Division of Gastroenterology, Department of Medicine, University of California-San Diego School of Medicine, San Diego, California 92103-8414
Submitted 14 May 2003 ; accepted in final form 30 August 2003
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ABSTRACT |
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sodium-potassium-chloride cotransporter; cystic fibrosis transmembrane conductance regulator; calcium-activated chloride channel
Under inflammatory conditions, the epithelium is exposed to a plethora of cytokines and other soluble mediators that are secreted by lymphocytes, macrophages, and epithelial cells themselves. One of these, IFN-, a proinflammatory cytokine, is secreted by activated T helper 1 lymphocytes and natural killer (NK) cells and plays a central role in inflammation (5, 11), in that it targets and activates macrophages, endothelial cells, and NK cells as well as epithelial cells, resulting in increases in major histocompatability complex class I/II expression (7). In patients with Crohn's disease, which can affect both the small and large intestine, mucosal production of IFN-
is strongly increased (14, 24, 27). The physiologically relevant targets of IFN-
are not well understood, but this cytokine has been shown to increase paracellular permeability (20, 28) and to downregulate ion transport functions (28) in colonic epithelial cell lines. Moreover, IFN-
has recently been identified as a key homeostatic mediator that may prevent excessive fluid loss by the small intestine in the setting of diarrhea (30).
Transepithelial ion transport is based on the activity of specific ion channels and transporters. Stimulation of colonic epithelial cell lines with IFN- has been shown to downregulate expression of the cAMP-dependent Cl- channel CFTR (4) and the Na+/H+ exchangers NHE2 and NHE3 (22), which also was verified in rat intestine (22). However, intestinal epithelial cell lines, although very useful reductionist models, are limited in that they consist only of cells with a secretory phenotype and lack a fully differentiated epithelium and other mucosal cells. Furthermore, most available transformed intestinal epithelial cell lines are of colonic origin, which might complicate extrapolation of data to the epithelium of the small intestine, which is frequently affected by Crohn's disease. Finally, ex vivo studies of human intestinal segments are restricted to experiments of short duration, whereas most findings in intestinal cell lines have indicated that many effects of cytokines on epithelial function are only seen after prolonged cytokine exposure.
To overcome these limitations, it is necessary to use in vivo models of human intestinal epithelium. Thus in the present study, we sought to develop a defined in vivo model of human small intestinal epithelium that could be used to understand persistent cytokine effects on physiological responses of the epithelium. We employed human small intestinal xenografts combined with studies of ion transport in modified Ussing chambers to demonstrate that both calcium- and cAMP-dependent Cl- secretion are downregulated in human small intestinal epithelium by prolonged treatment with IFN-, which may be due, at least in part, to decreased levels of Na+-K+-2Cl- cotransporter 1 (NKCC1) and the
-subunit of Na+-K+-ATPase. These data indicate that IFN-
can exert antisecretory functions in vivo.
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MATERIALS AND METHODS |
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Human fetal intestinal xenografts. Segments of human fetal small intestine (gestational age 1620 wk) were transplanted subcutaneously into the rear flanks of mice with severe combined immunodeficiency (SCID) as previously described (26). At 10 wk or more after transplantation, the intestinal xenografts resembled morphologically mature pediatric intestine, with an epithelium of entirely human origin (26). Experimental procedures were approved by the University of California-San Diego Human and Animal Subjects Committees. Fetal tissues were supplied by an independent procurement organization, and all studies were in accordance with US laws and APS regulations regarding fetal tissue research. SCID mice carrying mature xenografts were injected intraperitoneally with 10 µg human IFN- or PBS (controls) 24 h before xenograft harvest. Human IFN-
has no activity on mouse cells bearing IFN-
receptors (15), so the direct effects of the cytokine are restricted to human cells in the xenograft.
RNA isolation and detection of mRNA expression. Total cellular RNA was extracted from xenografts by using TRIzol reagent and was treated with ribonuclease-free deoxyribonuclease. For RT-PCR, 1 µg of total cellular RNA was reverse transcribed and complementary DNA was amplified as described previously (12). The primers for Mig were as described earlier (13). The primers for cytokeratin 8 were sense 5'-GAT CTC GGA CAC ATC TGT GGT GCT GTC-3' and antisense 5'-ATC CCC GTG CTT CCC AGC CAG GCT CTG C-3' and for GAPDH were sense 5'-ATC AAC GAC CCC TTC ATT GAC C-3' and antisense 5'-CCA GTA GAC TCC ACG ACA TAC TCA GC-3'. The amplification profile was 2535 cycles of 45-s denaturation at 95°C and 2.5 min annealing and extension at 60°C. Negative control reactions had no added RNA in the RT and subsequent PCR amplification.
