Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CD117), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEM16A), a Ca2+-activated Cl− channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Ano1 immunoreactivity compared with Kit and to establish whether Ano1 is a reliable marker for human and mouse ICC. Expression of Ano1 in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Ano1 alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Ano1 immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Ano1 positive. Some non-ICC in the mucosa and submucosa of human tissues were Ano1 positive but Kit negative. A few (3.2%) Ano1-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Ano1 labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells.
- mast cells
- chloride channels
- gastrointestinal motility
interstitial cells of Cajal (ICC) are mesoderm-derived mesenchymal cells that contribute to normal gastrointestinal motility (9). ICC generate pacemaker potentials that drive the electrical slow wave (19, 36) contribute to normal neuromuscular signaling (4), are involved in mechanotransduction (13, 31), and set gradients in smooth muscle membrane potential (10).
ICC were originally identified by Santiago Ramon y Cajal and were characterized by morphological criteria until the discovery that these cells express the receptor tyrosine kinase Kit (23). Subsequent studies determined that Kit immunoreactivity in the muscularis propria of the gastrointestinal tract is restricted to two cell types: mast cells (9, 25) and ICC. Kit-positive ICC are distributed throughout the gastrointestinal tract as well as in other smooth muscle tissues (24). All regions of the gastrointestinal tract contain ICC but the location within the muscularis propria varies according to region or species (15). In the myenteric plexus region, ICC form a network between the muscle layers forming a mesh around the ganglia (ICC-MY). A network of deep muscular plexus (DMP) ICC (ICC-DMP) is present in the small intestine between the inner and outer circular muscle layers (15). In the colon and parts of the gastric antrum, submuscular ICC (ICC-SM) are located outside the circular muscle layer (2, 3). Intramuscular ICC (ICC-IM) are distributed through the longitudinal and circular muscle layers. Septal ICC are found between the fascicles of muscle in humans and other large species. These can be considered a type of ICC-IM (29, 37). Stellate, subserosal ICC are observed on the boundary between the longitudinal muscle and the serosa in the colon of mice (34).
At present, Kit is the only reliable antigenic marker for ICC. Antibodies to Kit have been used extensively to characterize changes in ICC networks in human and animal tissue. Several human gastrointestinal motility disorders have been associated with depletion of Kit-positive ICC (11, 12, 16, 22, 26); however, these observations have not been confirmed by using antigenic markers independent of Kit signaling. Also, residual ICC-like function in ICC deficient mutants has been linked to Kit-negative cells (35). These cells may be related to ICC and may be revealed by a Kit-independent marker. Advances in gene expression profiling have been applied to identifying markers for ICC (6, 40), resulting in the demonstration that the Na+/K+/2Cl− cotransporter, NKCC1 (39), the neurokinin-1 receptor (7), and CD44 (21) are all proteins expressed selectively on some or all subtypes of ICC. At present, no comprehensive survey of the gastrointestinal tract has been published using antisera to any of these targets.
One protein identified recently as expressed on ICC is Ano1 (previously known as FLJ10261, DOG-1, and TMEM16A) (8, 38). Ano1 is part of a family of 10 gene products (Ano1–Ano10 or TMEM16A to TMEM16K) with similar primary sequences and predicted secondary structures (20, 41). Ano1 expression is upregulated in gastrointestinal stromal tumors (8, 38) and other tumors (20). In nontransformed cells, Ano1 is expressed in many organs including epithelia from the lung, foregut (28), and kidney, as well as pancreas and salivary glands (41). Knockout of Ano1 leads to death of the pups early after birth (28). Expression studies have determined that the cloned Ano1 gene product contributes to a Ca2+-activated Cl− conductance (5, 30, 41).
ICC in human colon and small intestine also appear to express Ano1 (8, 14, 38). It is not known whether Ano1 is expressed in all regions of the gastrointestinal tract, whether all types of ICC express Ano1, whether all Kit-positive ICC express Ano1 and vice versa, or whether Ano1 is expressed on mouse ICC. Therefore, the objective of this study was to investigate the potential utility of Ano1 as a Kit-independent marker of ICC by examining the distribution of Ano1-positive cells in the gastrointestinal tract of human and mouse tissue and determine the degree of colocalization of Ano1 with Kit on ICC.
