ICC in rodent gastrointestinal tract
The general organization of ICC networks in the rodent gastrointestinal tract was illustrated schematically by Hanani et al. based on ultrastructural and KIT immunohistochemical data from a large number of reviews. ICC are in contact with each other and form networks in four locations: (1) ICC with a stellate morphology are located in the serosa (ICC-SS); (2) between the longitudinal (LM) and circular (CM) muscle layers at the level of Auerbach’s plexus (ICC-AP) where they form a network around the ganglia; (3) in the small intestine, where bipolar-shaped ICC are located at the level of the deep muscular plexus between the inner and outer circular muscle layers (ICC-DMP); (4) in the stomach and colon, where they are located at the border between the submucosa and the circular muscle and are called ICC-SM in the stomach [53, 54] and ICC-SMP in the colon . In the oesophagus, stomach and colon, solitary bipolar ICC are seen in the circular and longitudinal muscle layers, ICC-CM and ICC-LM, respectively, or combined: ICC-IM. The oesophagus and fundus of the stomach are completely devoid of ICC at the level of AP [3, 53], but ICC-AP are present in the corpus, antrum and pylorus , in the small intestine [1, 2] and colon .
ICC in human gastrointestinal tract
In the human gastrointestinal tract, our knowledge of the distribution and morphology of ICC is based on KIT immunohistochemistry and electron microscopy and has been reviewed by several authors [22, 57, 58]. In human oesophagus ICC-LM and ICC-CM appear as elongated cells within both muscle layers but are absent at the level of AP; at the submucosal border the ICC are in close contact with nerve endings [57, 59]. In the human stomach KIT+ bipolar cells are present in CM and LM of the corpus, but in the antrum they are also present at the level of AP [57, 60]. ICC are also found at the submucosal border, ICC-SM. They lie close to naked nerve terminals and muscle cells, and are more frequent in antrum and corpus than in the fundus . In the human small intestine, KIT+ ICC are present in the longitudinal and circular muscle layers and at the level of AP [57, 62] whereas opinions differ as to whether ICC-DMP are KIT immunoreactive or not. Ultrastructurally ICC-AP have close associations with nerve varicosities  and ICC-DMP form contacts with both nitrergic and cholinergic nerves, they also form gap junctions with the outer CM . In the normal colon many KIT+ ICC are distributed regularly in the circular muscle layer. In the taenia of the longitudinal muscle layer, their long axes run parallel to those of the muscle cells, and they embrace the ganglion cells in AP . Ultrastructurally, only ICC at AP [65, 66] and at the submucosal border have been described .
In the mouse, slow waves that represent pacemaker activity are generated in ICC-AP in the small intestine [1, 2]; and in ICC-AP and ICC-IM in the stomach [67, 68]. In canine colon the prominent pacemaker is associated with ICC-SMP ; in rat colon both ICC-AP and ICC-SMP may be associated with pacemaker activity [55, 70]. Neurotransmission to the muscles is believed to be mediated via ICC-IM in oesophagus, in antrum and corpus of the stomach , and via ICC-DMP in the small intestine .
Macrophages in rodent gastrointestinal tract
The morphology and distribution of macrophages in the muscularis externa have been studied in detail in the mouse small intestine, and human small intestine and colon (see review ). In the last decade many papers have been published on experimental inflammation in animal models in which it has been suggested that macrophages are involved. However, in contrast to the many studies and reviews on ICC, there are very few studies on macrophage distribution, location and morphology in normal tissue. Macrophages have mostly been reported in control tissue in motility studies on inflammation, or ICC studies associated with various experimental and/or pathological conditions without any reference to the precise location of the macrophage.
Small intestine: In the mouse, macrophages have been described at three levels: (1) in the serosa as bipolar cells with two to three ramifications; (2) at the level of AP where the cells have between three and five ramifications; (3) a few bipolar cells at the level of the DMP between the inner and outer CM. The cell populations at levels (1) and (2) are regularly distributed, but do not create regular networks with cell contacts similar to those of the ICC, but the macrophages are often enveloped by and in close spatial contact with ICC. At the level of AP the macrophages line the vessels as well as the plexus. At the level of the DMP (level 3) the bipolar macrophages are also enveloped by processes of ICC or fibroblast-like cells, but close spatial contacts between macrophages and nerves have not been observed [5, 7, 72] .
Ultrastructurally, macrophages can be characterized by having a centrally situated, deeply indented nucleus and long slender cell processes, they do not have a basal lamina nor do they form specialized contacts with other cells, but they are often enveloped by ICC. In adult conventional mice they harbour several types of vesicles which vary in size, shape, content and membranes [4, 5] (Fig. 1A and B). However, the number, form and type of vesicles seem to be dependant on the activation state of the macrophage.