Measurement of mucosal ion transport in human intestinal xenografts. Xenografts were harvested, stripped of seromuscular layers by blunt dissection, and mounted in modified Ussing chambers (aperture = 0.07 cm2). The mucosal sheets were bathed in circulating Ringer solution [composition (in mM): 141.0 Na+, 4.2 K+, 3.0 Ca2+, 1.2 Mg2+, 125.8 Cl-, 27.6 , 3.2 H2PO4, and 10 glucose or mannitol] and gassed with 95% O2-5% CO2. The tissues were allowed to equilibrate for 60 min and were voltage-clamped to zero PD by the continuous application of a short-circuit current (Isc). Changes in ion transport across the mucosa in response to agonists were recorded as changes in Isc (
Isc) required to maintain a zero voltage clamp. In some experiments, intermittent measures of changes in open-circuit PD were used as a more sensitive surrogate for active ion transport according to Ohm's law.
Immunoprecipitation and Western blotting. Human intestinal xenografts were stripped of seromuscular layers by blunt dissection. The tissue was placed in ice-cold lysis buffer [consisting of 20 mM Tris (pH 7.4), 20 mM NaCl, 10 mM MgCl2, 1 mM NaVO4, 1 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride], homogenized, incubated at 4°C for 30 min, and then centrifuged at 10,000 rpm for 10 min, and the pellet was discarded. An aliquot was removed from each sample to determine protein content, and samples were adjusted so that they contained equal amounts of protein. Samples were boiled for 4 min, and proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (DuPont NEN) overnight at 4°C. The membranes were washed in 1% blocking buffer for 30 min, incubated with the appropriate dilution of primary antibody, and washed in Tris-buffered saline with 1% Tween (TBST). Following the washes, horseradish peroxidase-conjugated secondary antibodies were added to the membrane in 1% blocking buffer and were allowed to incubate for an additional 30 min. This was followed by a further washing in TBST. Immunoreactive proteins were detected by using an enhanced chemiluminescence detection kit (Amersham) and exposure of the membrane to X-ray film. The intensity of protein bands was determined by densitometry by using NIH Image software.
Histological analysis of xenografts. For immunohistochemical analysis, intestinal xenografts were embedded in optimum cutting temperature compound (TissueTek; Miles Scientific, Naperville, IL) and snap-frozen in methylbutane/dry ice or fixed in 10% neutral buffered formalin. Formalin-fixed tissues were embedded in paraffin, and 5-µm sections were prepared and stained with hematoxylin and eosin. From snap-frozen tissue samples, cryostat sections (5 µm) were prepared, fixed in 10% formalin for 10 min, and blocked (1% BSA, 0.2% blocking reagent, 0.3% Triton X-100). These sections were incubated with a polyclonal antibody to NKCC1 or a monoclonal antibody to Na+-K+-ATPase 1-subunit, washed in PBS, and incubated with Cy3-labeled donkey anti-goat IgG or Cy2-labeled anti-mouse IgG secondary antibodies (Amersham Pharmacia Biotech).
Statistical analysis. All results are expressed as means ± SE for a series of experiments. Student's t-tests were used to compare paired data. One-way ANOVA with a Student-Newman-Keuls posttest was used when three or more groups of data were compared. P values <0.05 were considered significant.
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RESULTS |
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To study ion transport function in human intestinal xenografts, we mounted the tissue, stripped of seromuscular layers, in modified Ussing chambers (aperture = 0.07 cm2). After equilibration, the tissues were stimulated with the cAMP-dependent agonists PGE2 (3 µM) or IBMX (1 µM) or the Ca2+-dependent agonist CCh (300 µM). As seen in Fig. 2, A and B, the tissue responded to the consecutive addition of agonists with an increase in Isc. The cAMP-dependent agonists induced ion transport responses with sustained kinetics, whereas CCh induced transient responses. The functional polarity of the tissue was demonstrated by mucosal vs. serosal additions of CCh, whose receptors are located on the epithelial basolateral aspect. As seen in Fig. 2C, the ion transport response obtained with mucosal addition of CCh was significantly less than that obtained with serosal addition.
