Over the last several years it has become increasingly appreciated that products derived from immune and inflammatory cells are important regulators of the electrolyte transport functions of the intestinal epithelium. The purpose of this presentation is to review briefly the evidence that has led to this appreciation, as well as some pathways, both direct and indirect, whereby immune and inflammatory mediators can interact with the epithelium. Due to limitations of space, this presentation will focus predominantly on work performed in the author's own laboratory. The reader is also referred to some excellent reviews in this area which will provide a more comprehensive overview [1-3].
The first immune/inflammatory cell type that was studied for its ability to alter intestinal transport was probably the mast cell . The basis for these studies was derived from the knowledge that mast cells are located very close to the intestinal epithelium, that these cells are activated in a variety of disease states that are associated with alterations in electrolyte transport, and that mast cells contain numerous mediators that had previously been shown to alter transport function when added to tissues exogenously [5,6]. To date, the ability of mast cell mediators to alter intestinal transport has focused on two main areas; chloride and bicarbonate secretion.
Regulation of chloride secretion by mast cell mediators
The activation of sub-epithelial mast cells in intestinal segments derived from a variety of species, including humans, appears to lead almost universally to a stimulation of active chloride secretion [7-11]. This is the case whether mast cells are activated by typical immune stimuli, such as specific antigen in a sensitized animal or antibodies directed against cell-fixed IgE, or if mast cells are activated by substances not ordinarily considered as immune agonists, including neuropeptides or secretory bile acids.
Mast cell activation leads to the rapid stimulation of chloride secretion, a response that could be considered to be a primitive defense mechanism directed against enteric pathogens. Thus, being able to call upon a rapidly increased rate of chloride (and correspondingly, fluid) secretion could be advantageous to the host in that it might then result in the "flushing" of the offending microorganism from the intestinal lumen. However, this response can also be deleterious to the host if activa ted inappropriately, such as in response to otherwise innocuous substances (dietary antigens) or if activated over a prolonged time scale such as in the setting of mucosal inflammation.
A number of mast cell-derived mediators have been identified as contributors to the secretory response that ensues when intestinal mast cells are activated in situ. Among such mediators, histamine, adenosine and mast cell-derived prostaglandins appear to play important roles, although the extent to which each contributes to the overall response may differ depending on the species and/or the precise intestinal segment that is being studied . Both direct and indirect pathways whereby mast cell mediators regulate intestinal chloride secretion have been identified . All of the listed mediators can bind to receptors located on intestinal epithelial cells that are, in turn, coupled to the stimulation of chloride secretion. For example, histamine binds to basolaterally-localized H1 receptors that are linked to an increase in cytosolic calcium and thereby causes a transient increase in chloride secretion . Prostaglandins, in contrast, stimulate basolaterally-located receptors that activate adenyly l cyclase, and subsequently induce sustained chloride secretion via cAMP-dependent pathways .
Adenosine represents an unusual neurohumoral chloride secretagogue in that receptors for this substance are located on both sides of polarized intestinal epithelial cells, with both populations of receptors linked to the stimulation of chloride secretion . It is possible that the existence of apical adenosine receptors may allow the epithelium to sense that its defenses have been breeched, since ordinarily one would not expect that adenosine generated in the lamina propria would be able to traverse an intact epithelium .
In addition, apical adenosine receptors may be critical in allowing the epithelium to respond to adenosine precursors which are produced by inflammatory cell types, such as eosinophils and neutrophils, that are capable of migrating across the intestinal epithelium in response to luminally-derived stimuli (e.g., bacterial chemotactic peptides) [17-19]. The mechanism whereby adenosine stimulates chloride secretion is still a matter for some debate. While the characteristics of the response suggest a cAMP-mediated mechanism, and events related to protein kinase A are clearly involved (such as phosphorylation of the CFTR chloride channel), adenosine causes a relatively modest increase in cAMP at concentrations of the agonist that cause almost maximal effects on chloride secretion [20,21]. This has ked to the suggestion that additional intracellular messengers, perhaps related to arachidonic acid mobilization, may amplify the actions of cAMP and thereby account for chloride secretion in cells stimulated wi th adenosine .
