MORPHOLOGY AND LOCATIONS OF ICC WITHIN THE GI TRACT
In the later part of the 19th century, a network of small anastomosing cells located within the gut muscle layers was described by the Spanish neuroanatomist Ramon Santiago y Cajal, who was awarded the Nobel Prize in 1906. These cells, which appeared to be situated between the nerve endings and smooth muscle, would later become known as interstitial cells of Cajal (ICCs). Until recent times, the major difficulty in studying ICCs was the lack of specific histological stains or immunohistochemical markers that would enable ICCs to be distinguished from the enteric neurons and smooth-muscle cells that comprise the neuromuscular apparatus within the gastrointestinal (GI) tract. Consequently, for many years ICCs were mostly identified by their ultrastructural characteristics (the gold standard), or by certain histochemical stains, such as vital methylene blue, each of which have their drawbacks in terms of the painstaking nature of the analysis or lack of cell specificity, respectively. Because of these deficiencies, until the early 1990s there was considerable controversy as to the identity of different classes of ICC, their roles within the gut, their interactions with other cell types, and their embryologic origin and development. However, a breakthrough in ICC analysis came with the finding that the protooncogene c-kit, which encodes a receptor tyrosine kinase (1), the ligand for which is stem cell factor (2), is expressed by ICCs and that anti-Kit antibody specifically labels these cells (3–5). Subsequently, using anti-Kit antibody, ICCs have been shown, in humans and laboratory animals, to have a range of different morphologies in different regions of the gut (6,7). For example, long, thin, spindle-shaped ICCs are distributed within the plane of the muscularis (ICC-IM), whilst highly branching, network-forming ICCs are closely associated with the myenteric plexus (ICC-MY) of the enteric nervous system. The distribution of morphologically different classes of ICC within various layers of the GI musculature (8) strongly suggested that different ICCs may perform distinct physiological roles in the gut.
PHYSIOLOGICAL ROLES OF ICC
Because Cajal and subsequent researchers observed ICCs in close association with enteric neurons and smooth muscle, it was logical to predict that ICCs could play a role in controlling gut motility (9,10). An important breakthrough in demonstrating such a physiological role was achieved by blocking the development of ICC networks using either injection of anti-Kit antibody into newborn mice, or genetically using W mutant mice that have loss-of-function mutations in c-kit. In such experiments, animals developed that lacked ICC-MY within the small intestine and also lacked intestinal pacemaker activity (3–5,11). These results demonstrated that ICCs are necessary for pacing electrical slow-wave activity and contractions within GI muscles. Similar results, showing loss of ICCs and loss of regular intestinal activity, were obtained in steel mutant mice (12), which lack the stem-cell factor ligand for the Kit receptor.
In W mutant mice, which lack ICC-MY in the pacemaking region of the small intestine, intramuscular ICCs also are deficient. Because ICC-IM are normally closely apposed to varicose nerve terminals and electrically coupled via gap junctions to neighbouring smooth-muscle cells (13), a role for ICCs in the mediation of neurotransmission seemed likely, as originally proposed by Cajal. This theory was confirmed by analysing stomach tissues from W mutant mice, which are deficient in ICC-IM but have normal patterns of enteric nerve fibres and smooth-muscle cells. Experiments clearly demonstrated a lack of nitric oxide–mediated neuroregulation of smooth muscle, because the muscle relaxed in the presence of an exogenous NO donor, but the membrane potential effects were greatly attenuated (13). These and more recent findings (14) confirm that ICC-IM play a fundamental role in the reception and transduction of both inhibitory and excitatory enteric motor neurotransmission (15).
ICC IN HUMAN GI MOTILITY DISORDERS
The findings outlined above, obtained from laboratory animals, highlight the key roles played by ICCs in the regulation of GI motility, and suggest that alterations in ICC networks in humans also could have an impact on gut motility and/or GI diseases. Indeed, loss of ICCs or disruption of ICC networks has been reported in a wide range of GI diseases, including achalasia, chronic intestinal pseudoobstruction, Hirschsprung disease, inflammatory bowel diseases, slow transit constipation, and others (6,16) (Table 1).