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To investigate whether the observed changes in ion transport were due to Cl- secretion, mounted tissues were incubated with the NKCC inhibitor bumetanide (100 µM) for 20 min before stimulation. Furthermore, to determine the involvement of the M3 muscarinic receptor, known to mediate the effects of CCh on Cl- secretion in intestinal epithelial cell lines, an antagonist to this receptor, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 100 nM) was used (10). As shown in Fig. 2C, both bumetanide and 4-DAMP decreased Isc after CCh stimulation by 78.2 ± 13.3% (n = 5) and 81.7 ± 7.6% (n = 4), respectively, indicating that the ion transport responses were largely due to electrogenic Cl- secretion and were evoked by muscarinic M3 receptor occupancy. These data demonstrate that the voltage clamp technique could reliably detect Cl- secretory responses in human intestinal xenografts, which provided the basis for examining the effects of long-term exposure to cytokines, specifically IFN-
, on ion transport function.
Effect of IFN- on xenograft ion transport responses. To examine the effects of IFN-
on histological appearance of the xenografts, mice were injected with IFN-
and the morphology of the xenograft tissue was observed. Treatment of intestinal xenografts with IFN-
for 24 h did not induce any visible morphological changes compared with control tissues (Fig. 3), although a slight decrease in the goblet cell census may occur in IFN-
-treated tissues. To show that IFN-
had specific effects on the xenografts, we determined the expression of a known IFN-
-responsive gene, the chemokine Mig (13). As can be seen in Fig. 3, IFN-
treatment induced increased levels of mRNA for Mig. In contrast, mRNA for two controls, cytokeratin 8, which is an epithelial cell-specific gene, and GAPDH, which was assessed as a housekeeping gene that is not regulated by IFN-
, showed no changes in expression after IFN-
treatment, indicating that IFN-
is able to exert a specific effect on small intestinal xenograft tissue in vivo.
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We next examined whether IFN- altered basal epithelial electrophysiological properties. Twenty-four hours after intraperitoneal injection of IFN-
, xenografts were harvested, stripped, and mounted in modified Ussing chambers. No difference was observed in baseline PD between control and IFN-
-treated tissues (Fig. 4). On the other hand, conductance was enhanced in IFN-
-treated tissues to a small but statistically significant extent compared with controls. Tissue conductance was 154.7 ± 11.8 vs. 186.3 ± 9.4 mS/cm2 in control and IFN-
-treated tissues, respectively (means ± SE; P < 0.05; n = 23).
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We subsequently studied whether stimulated ion transport responses were altered in xenografts exposed to IFN-. The effects of CCh and PGE2 on ion transport were measured as decreases in PD in these experiments to improve sensitivity. Responses evoked by CCh (300 µM) were significantly reduced in IFN-
-treated xenografts compared with control tissues (Fig. 4A). Figure 4B shows a typical time course of responses to CCh in control and IFN-
-treated tissues. We also tested whether the cytokine altered responses to PGE2. As shown in Fig. 4A, PD responses to PGE2 (3 µM) were also significantly reduced in IFN-
-treated tissues compared with untreated tissues. Again, Fig. 4C illustrates a typical time course of the PD response to PGE2 in IFN-
-treated and control xenografts. Note that for both CCh and PGE2 the initial PD responses were similar in test and control xenografts but the cytokine-exposed tissue could not sustain normal levels of ion transport over an extended period of time, as illustrated with the prolonged secretory response induced by the cAMP-dependent agonist PGE2.
Levels of membrane transporters in IFN--treated xenografts. To explore the mechanisms of decreased ion transport responses in IFN-
-treated xenografts, we assayed protein levels of key membrane ion transporters. Western blotting experiments revealed that IFN-
treatment decreased expression of the NKCC1 by 57% compared with controls (Fig. 5A). In contrast, levels of the calcium-activated CLCA and cAMP-activated CFTR were similar in IFN-
-treated and control human intestinal xenografts (Fig. 5, B and C). These data indicate that IFN-
does not exert a general effect on membrane protein expression.
