Journal of Pediatric Surgery
Volume 38, Issue 1 , Pages 73-77, January 2003

Immunocolocalization of the heme oxygenase-2 and interstitial cells of Cajal in normal and aganglionic colon☆

Presented at the 49th Annual Congress of the British Association of Paediatric Surgeons, Cambridge, England, July 23-26, 2002.

Children's Research Centre, Our Lady's Hospital for Sick Children, Dublin, Ireland. Dubin, Ireland

Article Outline

Abstract 

Purpose: Interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the control of gut motility. Carbon monoxide (CO) has been proposed as an endogenous messenger molecule between ICC and smooth muscle cells in the gastrointestinal tract (GIT). Heme oxygenase-2 (HO-2) is the main physiologic mechanism for generating CO in human cells. The aim of this study was to investigate the immunocolocalization of the HO-2 and ICCs in normal and aganglionic bowel of Hirschsprung's disease (HD). Methods: Full-thickness specimens were obtained from aganglionic colon during pull-through operation from 10 patients diagnosed as having HD. Normal control large bowel specimens were collected from 4 patients during bladder augmentation procedures. Double immunostaining was carried out using c-kit and HO-2 antibodies. Immunolocalization was detected by means of confocal laser scanning microscopy. Results: HO-2 immunoreactivity (IR) was found in many ICCs present around the myenteric plexus, within the longitudinal and circular muscle layers and at the innermost part of the circular muscle layer in normal colon. In the aganglionic colon there was absence of HO-2 IR in the sparsely found ICCs. In the transitional zone of HD bowel the colocalization of HO-2 IR and ICCs was much reduced compared with controls. Conclusions: The results of this study provide the first evidence for the presence of HO-2 immunoreactivity in the ICCs in normal human colon and absence of HO-2 immunoreactivity in sparsely appearing ICCs in the bowel of HD patients. The lack of HO-2 in the ICCs in the bowel of HD patients may result in impaired intracellular communication between ICCs and SMCs causing motility dysfunction. J Pediatr Surg 38:73-77. Copyright 2003, Elsevier Science (USA). All rights reserved.

Keywords:  Interstitial cells of Cajal, carbon monoxide, heme oxygenase, Hirschsprung disease

 

Normal peristaltic and segmental motor activity of the bowel requires communication between the enteric nervous system (ENS), smooth muscle cells (SMCs), and interstitial cells of Cajal (ICCs).1, 2, 3, 4, 5 ICCs are nonneuronal cells that serve as pacemaker cells and are responsible for the spontaneous, rhythmic, electrical excitatory activity of the gastrointestinal SMCs.4, 5, 6, 7 Recent studies have found that the c-kit receptor is essential for the development of the ICCs.4, 6, 7 Mesenchymal ICCs precursors that carry the c-kit receptor require the kit ligand (KL), which can be provided by neuronal cells or smooth muscle cells. According to the influence of the KL from either neuronal or smooth muscle cells, the ICCs develop as either myenteric ICCs (ICCs-MY) or intramuscular ICCs (ICCs-IM).2, 4, 6, 7 ICCs-IM are bipolar cells with usually one or 2 processes and scattered throughout the circular and longitudinal muscle layers and at the innermost part of the circular muscle.1, 4, 5, 8 These cells are associated closely with nerve fibres. Functionally, they modulate communications between nerves and muscles. Additionally, they serve to modify slow-wave activity.1, 4, 5, 8 ICCs-MY are triangular or stellate-shaped cells with multiply processes forming dense networks around myenteric plexus. They are pacemaker cells that are involved in pacemaker generation in the gastrointestinal tract (GIT).1, 4, 5, 7

It has been suggested recently that carbon monoxide (CO), a gaseous neurotransmitter in the GIT causes smooth muscle relaxation.9, 10, 12, 13, 14, 16, 20, 22, 23 CO is produced endogenously by the interaction between heme and heme oxygenase (HO).9, 10, 11, 12, 13, 14, 15, 16, 20 Three different isoforms of HO have been described: HO-1, HO-2, and HO-3.9, 12, 13 HO-1, also known as heat-shock protein 32K, is distributed widely throughout the body with high levels of expression in the liver, spleen, and bone-marrow. HO-2 is localized primarily in the neural tissues and is expressed highly in the brain and GIT. HO-3 has been proposed as a regulator of genes that are heme dependent. Recently, Miller et al16 reported HO-2 expression in ICCs of the murine intestine, thus, suggesting that CO is produced in ICCs and functions as a messenger molecule between ICCs and gastrointestinal SMCs.