Figure 1. Muscularis externa from mouse small intestine. (A)–(B): Conventional mice, (A) Serosal macrophage containing dense bodies (lysosomes) (D), light vesicles (L), coated vesicle (arrow head), doughnut-shaped vesicle (arrow), nucleus (N), bar: 0.5 μm. B. Macrophage at the level of AP, between the longitudinal muscle layer (LM) and the circular muscle layer (CM), is enveloped by ICC processes (ICC), bar: 1 μm. (C)–(D): Germfree mice, (C) Process of a serosal macrophage between the mesothelium (Me) and the longitudinal muscle layer (LM), bar: 0.5 μm. (D) Macrophage between LM and CM, the macrophages in the germfree mice contain mostly dense bodies, bar: 1 μm. (E) Dextran injected mouse (after 4 days), serosal macrophage between the mesothelium and the longitudinal muscle layer, the macrophage contain large dextran-filled vacuoles (V), light vesicles (L), dense bodies (D) and nucleus (N), bar: 1 μm. (F) Day 15 after infection with Trichinella spiralis, a macrophage (Ma), at the level of AP between LM and CM, is enveloped by ICC cytoplasm characterized by the content of mitochondria (m), the cell borders are marked by a large arrow. The macrophage contains phagocytic vesicles (pv) some lysosomes (L) and coated vesicles (small arrow), bar: 1 μm. Reproduced with kind permission from Wiley-Blackwell: (A–C) , Springer: (D) and (E)  and American Society for Investigative Pathology: (F) .
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In germfree mice the macrophages appeared to contain a similar if not higher number of vesicles with electron-dense granular content (lysosomes) and less light vesicles (vacuoles) in the cytoplasm  (Fig. 1C and D).
In mice that had been treated with FITC-dextran intravenously, the macrophages contained many dextran-filled vesicles. Notably serosal macrophages, which were not in the vicinity of vessels, contained dextran already after 1 hr. After 1 and 4 days the macrophages contained large dextran-filled vacuoles  (Fig. 1E). After intraperitoneal injection mesothelial cells also contained dextran-filled vesicles . This suggests that dextran can be transported from the peritoneal cavity via the mesothelium to the serosal macrophages and that these may have a function as sentinels against external exposure.
After 15 days of Trichinella spiralis inflammation some macrophages contained numerous large phagosomes and lysosomes  (Fig. 1F).
The macrophages distribution and morphology have been demonstrated immunohistochemically with several antibodies: F4/80, CD11b, class A scavenger receptor and recently CD169 [31, 33, 34, 36]. In adult mouse small intestine the macrophages are constitutively MHCII+[31, 34, 36, 74, 75], but MHC– in germ-free and newborn mice . This suggests a possible up-regulation/activation due to non-pathogenic, commensal bacterial antigens in the chow or the surroundings. In the MHCII+ cell population in mouse muscularis presence of subsets of dendritic cells has also been described . However, in a recent study only one cell population was found, expressing a MHCIIhigh, CD11clow CD103÷, CD11b+, F4/80+ phenotype, being derived from monocytes, with a monocyte/macrophage morphology and responsive to M-colony stimulating factor . Thus a subgroup of classical dendritic cells is unlikely to reside in the muscularis externa along with macrophages.
In an immunohistochemical study on rat small intestine, macrophages (ED2+ cells) were described within LM, between the muscle layers at the luminal side of the plexus and within CM . However, ultrastructural studies of rat small intestine show macrophages located only between the mesothelial cells and LM [40, 77]. Furthermore, cross-sections as well as double staining with ED2 and KIT together with confocal microscopy confirm that the macrophages are located at the same three levels as in the mouse (Fig. 2). Kalff et al. describe a few ED1+ monocytes in rat muscularis . However, in our unpublished observations we only found ED1+ granules in cells with a ramified morphology located in the serosa, between the muscle layers at AP, and at DMP (Fig. 2). The ED1+ cells therefore seem to be macrophages too. MHCII immunoreactivity seems to depend on the antibody used, as 70% of the macrophages are described to show MHCII immunoreactivity when using OX76 , whereas only a few ramified cells show MHCII imunnoreactivity when using OX6 . The structure of mouse and rat small intestinal muscularis externa is well documented. They are almost similar to one another with regard to their muscle layers, nerve plexuses, ICC networks and the location of macrophages. However, there are some differences between the two species in that the structure is simpler in mouse small intestine, where vessels are only found at AP and not in serosa , whereas in the rat both LM and serosa contain vessels . In mice, other immune cells (B and T lymphocytes) – apart from an occasional mast cell – are absent [4, 73], but rat muscularis externa is also reported to contain neutrophils, mast cells, T-cells, natural killer cells and dendritic cells . In mouse colon, CD 168+ cells were found in serosa and at AP as ramified cells. In addition more rounded cells were present near the mesenterial attachment (our unpublished observations). In proximal rat colon, ED2+ macrophages at the level of AP seem to have the same morphology and distribution as in the small intestine and are ultrastructurally in close spatial contact with ICC and ganglion cells [12, 78]. Some ramified cells were faintly ED1+. In another ultrastructural study on rat colon, macrophages were found at the level of SMP as well as at AP. There is no description of any close contact to ICC or any macrophage-to-nerve relationship . The distribution of macrophages in the gastric muscularis externa has not yet been documented in detail. In a study on the pathogenesis of septic ileus, CD169+ macrophages have been described in the circular muscle layer of the murine gastric corpus. These cells seem to be bipolar in shape . In a recent study, another population of F4/80+ macrophages was also described in the muscularis externa of the gastric corpus . The cells look ramified as macrophages at the level of AP in mouse small intestine. These studies suggest that there are macrophages with different morphologies and in various locations in the stomach as well. In a study of the ultrastructure of the circular muscle layer of antrum and corpus, Ishikawa et al.  described scattered nerve bundles and free cells (among them eosinophils and macrophages), which were occasionally observed in the interstitium around the nerve bundles. However, there is still a considerable lack of information on these macrophages in the stomach at both light and electron microscopic levels, especially with regard to their relation to ICC, and reports on serosal macrophages seem to be absent.