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Having demonstrated that 24-h IFN- treatment in human intestinal xenografts leads to a decrease in NKCC1 levels but not in Cl- channel levels, we also carried out experiments to determine whether 24-h IFN-
treatment had any effect on Na+-K+-ATPase levels in intact human tissues. The levels of the
- and
-subunits of Na+-K+-ATPase were determined by Western blotting. As seen in Fig. 6A, 24-h IFN-
treatment reduced the levels of the catalytic
-subunit of Na+-K+-ATPase by 35% compared with control tissues. In contrast, no changes were observed in the levels of the
-subunit of Na+-K+-ATPase between IFN-
-treated and control tissues (Fig. 6B).
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Immunohistological analysis of xenografts verified the decrease in ion transporters. To investigate whether the decrease in ion transporters was apparent by histological analysis and to define the site(s) where protein levels were decreased, snap-frozen tissue was cut and stained for either NKCC1 or Na+-K+-ATPase -subunit. As illustrated in Fig. 7, the decrease in both NKCC1 (Fig. 7, A and B) and Na+-K+-ATPase
-subunit (Fig. 7, C and D) could in fact be visualized by using immunohistochemical analyses. As illustrated by the figure, both NKCC1 and Na+-K+-ATPase
-subunit were strictly limited to the epithelium, with NKCC1 being predominantly in crypt epithelial cells. Immunofluorescent labeling for both transport proteins was appreciably diminished in tissues harvested from mice treated with IFN-
, verifying that the effects of the cytokine preferentially target the epithelium, at least for these proteins and in this xenograft model.
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DISCUSSION |
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The gross morphology of the intestinal xenografts was not altered after 24 h of IFN- treatment, as also observed in murine tissues (31), although in IFN-
-treated tissue a slight decrease in goblet cells may be present. The conductance of IFN-
-treated tissues was also elevated. Prior studies have yielded conflicting findings on the effect of IFN-
on barrier function between cell lines and tissues. Barrier function in cell lines has been reported to be markedly reduced by IFN-
(1, 16, 20), whereas mucosal resistance in tissues appeared to be unaltered, at least in some studies (8, 31). The findings in intact tissue are, however, in contrast to the findings in this study, where 24-h IFN-
treatment induced a small but significant reduction in mucosal resistance (or increase in conductance). It is possible that the time frame and dose of IFN-
are both critical to outcome and that longer periods of cytokine exposure might induce larger changes in this particular epithelial parameter (or that these findings are specific to the small intestine). Notably, the studies of Bode et al. (8), which used human colonic surgical specimens mounted in Ussing chambers, only followed responses to IFN-
for
2 h, likely the longest time that was feasible. Species differences or segmental variation may also explain differences between our findings and those obtained by using murine colon in organ culture (31).
In this study, both Ca2+- and cAMP-dependent secretory responses were decreased in IFN--treated xenografts compared with controls. With short-term exposure (2 h), IFN-
had no influence on active ion transport in human distal colon (8). On the other hand, several reports have implicated IFN-
as an important factor involved in downregulation of active anion secretion in intestinal epithelial cell lines after longer exposure times (1, 4, 9, 20, 28), where Na+-K+-ATPase (9, 28), K+ channels, and NKCC1 (9) have been suggested to be involved. The inhibition of Isc by IFN-
was also suggested (29) to be mediated by nitric oxide (NO) in experiments conducted in one epithelial cell line (Caco-2BBE), but in murine small intestine NO played no role in the attenuation of Isc (31). In our study, elevated levels of inducible NO synthase (iNOS) protein were found in IFN-
-treated tissues compared with controls (data not shown), but concomitant treatment with iNOS inhibitors for 24 h was not feasible, so the functional significance of this observation for ion transport requires further investigation. Yoo et al. (31) have shown that in murine small intestine, IFN-
downregulates Na+-coupled glucose absorption as well as cyclic nucleotide-activated Cl- secretion. Although these findings in mice, where IFN-
treatment downregulated Cl- secretion and Na+-K+-ATPase activity, are consistent with our work reported here, cytokines may in fact exert distinct effects in different species and organs. For example, TNF-
has been shown to stimulate ion secretion in human (8) and porcine (19) intestine but to attenuate Cl- secretion in canine tracheal epithelium (25). Since this might point to species and/or organ differences in the epithelial response to inflammatory mediators, a human-derived model system for studying cytokine-induced alterations in intestinal function over prolonged periods of time was greatly needed.