MATERIALS AND METHODS
This study was approved by the Institutional Review Board and Institutional Care and Use Committee of the Mayo Clinic.
Human gastric midbody tissues (n = 3) and jejunal tissues (n = 3) were obtained from six patients undergoing surgery for morbid obesity. Normal human colon (n = 3) was obtained from three patients undergoing resection for nonobstructing colon cancer (details in Table 1). Tissues were placed in ice-cold F12 medium (Invitrogen, Carlsbad, CA). A piece of tissue 2 cm × 2 cm was dissected, pinned out, flash frozen in isopentane cooled with dry ice, and frozen in OCT embedding compound (Sakura Finetek, Torrance, CA). Fresh frozen tissues were stored at −80°C until sectioned.
Adult BALB/c mice (4–8 wk old; n = 6) were purchased from Harlan Laboratories (Madison, WI). The animals were anesthetized by isoflurane (Aerrane; Baxter Healthcare, Deerfield, IL) inhalation and killed by decapitation. Mouse tissues (gastric fundus, gastric corpus and antrum, jejunum and ileum, proximal colon, distal colon) were excised, placed in ice-cold Krebs-Ringer bicarbonate buffer (21), and opened along the lesser curvature of the stomach or the insertion of the mesentery and their contents were washed away with ice-cold Krebs-Ringer bicarbonate buffer. The mucosa and submucosa were removed by peeling and only the muscularis propria was used for immunolabeling experiments.
For detail about the primary and secondary antisera used, see Table 2. The specificity of rabbit Ano1 antibody was demonstrated by the absence of labeling in colon tissue from Ano1 knockout mice compared with wild-type animals provided by Dr. Brian Harfe, University of Florida (27, 28).
Controls for each antibody used were carried out by incubating the sections with secondary antibodies but no primary antibodies, by applying secondary antibody directed against IgG from a species that was not the host for raising the primary antibody and by examining singly labeled tissues under illumination with the filter sets designed for the wrong fluorophore.
Human tissues were cut in 12-μm-thick sections whereas the immunolabeling for mouse tissues were carried out in whole mounts devoid of mucosa and submucosa. Tissues from human were fixed in 25% acetic acid-75% ethanol (vol/vol) solution for 10 min. After blocking for 2 h at room temperature in 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) in PBS, the sections were incubated overnight at 4°C with the primary antibodies for Kit and Ano1 (see Table 2) in 0.3% (vol/vol) Triton X-100 plus 1% BSA in PBS. After washing, the tissue was incubated for 1 h with the appropriate secondary antibodies (see Table 2), washed, and counterstained with 4′,6-diamidino-2-phenylindole dilactate (DAPI dilactate, Invitrogen, Carlsbad, CA) to label nuclei. The murine whole mounts were stretched over the surface of a Sylgard 184 (Dow Corning, Midland, MI)-coated petri dish, fixed with cold acetone (4°C, 10 min), washed with PBS (4°C, overnight), and blocked with 1% BSA (1 h at room temperature). The tissues were then incubated for 48 h at 4°C with a rat monoclonal anti-murine Kit antibody (ACK2; see Table 2) in 0.3% (vol/vol) Triton X-100 plus 1% BSA in PBS. After a second fixation step with 4% paraformaldehyde-PBS (10 min at room temperature), the tissues were washed with cold PBS overnight, blocked again with 1% BSA in PBS for 1 h, and labeled with the rabbit polyclonal anti-Ano1 also used in human tissues (48 h at 4°C in PBS containing 1% BSA). Secondary antisera (Table 2) were applied after an overnight wash with PBS at room temperature for 1 h. To label mast cells by using an antibody to mast cell tryptase, antigen retrieval was required. Human colon was fixed overnight in 4% paraformaldehyde and the next day the tissue was washed and then incubated overnight in 30% sucrose, and 12-μm sections were cut and subjected to antigen retrieval. This was carried out by the steamer method with the use of preheated antigen retrieval solution (Dako, Carpinteria, CA) at 90–96°C. The slides were maintained at this temperature for 15 min. After antigen retrieval, tissues were blocking for 2 h at room temperature in 1% BSA and incubated overnight at 4°C with the primary antibodies for mast cells tryptase and Ano1 (see Table 2) in 0.3% (vol/vol) Triton X-100 plus 1% BSA in PBS. The next day, the tissue was incubated for 1 h with the appropriate secondary antibodies (see Table 2), washed, and counterstained with DAPI dilactate (Invitrogen) to label nuclei.