In the current study we hypothesized that the lack of ICCs in the aganglionic colon may inhibit production of CO thus impairing communication between nerves and smooth muscle cells. To test this hypothesis we investigated immunocolocalization of HO-2 and ICCs in the normal colon and aganglionotic colon of HD patients.

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Materials and methods 

Full-thickness large bowel specimens were obtained from 10 patients, aged one to 20 months with classical rectosigmoid HD during pull-through operation. Normal control large bowel specimens were collected from children aged 4 to 12 years undergoing bladder augmentation. Specimens were fixed in Zamboni's solution for more than 24 hours and subsequently rinsed in phosphate-buffered saline (PBS). Then specimens were rinsed in PBS with 10% sucrose at 4°C overnight and subsequently frozen in OCT compound in liquid nitrogen. Double staining using c-kit/HO-2 antibodies was performed for confocal laser scanning microscopy. To prevent nonspecific absorption of immunoglobulin, the 8-μm tissue sections were incubated for 30 minutes in 10% goat serum (DAKO, Ely, England), in Tris-HCl buffer (0,05% Tween-20, 1% BSA; pH 7,6). Subsequently, sections were incubated with the primary mouse monoclonal antibody anti–c-kit (Novocastra, Newcastle-upon-Tyne, England), diluted 1:100, and rabbit polyclonal heme oxygenase-2 (HO-2)-antibody (Stress Gene Biotech Corp, York, UK) diluted 1:500 in the antibody diluent with background reducing components (DAKO), for 24 hours at 4°C. Sections then were incubated at the room temperature for 1 hour with secondary antibodies: Alexa Fluor 488 goat antirabbit and Alexa Fluor 594 goat antimouse (Molecular Probes, Netherlands) diluted 1:100 in Tris-HCl buffer. Each incubation period was followed by gently washing in Tris-buffer saline. The section were coverslipped with fluorescence mounting medium (DAKO).

Nonspecific labelling was checked by omitting the respective primary antibody. Staining results were evaluated using confocal laser scanning microscopy

Laser confocal microscopy 

An upright laser scanning confocal microscope (BIO-RAD 2000; Hemel Hempstead, England) with immersion objective (×40 Numerical aperture 0.45). Tissue sections were excited using a Krypton/Argon laser with excitation and barrier filters set for individual fluorophores according to their specific excitation-emission spectra (ë = 594 nm, 488 nm). The emitted light was detected by a photomultiplier tube and converted, via an analogue-to-digital converter (BIO-RAD, MRC 1024 Hamil Hamsted), into digital pixelated images (512 × 512 picture elements). The detection pinhole was set for use with ×40. Offset and gain setting were determined at the start of each experiment and kept constant throughout, with laser power recorded each time.

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Results 

Normal colon and ganglionic colon of HD patients 

ICCs-IM were long, thin, bipolar cells with only one or 2 long processes and many short processes. ICCs-IM were in abundance in the circular and longitudinal muscle layers and in the innermost part of the circular muscle (Fig 1A).

  • View full-size image.
  • Fig. 1. 

    Differences in the distribution of c-kit–immunopositive ICCs in the normal and HD colon. (A) ICCs-MY formed a dense network surrounding the myenteric plexus in the normal colon (other arrows). ICCs-IM in the circular muscle (top arrows) (B) ICCs-MY as a single cells in the transition zone. (C) Lack of ICCs-IM and ICCs-MY in the aganglionic colon.

