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GASTROENTEROLOGY Vol. 109, No. 2
formulation used by the authors) has immune-enhancing properties (i.e., less infection). Let us consider those data in more detail. The major differences in infections between the two groups were in the incidences of pneumonia (5 of 64 [8%] vs. 15 of 60 [25%] ). It should be noted that pneumonia was defined as a positive culture in association with "pneumonic" or "atelectatic" changes on the chest radiograph. The assessment of the presence of this complication was made by an "independent observer," but he or she was apparently not blinded to the treatment regimen. Perhaps even more importantly with regard to the diagnosis of pneumonia, the other features that are in most standard definitions (fever, leukocytosis, white blood cells in the sputum) did not have to be present in these patients. Because the patients in the control group had a more dramatic problem with ascites and because a positive sputum culture alone can be seen simply with bacterial colonization, one could speculate that the chest radiograph changes, atelectasis in particular, were actually caused by secondary effects of the intra-abdominal fluid. When Sam Klein and I discussed this report recently, we both wondered why there was so much more ascites in one group than in the other. In fact, this was the most dramatic clinical difference found. The authors took the time to note that the two groups were matched for (at least intravenous) fluid and sodium intake; without this comment, it would have been easy to hypothesize that the major difference was simply that the control patients were salt and water overloaded. Certainly if the control group did inadvertently receive more sodium, this alone could account for the clinical observations. A colleague of mine, who comes from Hong Kong, recently told me that the dietary intake of patients hospitalized there depends on what the family provides. Because the study was not blinded and because the general population may very well believe that nutrient intake is beneficial (all of the data from PRCTs not withstanding), it is not beyond the realm of possibility that the "unfed" control patients' relatives had more concern about nutrition and (overtly or covertly) brought in food from home (which, given my experiences with the standard Asian diet, was not likely to be low in sodium content). It would have been helpful if the authors had provided the total sodium intake data for each group. On the other hand (and my skeptical nature aside), while it is hard to understand why the PNS formulation resulted in less ascites formation, one must consider the possibility that this particular PNS solution is immune enhancing, helpful to a sick liver, or, in some other way, efficacious for these patients. At this time, there are no other data to use for comparison because this is a unique formulation. This treatment differs from standard PNS at least with regard to the branched-chain amino acids, the type (or perhaps quantity) of fat, and the initial intermittency of the infusions. Perhaps one or more of these factors (or others I haven't considered) will prove to be important therapeutic interventions. Nutritional support is entering a new era; investigators are attempting to identify nutrients that have specific biological effects. We have reviewed some of these efforts before (Gastroenterology 1993; 104:936-938, Gastroenterology 1994; 106:1393-1394, Gastroenterology 1994; 107:1208-1209). Given the generally poor record (regarding attempts to establish efficacy in PRCTs) that standard nutritional support has accumulated in the past, these new nutritional modalities may prove to be far better things that we have or have ever had before. But we must also remember what Charles Dickens wrote in Great Expectations: "Take nothing on its looks; take everything on evidence." RONALD L. KORETZ, M.D.
IMMUNE REGULATION OF CYSTIC FIBROSIS TRANSMEMBRANE REGULATOR Besancon F, Przewlocki G, Baro I, Hongre A, Escande D, Edelman A (Institut National de la Sant6 et de la Recherche MSdicale Unit6 245, H f p i t a l St.-Antoine, Paris; Institut National de la Sant~ et de la Recherche M6dicale Unit8 323, Centre Hospitaller Universitaire Necker, Paris; and Laboratoire de Physiologie Cellulaire, Centre National de la Recherche Scientifique Unit8 de Recherche Associ6e 1121 Universit~ Paris XI, Orsay, France). Interferon-y downregulates C F T R gene expression in epithelial cells. A m J Physiol 1 9 9 4 ; 2 6 7 : C 1 3 9 8 - C 1 4 0 4 (November). There is now convincing evidence that cyclic adenosine m o n o p h o s p h a t e - m e d i a t e d C1- conductances in a variety of epithelial cell types, including those in the gut, are mediated by the ~ 170-kilodalton cystic fibrosis transmembrane regulator (CFTR). This important protein, which is responsible for vectorial salt and water transport in epithelia, continues