Another facet of the direct effects of mast cell mediators on chloride secretion relates to the potential for interactions between secretagogues. These interactions are likely to have substantial clinical implications, since mast cell mediators are usually released simultaneously upon cell activation, and thus the epithelium would be exposed to a barrage of multiple substances. Mediators that act through diverging intracellular pathways can have synergistic effects on chloride secretion, with a greater response to the combined stimuli that exceeds that which would be predicted on the basis of simple summation of individual effects. Examples of such synergistic interactions have been provided for the effects of histamine in combination with adenosine, or with prostanoids [13,21]. The subcellular basis for such synergistic interactions relates in part to the different membrane transport pathways that are targeted by different signal transduction cascades. For example, increases in cytosolic calcium stim ulate chloride secretion predominantly by opening a basolateral potassium conductance, whereas cAMP targets the apical CFTR chloride channel . Thus, when calcium and cAMP are elevated simultaneously, the rate-limiting steps for both modes of chloride secretion are removed and thus an increase in the overall secretory rate occurs . However, more recent data suggest that there may also be interactions at the level of second messenger generation, with cAMP capable of modifying calcium-mobilization responses .
Mast cell mediators also have a number of important indirect effects on chloride secretion which likely are important in amplifying the overall response in vivo. For example, at least part of the chloride secretory response to mast cell activation in intact tissues is secondary to the stimulation of enteric nerves, in that it is sensitive to neurotoxins such as tetrodotoxin [25-27]. In fact, there is a bi-directional functional communication between mast cells and nerves in the gastrointestinal mucosa, with part of the chloride secretory response to nerve stimulation being dependent on the presence of tissue mast cells (as demonstrated in studies using mice displaying a genetic deficiency in mucosal mast cells) . There is a structural correlate for these observations, in that mast cells and enteric nerve endings are very intimately associated within the mucosa . Another indirect effect of mast cell mediators on intestinal chloride secretion is provided for by another mucosal cell type, the sube pithelial myofibroblast that forms a continuous sheath underneath the epithelium and may thereby serve a role as a "relay station" to convey and coordinate the transmission of information from lamina propria to the overlying epithelium [30,31]. Myofibroblasts appear to be important sources of E-series prostaglandins which are known to be potent chloride secretagogues. Mast cell mediators such as histamine, as well as other inflammatory mediators, can stimulate production of PGE2 by these cells and this likely serves to amplify the several chloride secretory response that occurs in response to mast cell activation . Furthermore, in response to infection or inflammatory cytokines, these myofibroblasts acquire an enhanced capacity for prostanoid generation secondary to the stimulation of cyclooxygenase-2 expression . This could certainly account for significantly enhanced rates of chloride and fluid secretion occurring in the intestine in the setting of chronic inflammation.
Studies from our laboratory have also indicated that mast cells and their mediators may also play an important role in transducing secretory responses to substances not commonly considered to be immune agonists . Bile acids are present normally in the small intestinal lumen at millimolar concentrations, but concentrations in the colonic lumen are low due to active re-uptake in the terminal ileum. If this re-uptake is interrupted, such as in the setting of surgical resection and /or ileal injury, then bile acids can escape into the colon where they act as potent secretagogues [33,34]. Diarrhea associated with bile acid malabsorption is almost certainly multifactorial. Nevertheless, a complete understanding of the mechanisms that underlie this disease state has proven somewhat elusive. We showed that secretory bile acids released histamine (and likely other mast cell mediators) from colonic mast cells at clinically relevant concentrations . Moreover, the chloride secretory response induced by suc h bile acids was markedly reduced by antagonists to histamine H1 receptors and the cyclooxygenase inhibitor indomethacin, but not by antagonists to H2 receptors nor by anti-cholinergics . Finally, the secretory response to bile acids was delayed and diminished in tissues derived from mast cell-deficient mice, further supporting the concept that mast cell-derived mediators are likely to be important contributors to bile acid-induced diarrhea . It is notable that the responses to bile acids were more pronounced when the compounds were added to the serosal face of tissue specimens. This implies that the effects described may be more significant in vivo in the setting of mucosal injury and a breakdown of the epithelial barrier function, such as might be expected in patients with inflammatory bowel disease.
