown in Fig. 4, exposure to CM from SEB-activated LPL obtained from resected ileal or colonic tissue from patients with active Crohn's disease (n = 3) or colonic cancer (n = 1) resulted in STAT-1 activation in T84 cells.


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Fig. 4.   EMSA showing activation of a nuclear STAT-1 (arrow) response in T84 cells treated with CM (50%, 30 min) from SEB-activated lamina propria lymphocytes from patients with active Crohn's disease (n = 3, i.e., CM 1-3) or a control patient with colonic cancer (CM 4). EMSA shows that the STAT-1 band could be competed out with a cold competitor and supershifted with an anti-STAT-1 antibody (arrowhead).

Pharmacological Assessment of SEB-PBMC CM Evoked Drop in Transepithelial Resistance

Having shown a clear activation of STAT-1 (±STAT-3), we proceeded to examine the putative involvement of STATs in the SEB-PBMC CM-evoked drop in transepithelial resistance. Also, since an interaction of STAT proteins with PI 3-K has been suggested (37), we assessed the effect of PI 3-K inhibition on SEB-PBMC CM effects on T84 resistance.

Genistein. STAT activation is dependent on tyrosine phosphorylation; thus, we first employed a general inhibitor of tyrosine phosphorylation. Addition of genistein to T84 cells did not evoke a STAT response (Fig. 5A). In contrast, genistein treatment of T84 cells concomitantly exposed to SEB-PBMC CM led to reduced STAT-1- and STAT-3 DNA-binding activity on EMSA (Fig. 5A; n = 2). Figure 5B shows that genistein (5 µM), but not the inactive isomer daidzein, partially prevented the SEB-PBMC CM-induced drop in epithelial monolayer resistance.


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Fig. 5.   A: a representative EMSA showing that genistein (GEN) alone did not evoke a STAT response in T84 cells but did diminish the STAT activity in nuclear extracts from T84 cells treated 12 h previously with a 50% SEB-PBMC CM [arrows indicate STAT-1 homodimer (1-1), STAT-1/3 heterodimer (1-3), and STAT-3 homodimer (3-3); n = 2]. B: genistein (5 µM, 1 h pretreatment and 12 h concomitant with CM), but not the inactive analog daidzein (DAIZ), partially prevents the drop in transepithelial resistance across T84 monolayers observed 12 h after treatment with 50% SEB-PBMC CM. * and delta P < 0.05 compared with control and other groups, respectively; control resistance range was 1,005-1,818 Omega /cm2; data are means ± SE and are presented as % of the control response; n = 4-7 (except daidzein, where n = 2 experiments, 6 epithelial monolayers).

TFDs. EMSAs conducted with the TFDs as a cold competitor (i.e., added to the reaction mixture and not treatment of the epithelial cells directly) confirmed that the phosphorothioation process did not significantly impair the ability of the TFD to bind the STAT proteins (see Fig. 6A, lane 3). Subsequently, T84 cells were exposed to TFDs for 20 h and then were treated with 25% SEB-PBMC CM for 30 min. The T84 cells transfected with the TFDs showed a significant reduction in, but not a complete block of, the STAT-1 activation elicited by a subsequent exposure to SEB-PBMC CM (n = 3; STAT identification on EMSA blocked by ~40-70%; Fig. 6B). However, with the use of the same treatment regime and SEB-PBMC CM from the same blood cell donors (+3 additional experiments), the TFDs had no effect on the drop in transepithelial resistance evoked by a 24-h exposure to SEB-PBMC CM (Fig. 6C). Use of a mutated sequence of the hSIE probe revealed that the mutant TFD did not function as a cold competitor on EMSA (data not shown) and did not affect transepithelial resistance (Fig. 6C). These data neither support nor conclusively refute STAT-1/-3 modulation of epithelial paracellular permeability, so we utilized a pharmacological approach to assess the possible role of PI 3-K and p38 MAPK in the SEB-PBMC CM-induced drop in transepithelial resistance.