to be the focus of intense research activity. Much of the work to date has provided details of how C F T R expression in a given cell type is regulated at the biophysical and molecular levels. A critical line of research that augments and expands these levels is the regulation of C F T R expression by other cell types that interact with CFTR-producing ceils. To this end, Besancon et al. present an intriguing report of the effect of interferon g a m m a (IFN-y) on expression of the CFTR gene in two gutderived epithelial cell lines: H T - 2 9 and T84. To assess C F T R gene and protein expression, H T 2 9 and T84 ceils were incubated with or without human recombinant I F N - 7. After a specified incubation period, total R N A was isolated for Northern analysis. The authors found that I F N - y reduced C F T R messenger R N A ( m R N A ) at concentrations as low as 0.1 U / m L with marked reductions at 100 U/mL. Incubation of cells with other interferons ( I F N - ~ or IFN-~) had no effect on C F T R m R N A , which established that the effect of I F N - y was distinct from others in the I F N family. The effect of I F N - y was time dependent: C F T R m R N A content of cells incubated with 100 U/mL I F N - y was 60% of controls after ~ 3 hours of incubation, and maximal inhibition occurred by 12 hours. Incubations of < 1 hour had no effect. Importantly, the reduction of C F T R m R N A by I F N - y was not due to a general effect of the cytokine on message levels because actin m R N A levels remained the same, and m R N A levels for 2 ' , 5 ' oligoadenylate synthase, an IFN-inducible gene, were increased by all three IFNs. The mechanism responsible for I F N - ~ - i n d u c e d inhibition of C F T R m R N A was investigated with nuclear run-on assays. Incubation of H T 2 9 cells with I F N - y had no effect on transcription rates of the CFTR, actin, or 28 ribosomal R N A genes. In contrast, I F N - y significantly reduced the half-life of C F T R m R N A (this was assessed by incubating control and I F N - y treated cells for up to 16 hours in actinomycin D to block transcription). Thus, it seems that at least part of the downregulation of C F T R m R N A by I F N - y occurs by destabilization of the transcripts, reminiscent of the effect of another cytokine,
August 1995
tumor necrosis factor (~, on CFTR gene expression (FEBS Lett 1992; 3 1 4 : 3 6 6 - 3 7 0 ) , That study prompted the authors to ask whether the combination of t u m o r necrosis factor and IFN-y, which are both produced during the inflammatory response, would result in a synergistic effect on down-regulation of CFTR. Incubation of H T 2 9 cells with 0.1 U / m L I F N - y and 5 ng/mL tumor necrosis factor was more effective in reducing the abundance of C F T R m R N A than either cytokine alone. Besancon et al. also showed that the effect of I F N - y on C F T R expression was paralleled by a down-regulation of C F T R protein as determined by Western blot analysis of cell lysates. However, longer incubation periods (more than 24 hours) than those required to significantly suppress C F T R m R N A levels were needed to alter protein levels. The effect of I F N - T on C F T R gene expression and protein abundance was supported by functional studies using T84 cells. 36C1- efflux from T84 cells was inhibited by 42% in cells treated for 48 hours with 100 U / m L I F N - y and by 54% in ceils treated with 300 U / m L I F N - 7. Pretreatment with the cytokine markedly reduced the C1- current activated by a cocktail that increased intracellular cyclic adenosine monophosphate levels (containing forskolin, 8-bromo cyclic adenosine monophosphate, and isobutyl-methylxanthine). In contrast, I F N - y pretreatment did not alter outward currents that mainly reflected the activity of K + currents. These studies support the notion that the down-regulation of C F T R m R N A and protein abundance by I F N - 7 reduces C1- permeability of epithelial cells. Comment. IFN-y plays a key role in cell-mediated immune responses. Among other effects, it amplifies the immune response by enhancing expression of major histocompatibility complex class II molecules on macrophages and B lymphocytes and on dendritic, endothelial, mesenchymal, and epithelial cells (Gastroenterology 1994;106:533-539). Like other cytokines, IFN-y has also been shown to influence the growth and function of nonimmune cells in the intestinal mucosa. Besancon et al. now provide evidence that IFNy can alter the expression of an epithelial cell protein involved in ion transport. A role for cytokines in the regulation of intestinal ion transport is not new; for example, interleukins 1 and 3 have been shown to stimulate intestinal CI- secretion, primarily through the generation of arachidonic acid metabolites. With regard to IFN-y, Hiribarren et al. (Gut 1993;34:616-620) produced a cytokine-enriched supernatant (including IFN-7) by incubating