Regulation of bicarbonate secretion by mast cell mediators
The proximal duodenum of the mammalian gastrointestinal tract is subjected to rather hostile conditions, since it is continually exposed to gastric acid and pepsin . To defend itself against these aggressive factors, this segment of the intestine engages in high rates of active bicarbonate secretion, which neutralizes gastric acid and thereby protects against ulceration. There is evidence that a failure by the proximal duodenum to secrete appropriate amounts of bicarbonate may be a precipitating factor in the development of duodenal ulcer disease . Because of similarities between chloride and bicarbonate secretory mechanisms we hypothesized that mast cell mediators might also be responsible for regulating bicarbonate secretion. To test this hypothesis we initially examined the ability of exogenous histamine to stimulate bicarbonate secretion in segments of rabbit proximal duodenum, using a combination of Ussing chamber and pH-stat techniques. Much to our surprise, histamine had no stimulatory eff ect on bicarbonate secretion by this tissue, although the amine did cause a prompt increase in short circuit current across the tissue, presumably reflective of chloride secretion .
However, histamine was not without effect on bicarbonate secretion altogether since it profoundly suppressed the ability of the tissue to secrete bicarbonate in response to a known agonist of this process, PGE2 [37,38]. This inhibitory effect of histamine was specific, in that it was not observed if bicarbonate secretion was activated by alternative stimuli such as vasoactive intestinal polypeptide, carbachol, dibutyryl cAMP or electric field stimulation .
Pharmacological studies also suggested that, unlike the stimulatory effect of histamine on chloride secretion, the inhibitory effect of histamine on bicarbonate secretion was transduced by H2-histamine receptors . Furthermore, the effect of histamine on bicarbonate secretion was indirect, in that it could be completely reversed by tetrodotoxin and therefore presumably required the obligate intermediate step of activation of at least a subset of enteric nerve endings [37. The clinical significance of these observations was enhanced by the finding that two known injurious agents that lead to the production of duodenal ulcers, aspirin and ethanol, released histamine from proximal duodenal tissue specimens and inhibited PGE2-stimulated bicarbonate secretion at similar doses . Moreover, the inhibitory effects of aspirin and ethanol on stimulated bicarbonate secretion were reversed by antagonists of H2-histamine receptors as well as by tetrodotoxin . The simplest model that takes account of all of the data that have been gathered to date suggests that histamine released from subepithelial mast cells binds to H2-receptors localized to a subset of enteric nerve endings, and thereby stimulates these nerves to release an inhibitory neurotransmitter that can selectively inhibit PGE2-stimulated bicarbonate secretion.
Preliminary data from our laboratory suggest that somatostatin is a candidate to serve as this inhibitory neurotransmitter . PGE2 is thought to be the major mediator of duodenal bicarbonate secretion in response to luminal acidification , and data also suggest that mast cells are activated in the setting of infection with H. pylori, the major cause of duodenal ulcer disease . Thus, it is tempting to speculate that the "anti-defensive" effects of histamine (and perhaps other mast cell-derived mediators), inherent in the indirect ability of the amine to reduce duodenal bicarbonate secretion, may contribute to the development of duodenal ulcer disease by disrupting the balance between aggressive and defensive factors.
Summary and conclusions
It is evident that mast cell mediators, as well as mediators derived from other inflammatory cell types, have complex and intersecting actions on a variety of intestinal transport responses. While much has been learned of the mechanisms underlying certain of these responses, there is still a considerable lack of information regarding the precise contributions of specific mediators to pathophysiological responses in vivo. Moreover, we have only a limited understanding, at best, as to whether immune or inflammatory mediators are important in regulating the normal electrolyte transport functions of the intestine. For these reasons, further investigation in this area seems warranted, particularly since modulation of transport responses by immune and inflammatory cells is likely central to disease pathogenesis in a variety of intestinal disorders, including inflammatory bowel diseases, food allergies, enteric infections and peptic ulcer disease. An improved understanding of these regulatory pathways may in turn lead to improved therapies for such conditions.
Acknowledgments: I am grateful to the following current and former members of my laboratory, as well as collaborators, whose studies are summarized in the foregoing presentation: Cornelia Gelbmann, Alan Hofmann, Mana Vajanaphanich, Christopher Myers, Jon Isenberg, Daniel Hogan, Biguang Yao, Stephen Thompson, the late Kiertisin Dharmsathaphorn and Timothy Bigby. Studies from the author's laboratory have been supported by grants from the National Institutes of Health and the Crohn's and Colitis Foundation of America.
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