Three to ten sections from each tissue were examined by use of an Olympus BX51WI epifluorescence microscope (Olympus America, Center Valley, PA) or by confocal microscopy (Olympus FV300, Melville, NY). The confocal images were collected using the optimal pinhole size for the ×60 1.2-NA water objective at 633 nm (z-axis step 0.48 μm; human tissues) or for the ×40 1.0 NA oil objective at 543 nm (z-axis step 0.7 μm; mouse tissues). The following lasers and emission filters were used to visualize the labeled structures and collect images: multiline Ar laser at 488 nm (used for the excitation of Alexa Fluor 488); emission filter 535 ± 15 nm; 543 nm HeNe laser (used for Cy3 and Alexa Fluor 594); emission filter 575–630 nm; and 633 nm HeNe laser (used for Cy5); emission filter HQ660 nm (650–700 nm).
Quantification of Ano1-positive cells in human stomach.
The numbers of Ano1- and Kit-positive cells in the circular muscle layer of the human stomach tissue were counted at ×200 magnification. Cells in 30 fields from three nonadjacent sections were counted (10 fields per slide). Ano1-positive cells were scored as either brightly Kit positive or weakly Kit positive (Kit-dim). Kit-positive cells were scored as either Ano1 positive or Ano1 negative.
Unless indicated, reagents were from Sigma-Aldrich (St. Louis, MO).
Ano1-positive cells in the human gastrointestinal tract.
In the human gastrointestinal tract (Fig. 1), Ano1-immunoreactive cells were detected using both the polyclonal rabbit antiserum (Abcam) and the monoclonal mouse antiserum (Applied Genomics) by labeling of acid-ethanol fixed sections. Use of other fixation conditions, including 2–4% paraformaldehyde (with and without antigen retrieval) and cold acetone, resulted in nonspecific labeling of cell nuclei and much dimmer signal. Double labeling with the two antibodies to Ano1 resulted in exactly coincident signals (Fig. 1A).
The Ano1-positive cells detected in the muscularis propria had the characteristic distribution pattern of ICC for each region. All types of ICC identified previously by Kit immunoreactivity (15) were identified by Ano1 immunoreactivity. In the body of the stomach, Ano1-positive cells were present in both the longitudinal and circular muscle layers as well as in the region of the myenteric plexus and in the submucosal plexus (Fig. 1B). As reported for Kit-positive ICC (32), myenteric networks of Ano1-positive cells were considerably less dense in the stomach than in other tissues. Ano1-positive cells were also observed in septa. In the human jejunum, Ano1-positive cells were observed in the myenteric plexus region, in septa, and in the DMP (Fig. 1C). Ano1-positive, ICC-like cells in the human colon were present in the longitudinal and circular muscle layers including septa, the myenteric plexus region, and the submucosal plexus (Fig. 1D).