lymphocytes with phytohemagglutinin. Long-term (48 hours) exposure of Ussing-chambered HT29 monolayers with the supernatants significantly decreased short-circuit current (a measure of Cl- secretion) and monolayer resistance compared with control supernatants nor enriched in the cytokines. Furthermore, Holmgren et al. (Scand J Immunol 1989;30:499-503) showed that pretreatment of T84 cell monolayers with IFN-y for 24 hours markedly decreased CI- secretory responses to vasoactive intestinal polypeptide, cholera toxin, and carbachol. There are several reasons why the studies of Holmgren et al. provide an important complement to those of Besancon et al. First, IFN-y reduced secretory responses to several secretagogues that act through a variety of second messenger pathways, including cyclic adenosine monophosphate and calcium, suggesting that the effect likely occurs at a step subsequent to receptor binding and elevation of intraeellular messengers. Second,
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the inhibition of C1- secretion evoked by IFN-y was not a short-term effect because addition of the cytokine directly to Ussing chambers did not cause any change in the peak secretory responses compared with control tissues. Thus, the inhibitory effect of IFN- T requires ample time, as Besancon et al. showed for IFN-T-induced reductions in CFTR mRNA levels. Third, Holmgren et al. showed that IFN-y pretreatment resulted in the loss or decrease in at least three T84 cell membrane proteins, yet IFN-y-treated ceils produced similar amounts of cyclic adenosine monophosphate in response to cholera toxin, as did untreated ceils. IFN-7 pretreatment also increased the expression of several membrane proteins, suggesting that the cytokine could affect a number of molecules involved in epithelial transport. Thus, these lines of evidence support the idea that IFN-y may inhibit CI- conductance in epithelial cells through alterations in expression of genes that encode membrane channel proteins, such as CFTR. The results of Besancon et al. raise intriguing questions regarding the possible physiological role of IFN-y in regulating normal intestinal ion transport. There is evidence that IFN- 7 is spontaneously secreted at a high frequency by human duodenal lymphocytes (J Clin Invest 1991; 88:143 - 148). It is difficult to know precisely the effective concentrations of IFN-7 in the intestinal mucosa; however, the concentrations used by Besancon et al. to achieve significant inhibition of CFTR expression (0.1-100 U/mL) and function (300 U/mL) were less than those used to inhibit C1- secretion in the studies of Holmgren et al. (1000 U/mL). It should be noted, however, that longterm incubation (72 hours) of T84 monolayers with a relatively high concentration of IFN- 7 (1000 U/mL) significantly increased tight junctional permeability (J Clin Invest 1989; 83:724-727), and IFNy (100 U/mL) as well as the combination of IFN-y and tumor necrosis factor have been shown to be cytotoxic to HT29 cells (Clin Exp Immunol 1991 ; 83:79- 84). Thus, much remains to be learned regarding the physiological and pathophysiological effects of IFN-y in the intestinal mucosa. The studies of Besancon et al. and others raise the interesting possibility that this cytokine may be a solid member of the group of molecules released from lamina propria cells that regulate intestinal ion and fluid transport in normal health. Yet, abnormally high or low levels of IFN-y, acting alone or in concert with other soluble mediators (such as tumor necrosis factor), could alter the normal secretory tone of the bowel and tip the balance towards intestinal pathology. HANNAH V. CAREY, PH.D. Reply. We thank Dr. Carey for her accurate summary of our report and her very important comments. We would like to add two comments. The first concerns the possible regulation by IFN-y of intestinal CI- transport under physiological conditions. In a recent report, Kaoutzani et al. (J Clin lnvest 1994;94:788-796) cocultured T84 epithelial cells together with human mucosa-derived lymphocytes with phenotypic features similar to intraepithelial lymphocytes and showed that the presence of mucosa-derived lymphocytes in the coculture significantly attenuated epithelial electrogenic C1 secretion. Attenuation was also observed when virgin epithelia were treated with soluble factors released from mucosa-derived lymphocytes and was largely, albeit not exclusively, attributed to IFN-y. Kaoutzani et al. concluded that physiological IFN- 7 levels secreted by intraepithelial lymphocytes may lead to tonic attenuation of epithelial CI- secretion. Our results showing that CFTR-related C1- current is decreased by IFN-y provide an explanation for the above conclusion and suggest that this cytokine tonically released from intraepithelial lymphocytes attenuates cyclic adenosine monophosphate-stimulated C1- secretion through the regulation of CFTR protein level.