Immunoreactivity for Ano1 detects depletion of Kit-positive interstitial cells of Cajal in patients with slow transit constipation

Abstract

Background: Depletion of interstitial cells of Cajal (ICC) is associated with several gastrointestinal (GI) motility disorders. Changes in ICC networks are usually detected by immunolabeling for the receptor tyrosine kinase Kit. Ano1 (DOG1 or TMEM16A) was recently described as a marker of ICC in GI tract. Our aim was to determine whether Ano1 immunoreactivity can be used as a reliable marker for ICC in tissues from patients with motility disorders.

Methods: Four tissues from patients with normal ICC numbers and four tissues from patients with slow transit constipation and loss of Kit-positive ICC were studied. Interstitial cells of Cajal were detected by double labeling using antisera to Kit and Ano1.

Key results: Both the processes and cell bodies of ICC in tissue from controls and slow transit constipation were immunoreactive for Ano1. There was a near complete overlap between Kit and Ano1 immunoreactivity. Tissues from patients with slow transit constipation contained significantly fewer Ano1-positive ICC than control tissues. The numbers of ICC identified by Ano1 and Kit immunoreactivity were nearly identical across the range of ICC numbers from an average of 1.64 to 7.05 cells per field and correlated with an R(2) value of 0.99.

Conclusions & inferences: Ano1 is a reliable and sensitive marker for detecting changes in ICC networks in humans. Labeling with antibodies selective for Ano1 reproducibly detects depletion of Kit-positive ICC in tissues from patients with slow transit constipation.

Fig 1

Ano1-immunoreactivity is seen in both the ICC cell body and in the processes (A) Ano1 and Kit-immunoreactivity in the ICC cell body and the processes in colon tissue obtained from a patient with non obstructive colon cancer. The merged image shows Ano1 and Kit-immunoreactivity were coincident. (B) Ano1 and Kit-immunoreactivity in an ICC cell body and its processes in colon tissue obtained from a patient with slow transit constipation. The merged image shows Ano1 and Kit-immunoreactivity were also coincident in slow transit constipation. These images were collected using a 60×, 1.2NA water immersion objective and represent a single section obtained from a confocal stack collected with 0.3 μm z axis steps. The signal obtained with the rabbit antiserum (Ano1) is shown in red and the signal from mouse antiserum (Kit) is shown in green.

Fig 2

Ano1-positive ICC are decreased in all layers of the colon wall in slow transit constipation. (A) In colon tissue from control patients, Ano1 and Kit-immunolabeling showed numerous ICC in the circular muscle, submucosal border of circular muscle, myenteric plexus and the longitudinal muscle. The merged image shows overlap of Ano1 and Kit-mmunolabeling. The arrow points to a mast cell in the mucosa which was Kit-positive but negative for Ano1. (B) In colon tissue from patients with slow transit constipation, Ano1 and Kit-immunolabeling show markedly reduced ICC in the circular muscle, submucosal border of circular muscle, myenteric plexus and the longitudinal muscle as compared to the control tissue. The merged images show overlap of residual Ano1 and Kit- immunolabeling. Both panels A and B are montages of images that were collected using a 10× water immersion objective. The signal obtained with the rabbit antiserum (Ano1) is shown in red and the signal from mouse antiserum (Kit) is shown in green. (C) There were significantly fewer ICC in the circular muscle layer of the colon in patients with slow transit constipation as detected by Ano1-immunolabeling (1.89±0.13 vs. 6.43±0.37 cells per field, P <0.05, n = 4, unpaired t test). Closed symbols represent mean number of ICC in each patient per field with the standard error of mean while the open symbols represent mean number of ICC per field from all 4 patients with standard error of mean. (D) The number of ICC per field detected as Kit and Ano1-positive are identical in control patients and patients with slow transit constipation. The left part of the graph shows the number of ICC per field detected by Ano1 and Kit-immunoreactivity in colon tissue from patients with slow transit constipation (1.8 cells/field) and the right part shows similar data for colon tissue from control patients (6.4 cells/field) showing near complete overlap.