Figure 2. . Whole mounts from rat jejunum. Confocal micrographs. (A–C) Double staining with ED2 (red) towards macrophages and KIT (green) towards ICC. (A) Macrophages in serosa. (B) Macrophages at the level of AP and ICC-AP. (C) Macrophages at the level of DMP and ICC-DMP. Arrows: macrophages, arrowheads: ICC. (D–F) ED1 (CD68) stained macrophages display a granular staining pattern and a ramified morphology. (D) In serosa. (E) At the level of AP. (F) At the level of DMP. Bars: 30 μm.
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Macrophages in human gastrointestinal tract
Very few studies of the muscularis externa of normal human oesophagus and stomach focus on macrophages. The muscularis of the small and large intestine do have immunoreactive CD68+ and CD11b+ cells which seem to have long cell processes. These cells were found in the serosa, in the muscle layers and in the submucosa. Some of the serosal macrophages appeared to line the longitudinal muscle layer. Macrophages also occurred in septa between the main muscle lamellae, in smaller intralamellar septa between the muscle cells as well as in the lining of AP. Moreover they were located in the connective tissue of the serosa and submucosa. HLA-DR+ cells (MHCII) were present in the muscle layers and seemed to co-stain with CD68+ and CD11b+ cells, but they were scarce in serosa and submucosa. Accordingly, macrophages in the muscularis differ from macrophages in serosa and submucosa with regard to their immunophenotype . Full-thickness whole mounts of jejunum have been studied, where CD163+ cells were suggested to be located in the septa , but they do not seem to have a ramified morphology. Ultrastructurally, human macrophages resemble those of mice and rats . However, in contrast to rodent small intestine, specific relations between macrophages and ICC have only been reported in a recent ultrastructural study on colonic ICC-AP, where macrophages were described as being frequent at the level of AP with intimate and close appositions to ICC-AP. Membrane-to-membrane contacts between macrophages and fibroblast-like cells also seemed to be a characteristic and frequent feature, but contacts to other cell types or nerves are not described . In CM and LM they were encountered as single cells . In the DMP of the small intestine there were many macrophages in close proximity to fibroblast-like cell processes, but they did not form special contacts. In serosa and submucosa the macrophages were surrounded by collagen and elastic fibres; in the septa they were often in close contact with fibroblasts and nerve fibres .
Mast cells in rodent gastrointestinal tract
Mast cells are mainly found in the lamina propria of the intestine, but have also been observed in the epithelium, in the submucosal layer and in serosa covering the peritoneal cavity. General studies of the distribution of mast cells in the normal rodent intestinal muscularis externa are lacking, and mast cells are only mentioned in connection with studies on other cell types or in models of inflammation. In general, rats are considered to have more mast cells than mice, and Wistar rats to have more than Sprague-Dawley rats . In the stomach Alcian-blue+ mast cells have been described in the submucosal layer of antrum and corpus, but very few were observed in the muscle layer . In the muscularis externa of mouse small intestine, mast cells were extremely rare at both light and electron microscopical levels . Significant differences have been reported between various murine strains: the mucosa, submucosa and muscularis externa of C57/B6 mice have hardly any mast cells, whereas several have been observed in NMRI mouse tissue stained with toluidine blue (our unpublished observation) and with an unspecific avidin stain in rat muscularis externa . Descriptions of mast cells in the muscularis externa of colon are lacking.