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Fig. 6.   A: EMSA showing that transcription factor decoys (TFDs; designated thiol cold comp; lane 3) specifically bind to the STAT proteins activated in T84 cells by a 30-min exposure to SEB-PBMC CM. *Super-shift; arrows, STAT-1, STAT-1/3, and STAT-3, respectively; IFN-gamma is included as a positive control for STAT-1. B: EMSA showing that the STAT-1 response evoked by 25% SEB-PBMC CM from 3 different blood donors is reduced by pretreating the T84 cells with TFDs (20 h at 10 µM). Specificity of the band is confirmed by inclusion of cold competitor (cold comp) or anti-STAT-1 antibodies in the reaction mixture before electrophoresis. Use of the antibody against tyrosine 701-phosphorylated STAT-1 also reduced the signal by competing for the site on STAT-1 that binds the high-affinity sis-inducible element probe. C: bar graph showing that the change in T84 transepithelial ion resistance induced by 24 h exposure to 25% SEB-PBMC CM is not affected by pretreatment with TFDs or mutant TFDs (mTFD). Control resistance range was 2,000-2,500 Omega /cm2; data are means ± SE and are presented as % of the control response; n = 6 (2 T84 monolayers/experiment). *P < 0.05 compared with control.

LY-294002. LY-294002 is recognized as a specific inhibitor of PI 3-K activity, a molecule that has been implicated in cytokine signaling and in regulation of the cytoskeleton (10, 41). Pretreatment with this agent significantly prevented the increase in epithelial permeability induced by 24 h of exposure to 25% SEB-PBMC CM (Fig. 7; n = 6). Also, the STAT-1 signal elicited by 1 h of exposure to IFN-gamma (10 ng/ml) was not affected by cotreatment with LY-294002 (personal observation).


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Fig. 7.   Bar graph showing that the drop in T84 transepithelial ion resistance induced by 24 h exposure to 25% SEB-PBMC CM is significantly reduced by 10 min pretreatment and concomitant treatment with the inhibitor of phosphatidylinositol 3'-kinase activity, LY-294002 (20 µM). * and delta P < 0.05 compared with control and other groups, respectively; control resistance range was 1,667-2,857 Omega /cm2; data are means ± SE and are presented as % of the control response; n = 6 (2 monolayers/experiment).

SB-203580. Finally, the specific p38 MAPK inhibitor SB-203580 was employed. These experiments served the following two roles: 1) to examine the role of p38 MAPK in the control of paracellular permeability and 2) to serve as a pharmacological control for comparison with PI 3-K inhibition. With the use of doses and treatment times defined in the literature (19), SB-203580 had no significant effect on the reduction in transepithelial resistance caused by exposure to 25% SEB-PBMC CM for 24 h [control = 1,339 ± 194; SB-20358 (10 µM) only = 1,274 ± 172; SEB-PBMC CM = 777 ± 135 (P < 0.05 compared with control); SEB-PBMC CM + SB-20358 = 599 ± 123 (P < 0.05 compared with control) Omega /cm2 (n = 4 monolayers from 2 experiments)].


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Interference with cytokine intercellular signaling cascades allows the modulation of physiological or pathophysiological processes by administration, or inhibition of, the appropriate cytokine. An alternative strategy is the extrinsic regulation of the cytokine's target cell. For example, adenoviral gene transfer of a superrepressor inhibitor protein-kappa B has been used to inhibit NF-kappa B activity and consequently the production of proinflammatory cytokines by epithelial cells (16). In the present study, we show that IFN-gamma , the prototypic activator of STAT-1, and SEB-PBMC CM activate STAT proteins in model gut epithelia. Pharmacological interventions suggest that the disruption of epithelial barrier function caused by exposure to SEB-PBMC CM occurs, at least in part, via a PI 3-K pathway, and we are unable to provide conclusive evidence in support of or refuting STAT-1 (or STAT-3) involvement in the regulation of epithelial paracellular permeability.