The morphology of the Ano1-positive cells in the muscularis propria of the tissues was characteristic of ICC. Cells within the muscle layers were oriented parallel to the long axis of the myocytes and had branching processes running in opposite directions from the cell body (Fig. 2A). In the septa, the Ano1-positive cells were located between the muscle bundles (Fig. 2B). Cells in the myenteric plexus regions were located around the periphery of the ganglia forming a dense network of Ano1-positive processes. These ICC-MY-like cells had triangular cell bodies and two or more processes (Fig. 2C). In the DMP, the Ano1-positive cells had bipolar morphology and two processes running parallel to the circular muscle layer (Fig. 2D). Ano1-positive cells characteristic of ICC-SM were observed close to the circular muscle in the submucosa of the colon and stomach. These cells were also predominantly bipolar in morphology (Fig. 2E).
Ano1-positive cells with a different morphology from ICC were also detected in areas that did not contain Kit-positive ICC. Specifically, Ano1-positive cells were located in the mucosa and submucosa of all regions studied (Fig. 2F).
Colocalization of Kit and Ano1.
To confirm the identity of the Ano1-positive cells as ICC, tissue was colabeled for both Ano1 and Kit. The rabbit polyclonal antiserum to Kit labeled the ICC-like cells identified by using the mouse monoclonal antibody to Ano1. Similarly, the mouse monoclonal antiserum to Kit labeled the ICC-like cells identified by using rabbit polyclonal antiserum to Ano1 (Fig. 3, A and B). All Ano1-positive cells in the external muscle layers were also Kit positive in all regions of the gastrointestinal tract (Fig. 3C–E), and the colocalization of Ano1 and Kit was complete within the spatial resolution of confocal microscopy (Fig. 3F). There was no labeling of ICC-like cells by any of the secondary antibodies in the absence of primary antiserum. There was also no labeling of ICC-like cells when the anti-mouse secondary antisera were applied to tissues incubated with the primary antisera raised in rabbit or vice versa. No signal was observed when singly labeled tissues were examined by using the excitation and emission filters for the wrong fluorophore.
The Ano1-positive cells in the mucosa and submucosa (see above, Fig. 2F) were all negative for Kit (Fig. 4A) and had the morphology and location of myofibroblasts. In the muscularis propria, Kit-negative, Ano1-positive cells were not observed.
Mast cells were not positive for Ano1, although mast cells (Kit positive, Ano1 negative) were detected in all layers of the gastrointestinal tract. The mucosa and submucosa contained the greatest numbers of mast cells (Fig. 4A), but mast cells were also observed in the muscle layers (Fig. 4B). The mast cells were distinguished by bright Kit labeling with round cell bodies and absence of processes. No Kit-positive cells with mast cell morphology were observed to be immunoreactive for Ano1. This was confirmed by double labeling human colon with antibodies to mast cell tryptase and to Ano1. Tryptase-positive mast cells were frequently found in the in mucosa and submucosa as well as in the muscularis propria (Fig. 4C). Ano1 immunoreactivity never colocalized with mast cell tryptase (Fig. 4C), confirming that mast cells do not express Ano1.
A small number of strongly Ano1-positive cells with clear ICC-like morphology were weakly positive for Kit when compared with adjacent Kit-positive ICC (Fig. 5A). These cells were observed most frequently in the circular muscle layer of the gastric body but were also detected in small numbers in the septa of the circular muscle layer of the small intestine. In the stomach, in three tissues, a total of 416 Ano1-positive ICC were counted, and 3.2 ± 1.5% (1.57, 1.88, and 6.2%) of Ano1-positive ICC were weakly positive for Kit. All Kit-positive cells with ICC morphology were positive for Ano1.
Ano1 immunoreactivity in mouse gastrointestinal tract.
The rabbit anti-Ano1 antiserum was used to detect ICC in mice. Colocalization of Ano1 immunoreactivity with Kit immunoreactivity was examined in whole-mount preparations of the muscularis propria from the fundus and body of the stomach, small intestine, and proximal and distal colon. As in human tissue, the pattern and distribution of Ano1 immunoreactivity was consistent with labeling of ICC and overlapped exactly with Kit immunoreactivity (Figs. 6–8). As in human tissues, we detected no labeling of ICC-like cells by any of the secondary antibodies in the absence of primary antiserum. We did not obtain specific immunolabeling with the mouse antiserum to Ano1 on mouse tissues. In the small intestines, ICC were also labeled with the chicken antibody (Abcam), but the labeling was inconsistent and of poor quality.