A number of important factors should be kept in mind when studying ICCs in disease states. For example, ICCs are derived from mesenchymal cells that express c-kit(17,18), and as outlined above antibodies against the extracellular domain of the Kit receptor have been used extensively to immunolabel and identify ICCs in different species, including humans. However, in disease states, a lack of kit-labelling may either indicate an actual loss of ICCs from the gut tissues or only a loss of kit-positivity from ICCs which remain in situ. In the latter case, transmission electron microscopy may be necessary to reveal ICCs based on well-defined ultrastructural criteria. However, there is little such information regarding normal ICCs in disease states, and if ICC features change under pathophysiological conditions, then it may be difficult to recognise cells as ICCs. Indeed, experimental findings support the idea that ICCs can change phenotype under certain conditions. For example, in studies in which Kit receptors were blocked during development, ICCs almost entirely disappeared from the small intestine. However, this loss of ICCs was not accounted for using assays for cell death, and closer examination revealed that remaining ICCs developed ultrastructural features similar to smooth-muscle cells (19). These findings point to an inherent plasticity between ICCs and smooth-muscle cells that is regulated by Kit signalling (19,20). ICCs have been shown not only to be capable of transdifferentiation (19) but also to have some capacity for regeneration. In experiments in which the mouse intestine was exposed to a chemical insult that induced loss of the myenteric plexus and the closely associated ICC-MY, a few weeks later, cells with ICC-like features began to reappear (21). These data, reporting on transdifferentiation and regeneration of ICCs, highlight the difficulty in distinguishing what is cause and what is consequence in the relationship between ICCs and the generation of certain gut motility disorders. Are defects in ICC networks the cause of motility disorders, or are disrupted ICC networks a consequence of gut dysfunction?
A good example of the conflicting evidence obtained regarding potential loss/reduction of ICCs and gut motility is the congenital gut defect Hirschsprung disease (HSCR), which presents as absence of enteric ganglia in the distal hindgut and associated obstructed gut, resulting in severe constipation (22). Some studies of HSCR tissues have reported a reduction in the cellular density of ICCs or “disrupted” ICC networks within the aganglionic segment (23,24), whereas others have observed normal ICC networks in the aganglionic bowel (25,26). One may argue that in the aganglionic segment of HSCR gut, which by definition is devoid of enteric neurons, ICC networks would not develop normally because these cells normally occur in close association with neurons or nerve fibres of the enteric nervous system. Animal models of gut aganglionosis have been used to further investigate this problem. In chick or mouse gut, deprived of neural crest cells that are the precursors of enteric neurons, ICC have been found to develop normally in the absence of neurons (17,18). Similarly, ls/ls−/− and Gdnf−/− mutant mice, which are deficient in enteric neurons in varying lengths of the gut, appear to have similar distributions of ICCs and normal slow waves (27), although in ls/ls−/− mice there appear to be different electrical activity and neural responses (28). Overall, these data from animal models provide strong evidence that ICCs can develop normally in the absence of enteric neurons. Translating these findings back to HSCR in humans is still problematic for the reasons outlined above, but they would imply that any deficiency in ICCs in HSCR gut would not likely be due to a loss of enteric neurons.
SUMMARY AND FUTURE DIRECTIONS
In the last 10 to 15 years, great progress has been made in our understanding of the morphology and physiological roles of ICCs, the small network-forming cells distributed throughout the smooth-muscle wall of the gastrointestinal tract. These advances have been made primarily because of the discovery that ICCs express c-kit, the protooncogene that encodes the receptor tyrosine kinase, Kit. Activation of Kit signalling is required for ICC development, and antibody against Kit can be used as a simple, efficient means of specifically labelling ICCs. Developmental biologists have determined that ICCs are mesenchyme-derived cells, belonging to the family of smooth-muscle cells. Morphological studies have identified different phenotypic classes of ICCs, which have different regulatory roles within the gut. ICCs associated with the myenteric plexus act as pacemaker cells within the phasic regions of the GI tract, whilst intramuscular ICCs act as mediators of neural inputs from the enteric nervous system.
Future work will likely be geared toward achieving a better understanding of the interrelationship between ICCs and the generation of human gut motility disorders. This will necessitate a range of investigations using morphological and physiological studies, using perhaps alternative markers to Kit; a better understanding of the genetics and/or cell biology of ICCs; further studies on animal models of GI disease states; and more detailed immunohistochemical investigations on tissues from patients with GI pathologies. With the advances such investigations should bring, it may be possible to restore functional populations of ICCs in patients experiencing loss of these highly specialised cells identified by Cajal >100 years ago.
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