Activation of the JAK-STAT signaling pathway has piqued the interest of the epithelial biologist (13, 17, 21, 29, 35). IFN-gamma mobilizes STAT-1 only (15), and, since IFN-gamma regulates many epithelial functions including permeability and expression of MHC II (26, 39), we first examined STAT-1 induction by IFN-gamma in gut-derived epithelia. Exposure to IFN-gamma resulted in detectable STAT-1 proteins in nuclear extracts 15 min posttreatment, and, although the intensity of the signal was considerably diminished, nuclear STAT-1 proteins were still apparent after 24 h. The significance of the longevity of this response is unclear but could be linked to the 24- to 72-h period required to observe the IFN-gamma (>10 ng/ml)-induced increase in T84 epithelial permeability (24). The specificity of the IFN-gamma response was shown by inhibition with a cold competitor, and inclusion of STAT-1-specific antibodies in the EMSAs confirmed the identity of the detected band as authentic STAT-1. IL-4 and TNF-alpha do not mobilize STAT-1, although both can affect epithelial permeability, and T84 cells treated with either cytokine showed no STAT-1 signal when nuclear extracts were reacted with the hSIE probe (indicates specificity of the probe and the IFN-gamma effect). IL-10 can activate STAT-1 and STAT-3 (37) but did not elicit a signal in T84 cells. Direct effects of IL-10 on T84 cells have been reported, albeit at a high dose (100 ng/ml) administered daily for 7 days (25). The discrepancy between these data and our findings suggests that either >10 ng/ml of IL-10 are required to elicit detectable STAT-1 or STAT-3 signals in T84 cells or that the IL-10 effect in the former study was via a STAT-independent mechanism, perhaps analogous to that proposed for IL-10 inhibition of lipopolysaccharide activation of macrophages (31). These studies underscore the need to precisely define STAT signaling in multiple cell types (cell lines and primary isolates) if pharmacological STAT regulation is to be a therapeutic goal (14).

Analysis of the effects of recombinant cytokines on epithelia has been complemented by coculture studies comprising immune cells (±specific stimuli) or CM (18, 27, 40). We showed that superantigen activation of T cells caused increased T84 permeability that was apparent 12 h after the initiation of the coculture and was maximal by 24 h (11, 27). In the present study, we extended these observations by assessing STAT protein activation in response to SEB-PBMC CM. EMSA revealed that SEB-PBMC CM treatment of T84 cells resulted in STAT-1 activation, but not STAT-5 or STAT-6, and this signal was still evident 24 h posttreatment. The kinetics (i.e., intensity of STAT bands, induction and longevity of the response) of this response were similar to those elicited by recombinant IFN-gamma , and neutralization of IFN-gamma completely abolished the epithelial STAT-1 response to SEB-PBMC CM. These data are consistent with in vivo and in vitro data showing that superantigen immune activation is initially dominated by IFN-gamma -driven events (5, 12). In addition, we found that exposure of T84 cells to SEB-LPL CM also resulted in an increase in STAT-1 DNA binding, and this may have disease ramifications (e.g., active participation in modulation of mucosal immunity) should superantigens gain access to the gut mucosa.

Exposure of T84 cells to some, but not all, SEB-PBMC CM resulted in STAT-1 and STAT-3 activation; STAT-3 mediates some of the effects of IL-6-type cytokines, IL-2, and IL-10 (15). IL-6 levels are increased in SEB-PBMC CM; however, neutralizing IL-6 antibodies failed to abrogate the loss of epithelial barrier function evoked by SEB-PBMC CM (27). The functional significance of induction of a STAT-1 vs. a STAT-1/-3 signal in response to SEB-PBMC CM is unclear. However, it is intriguing to speculate that the pattern of STAT protein responses elicited in an epithelium may be a determinant in the host's overall response to immune activation, such as the susceptibility to infection, time to recovery, etc. Increased activation of STAT-1 has been shown in bronchial epithelium from patients with asthma compared with controls, and this occurred in the absence of any significant increase in tissue levels of IFN-gamma or IFN-gamma -producing cells (38). This finding is complemented by the present examination of enteric epithelium showing that exposure to IFN-gamma or SEB-PBMC CM resulted in a nuclear STAT-1 signal that was still detectable 24 h posttreatment.

Analysis of STAT responses must be coupled to functional studies. With the exception of MHC II and intercellular adhesion molecule-1 expression (28, 43), there is little firm evidence relating to epithelial functions that are directly regulated by STAT proteins. Here, T84 cells treated with the tyrosine phosphorylation inhibitor genistein showed reduced STAT-1/-3 activation and a partial inhibition of the drop in transepithelial resistance caused by SEB-PBMC CM. Similarly, recent studies found that genistein or the unrelated tyrosine kinase inhibitor herbimycin can reduce immune-mediated disruption of epithelial barrier function (4, 7, 40); STAT proteins were not examined in these studies.