In mouse stomachs, doubly labeled, bipolar ICC were observed in the muscle layers of the fundus (Fig. 6A), body (Fig. 6B), and antrum (Fig. 6C). Networks of multipolar ICC-MY were labeled in the gastric body (Fig. 6D), and additionally a small number of ICC-SM in the gastric antrum were identified by double labeling (Fig. 6E). In the small intestine of mice, both ICC-DMP and ICC-MY were labeled clearly with both antisera (Fig. 7). The ICC-DMP were bipolar in shape (Fig. 7A), and the ICC-MY formed a dense network around the myenteric ganglia (Fig. 7B). ICC-IM and ICC-MY positive for Ano1 and Kit were also identified in the mouse proximal colon (Fig. 7, C and D). Doubly positive ICC with stellate morphology were observed on the serosal surface of the longitudinal muscle (Fig. 7E). In addition, ICC with large cell bodies and two or more widely branching processes were also doubly labeled for Ano1 and Kit in the submucosal plexus. In the distal colon, ICC-SM (Fig. 8A) and ICC-IM (Fig. 8, B and D) had more extensive processes than in the proximal colon but were also doubly labeled for Ano1 and Kit. Doubly labeled ICC-MY of the distal colon had larger cell bodies (Fig. 8C) than ICC-MY of other parts of the mouse gastrointestinal tract.
The brightness of the fluorescently labeled structures varied considerably for both Kit and Ano1-immunoreactive cells. No cells with ICC morphology were, however, detected that were Ano1 positive and completely Kit negative or Kit positive and completely Ano1 negative. No Kit-positive, Ano1-negative mast cells were detected.
This study identifies Ano1 as a new, selective molecular marker for all classes of ICC in the stomach, small intestine, and large intestine of humans and mice that permits the immunochemical identification of these cells independent of Kit.
The demonstration that Kit is a selective marker for ICC (23) permitted discoveries that explain basic mechanisms in the regulation of gastrointestinal motility in health and disease. In human disease, Kit has been used as a marker to follow loss of ICC and changes in network density associated with a variety of diseases (9). Use of neutralizing antibodies that inhibit signaling has demonstrated that Kit is required for normal development and maintenance of ICC networks (33). Kit function and regulation of ICC numbers and network density seem to be associated intimately. Although Kit remains an excellent marker for ICC, an advantage of Ano1 is that it represents a novel marker that has no known link to Kit, and so use of Ano1 should permit examination of ICC independent of Kit. Also, the Ano1 antisera do not label mast cells (Kit and tryptase-positive cells), so there is no need to take into account mast cells when quantifying ICC by using Ano1. Also, this increases the accuracy of ICC quantification when using Ano1 protein and/or mRNA compared with Kit. Ano1 also had the additional benefit of identifying ICC-like cells that were positive for Ano1 but weakly positive for Kit. These “Kit-dim” ICC were only marginally brighter for Kit immunofluorescence than the background fluorescence, and it is feasible that these cells would have been missed if labeled only for Kit. The only caveat is that, in mouse but not human tissues, immunolabeling for Ano1 using the commercially available antibodies was not quite so clear as immunolabeling for Kit.
The identification of Ano1-positive cells as ICC was based on the coexpression of Ano1 and Kit immunoreactivity as well as the ICC-like morphology of the doubly labeled cells. Also, the distribution of the Ano1-positive, Kit-positive cells in the gastrointestinal tract was exactly as expected for ICC in those tissues (as reviewed by Ref. 15). These observations were confirmed by using two different antisera to label human tissues and were repeated in studies using one of the antisera on mouse gastrointestinal tract. The Ano1 antisera labeled intramuscular, myenteric, septal, submuscular, subserosal, and DMP ICC. Therefore Ano1 labels all classes of ICC. Immunohistochemical evidence for Ano1 protein expression in ICC is supported by studies investigating differential expression of genes in ICC compared with overall expression of those genes in the mouse gastrointestinal tract. Ano1 mRNA was four- to eightfold more highly expressed in myenteric and DMP ICC compared with all cells in the mouse jejunum (6).