Furthermore, in this study, 24-h treatment of human intestinal xenografts with IFN- caused decreased levels of NKCC1, whereas no difference was observed in levels of the Cl- channels CLCA and CFTR. Some authors have shown downregulation of CFTR, at least in cultured epithelial cells treated with IFN-
, on the basis of either expression levels (4, 16, 17) or function (9), but this has not been a consistent finding (28). When effects of IFN-
on CFTR expression were seen in cell lines (4), they were both time and concentration dependent. The precise concentration of IFN-
in the xenograft is difficult to estimate, but it is conceivable that the intraperitoneal administration of 10 µg to mice may result in concentrations toward the low end of those used in other studies. In addition, the 24-h time point selected here may be too early for alterations in CFTR to occur. In other studies (3), however, we have observed that effects of various treatments on cultured epithelial cells are actually accelerated in intestinal xenografts, perhaps reflecting the cooperation of subepithelial elements. Moreover, both Western blot and histological studies revealed that the expression levels of NKCC1 and Na+-K+-ATPase
-subunit were already reduced after 24 h of IFN-
treatment, in contrast to our findings with Cl- channel expression. Others have shown that T cell activation in a mouse model decreased Na+-K+-ATPase activity but not levels of either subunit after 3 h (21). This is in accordance with other observations showing that T cell activation induced luminal fluid accumulation by increasing mucosal permeability and reducing Na+-K+-ATPase activity (21). This is also consistent with observations reported in T84 cells (28) and supports the hypothesis of Holmgren et al. (18), who suggested that the effects of IFN-
on transport are dependent on altered expression of transporters and not on changes in second messengers. At the very least, our data show that secretory hyporesponsiveness evoked by IFN-
in human intestine does not require changes in Cl- channel expression. Nevertheless, we cannot exclude the possibility that, in addition to decreased levels of NKCC1 and Na+-K+-ATPase
-subunit, reduced expression of CFTR (and perhaps CLCA) could contribute to secretory dysfunction with longer periods of IFN-
exposure.
Although the ion transport studies in this study were carried out in small intestinal xenografts and are therefore most directly relevant to Crohn's disease, in which IFN- production from lymphocytes is greatly elevated (6), the similarities of secretory transport mechanisms in the small intestine and colon suggest that our findings may also be relevant to ulcerative colitis, in which elevated levels of IFN-
also have been found (6). Furthermore, our findings are relevant to the recent observation that IFN-
may be selectively upregulated as an adaptive and homeostatic factor in a model of congenital diarrhea, the NHE3 knockout mouse, in which levels of CFTR but not NKCC1 or Na+-K+-ATPase were decreased (30).
Much still remains to be learned about both physiological and pathophysiological effects of IFN- and other cytokines on the intestinal epithelium. The seeming paradox that IBD, with increased levels of IFN-
, is associated with diarrhea but that IFN-
was found to have antisecretory effects may be explained by the possibility that IFN-
actually induces diarrhea by impairing absorption. Alternatively, the ability of this cytokine to reduce intestinal secretion could represent an adaptive response to injury, as also suggested by Shull and co-workers (30) in mice lacking NHE3. Because it allows for the study of cytokine effects over prolonged periods of time in human tissue, the model presented here enables studies that can provide novel insights into cytokine effects on intestinal epithelial transport and barrier function. Because of its ability to support chronic exposure, and because of the existence of species differences, the xenograft model may be the optimal one of those currently available to permit observations that are clinically relevant. Our findings from the present study suggest that elevated mucosal levels of IFN-
affect the small intestine in an antisecretory fashion, and thus treatments that might reduce elevated levels of IFN-
could in fact worsen diarrheal disease.
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ACKNOWLEDGMENTS |
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This work was presented, in part, at the 2000 and 2001 meetings of the American Gastroenterological Association (in San Diego, CA and Atlanta, GA, respectively) and has been published in abstract form (Gastroenterology 118: A3118, 2000; Gastroenterology 120: A1014, 2001).
GRANTS
This work was supported by grants to K. E. Barrett and L. Eckmann from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-28305, DK-35108 unit 5, and DK-35108 core B).
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FOOTNOTES |
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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.
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REFERENCES |
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