The correlation of genistein's ability to reduce the disruption in epithelial barrier function and STAT signaling elicited by SEB-PBMC CM suggests but does not prove that the two events are causally linked. Indeed, it is feasible that inhibition of tyrosine phosphorylation of non-STAT proteins contributes to the maintenance of epithelial barrier integrity. Endogenous off-signals for STAT proteins are being identified (30); however, specific pharmacological inhibitors of STAT proteins are not available. Therefore, we attempted to block STAT signaling using TFDs following methods that have been shown to block, or reduce, the effects of STAT protein activation or NF-kappa B signaling (6, 8, 44). T84 epithelia pretreated with TFDs showed reduced STAT-1 activation when subsequently challenged with SEB-PBMC CM, but the disruption in barrier function remained unaffected. At least three possibilities explain these findings. 1) The amount of STAT-1 that escaped the TFDs was enough to affect gene transcription, leading to opening of the epithelial tight junctions; 2) transepithelial resistance is a sensitive assay of permeability, and since TFD transfection will have been random, responses from cells that did not incorporate a significant amount of TFD could account for the ~50% drop in resistance; and 3) STAT-1 is not involved in the modulation of paracellular permeability. The investigative technique used here provides no conclusive evidence in support of, or refuting, STAT-1 involvement in the regulation of epithelial permeability. To unequivocally address this issue requires the development of an epithelium containing a stable (and preferably inducible) dominant-negative STAT-1 gene (46) that is also suitable for Ussing chamber/flux studies.

IFN-gamma does directly (24), or in the context of CM (11), affect epithelial permeability, and, although research has focused on STAT-1 as the major molecule that transduces IFN-gamma effects, data are emerging showing IFN-gamma - and STAT-1-independent signaling mechanisms (34). A common pathway in intracellular cytokine signaling involves the activation of PI 3-K (41). This molecule has been implicated in vesicular trafficking, modulation of the cytoskeleton (a determinant of tight junction activity; see Refs. 10 and 23), and in IL-13 regulation of HT-29 epithelial cell survival (45). Moreover, recent data from our laboratory (8) indicate that IL-4 disruption of T84 permeability is significantly ablated when the cells are cotreated with inhibitors of PI 3-K activity. Thus we assessed the ability of the specific PI 3-K inhibitor LY-294002 to affect SEB-PBMC CM-induced increases in epithelial permeability; LY-294002 consistently and significantly reduced the increased permeability caused by exposure to SEB-PBMC CM. These findings fit with the effects of genistein in the physiological experiments because PI 3-K activity is dependent on tyrosine phosphorylation. Moreover, we (personal observation and Ref. 8) and others (1) have shown that PI 3-K inhibitors do not prevent STAT activation as monitored by EMSA and Western blotting. Also, inhibition of p38 MAPK activity did not alter T84 responses to SEB-PBMC CM. This observation is noteworthy in the context of a recent report showing stimulus-dependent cross-talk between the STAT-1 and MAPK pathways in macrophages (19). Collectively, our pharmacological studies provide data in support of SEB-PBMC CM-induced activation of PI 3-K and indicate that a PI 3-K-dependent mechanism is at least partially responsible for the regulation of epithelial permeability in this model system. Additional studies are required to test this hypothesis and provide a fuller understanding of the kinetics of PI 3-K activity in enteric epithelia and downstream events after PI 3-K activation.

In summary: 1) IFN-gamma caused a rapid and specific induction of STAT-1 in three human colonic epithelial cell lines; 2) exposure to CM from SEB-activated PBMC or LPL evoked a STAT-1 or a STAT-1/-3 (but not STAT-5 or STAT-6) response in T84 cells, and we suggest that epithelial signal transduction is important in the homeostatic balance between physiological and pathophysiological events; and 3) regulation of epithelial paracellular permeability is at least partially dependent on a PI 3-K pathway, and the putative involvement of STAT-1 in the modulation of epithelial barrier function requires additional experimentation. The latter two observations raise important issues for future studies aimed at understanding the kinetics of epithelial STAT protein activation and the physiological events that they control and for elucidation of the full role of PI 3-K in the modulation of epithelial tight junction activity.


    ACKNOWLEDGEMENTS

The technical assistance of J. Lu and J. Brokenshire is gratefully acknowledged.


    FOOTNOTES

This work was funded by Medical Research of Canada grants to D. M. McKay and C. D. Richards and in part by a Crohn's and Colitis Foundation of Canada grant to D. M. McKay.

Portions of this work were presented at the American Association of Gastroenterology Meeting in New Orleans (May 1998).

Address for reprint requests and other correspondence: D. M. McKay, Intestinal Disease Research Programme, HSC-3N5, McMaster Univ., 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5 (E-mail:mckayd{at}fhs.mcmaster.ca).

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.

Received 22 November 1999; accepted in final form 8 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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