The authenticity of Ano1 immunoreactivity in our studies is confirmed by the use of antibodies raised in different species to different antigens derived from the sequence of Ano1 (8) and the lack of labeling in the colon from Ano1 knockout mice. A third antiserum raised in chickens was also tested. The signal from the chicken antiserum showed unacceptably high levels of background signal, but signal from ICC-like cells represented the only signal that overlapped with the signals from the rabbit and/or mouse antisera. The mouse antiserum used to identify Ano1-positive cells in human tissues was raised against peptides with the primary amino acid sequences (8) of regions of the protein predicted to be on the intracellular surface of the plasma membrane (5). These Ano1-derived antigens had no sequence homology to the peptide sequence of Kit (8), so it is very unlikely that the antibodies were cross-reacting with Kit on ICC. Also, in situ hybridization for Ano1 (DOG1) RNA correlates well with the Ano1 immunohistochemistry (38) and the probe sequences have no overlap with Kit. In addition, the failure of Ano1 antisera to recognize strongly Kit-positive mast cells further supports the conclusion that the antisera studied do not recognize Kit. Our data are in agreement with the assertion that Ano1 is not expressed on mast cells (8), and we have no evidence to support earlier suggestions that Ano1 immunoreactivity is present on mast cells (38).
It is possible that the antibodies were recognizing proteins containing peptide sequences similar to the Ano1-sequences. Therefore, we checked for similarities by BLAST search (1) and determined that the peptide sequences in the regions of the antigen were only similar to the sequences of analogous regions in other members of the Ano/TMEM16 family of proteins, and even then, the identity was less than 50%. Furthermore, mRNAs for members of the Ano/TMEM16 family of proteins that were not Ano1 were not overrepresented in ICC compared with the surrounding tissue (6), so it is unlikely that the antisera were cross reacting with any protein related to Ano1.
Ano1-positive, Kit-negative cells in the mucosa and submucosa were not in regions where ICC are detected, and unlike the weakly Kit-positive cells they did not resemble either mast cells or ICC. It is not clear what those cells might be, but other investigators have identified Ano1 in many other tissues (18), specifically in the epithelia of foregut and airways (28) and mammary and salivary glands (30, 41). The morphologies of the Ano1-positive, Kit-negative cells at the base of the crypts of the mucosa were similar to those of myofibroblasts, but confirmation of their identity was not obtained in this study. The distribution of Ano1 immunoreactivity in the submucosal and mucosal regions of the mouse gastrointestinal tract was not investigated in the present study.
The function of Ano1 in ICC is not known. The labeling pattern we observed in both mouse and human ICC is consistent with the recent identification of Ano1 as a membrane-associated, Ca2+-activated Cl− conductance (5, 30, 41). What function Ano1 plays in ICC physiology and development remains to be determined. Kit plays a critical role in the development and maintenance of ICC (4, 9, 19, 21, 23, 33, 36) so changes in its expression may temporally precede changes in ICC mass. If Ano1 did not have a similar developmental role, it could perhaps be utilized as a more accurate indicator of ICC numbers, especially in pathologies involving changes in Kit signaling (17).
In conclusion, Ano1 is expressed on Kit-positive ICC in the human and mouse gastrointestinal tract. Ano1 represents a new highly selective molecular marker for studying the distribution and fate of ICC.
Supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK68055, DK57061, and DK58185. P. J. Gomez-Pinilla and M. J. Pozo are funded by the Spanish Ministerio de Educación y Ciencia (BFU2007-60563) and Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RD060013/1012).
The authors thank Kristy Zodrow for secretarial assistance and Dr. Brian Harfe for providing tissues from Ano1 knockout mice. The authors thank Peter Strege for assistance with preparation of the figures.
- Copyright © 2009 the American Physiological Society