ICCs-MY were stellatelike cells with large cell bodies and several processes intermingling with each other and forming a dense network around the myenteric plexus. Numerous c-kit–positive ICCs-MY were observed in the normal colon and ganglionic colon from HD patients (Fig 1A). Strong HO-2 immunoreactivity (IR) was observed in ganglion cells throughout the myenteric (Fig 2A) and submucosus plexuses (Fig 2B) and endothelium of blood vessels (Fig 2C).
  • View full-size image.
  • Fig. 2. 

    Immunostaining of HO-2 in the normal bowel. (A) HO-2 IR in the ganglion cells in the myenteric plexus (arrow) and in the nerve fibres interconnecting ganglia. (B) HO-2 IR in the submucosal ganglion (arrow). (C) HO-2 IR in the endothelial cells in the blood submucosal vessels in the submucosa (arrow).

Occasionally, HO-2 IR was seen in fine nerve fibres in the circular and longitudinal muscle layers. Double-labelling with c-kit and HO-2 showed strong HO-2 IR in the ICCs-IM (Fig 3) and ICCs-MY (Fig 4).
  • View full-size image.
  • Fig. 3. 

    C-kit and HO-2 IR in the circular muscle in the normal colon. (A) C-kit–positive ICCs-IM in the circular muscle (arrows). (B) HO-2 IR in the circular muscle (arrows). (C) Double labelling with c-kit/HO-2 shows immunocolocalization HO-2 in the ICCs-IM (arrows).

  • View full-size image.
  • Fig. 4. 

    C-kit and HO-2 IR in the myentric plexus in the normal colon. (A) C-kit–positive ICCs-MY around ganglia cells in the myenteric plexus (arrows). (B) HO-2 IR in the ganglia cells in the myenteric plexus (arrows). (C) Double labelling with c-kit/HO-2 shows immunocolocalization HO-2 in the ICCs-MY (arrows).

Aganglionic rectosigmoid colon of HD patients 

There was lack or marked reduction of ICCs-IM and ICCs-MY in the aganglionotic colon compared with normal colon (Fig 1C). Sparsely observed ICCs-MY appeared as thin and bipolar cells closely related to hypertrophic nerves trunks. There was lack of HO-2 IR in the nerve fibres in the muscle layer and in hypertrophic nerve trunks in the submucosa and the intramuscular space. Positive staining for HO-2 was found in the endothelial cells in intramural blood vessels similar to the staining in normal colon. There was no c-kit/HO-2 immunocolocalization in the sparsely found ICCs.

Transitional zone of rectosigmoid colon of HD patients 

ICCs-IM were found between the smooth muscle fibres, but their number was reduced compared with the normal colon. ICCs-MY were evident as single or cell clusters related to small myenteric ganglia. They did not form the typical networks as seen in the normal colon (Fig 1B). Positive staining for HO-2 was found in the mucosal epithelial cells and endothelial cells in the intramural blood vessels similar to normal colon. Some staining of neuronal cell bodies in the single ganglion cells, mainly in the myentric plexus, appeared to display HO-2 IR. Occasional staining of HO-2–positive nerve fibers was seen mainly in the circular muscle layer. In the double c-kit/HO-2 staining HO-2 IR was present in the sparsely observed ICCs-MY and ICCs-IM.

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Discussion 

The ICCs are of fundamental importance in regulating gastrointestinal motility. Intestinal smooth muscle cells (SMCs) are depolarised in the absence of ICCs, and there are no spontaneous slow waves.2, 7 Altered distribution of ICCs has been reported previously in the HD.8, 17, 18 The distribution pattern of ICCs in our study was similar to that previously reported. It is currently accepted that deficient expression of ICCs in the aganglionic bowel may contribute to motility dysfunction in HD by defective generation of electrical pacemaker activity.8, 17, 18

ICCs and SMCs communicate through messenger molecules. However, the messenger molcules mediating communication between ICCs and SMCs in the human colon are unknown. It is currently accepted that ICCs are connected by gap junctions to each other and to SMCs.2, 19 Gap junctions are transmembrane channels that allow the exchange of irons, metabolites, and other small molecules between the cytoplasm of adjacent cells. Recently, it has been reported that CO is produced by ICCs and functions as a messenger molecule between ICCs and gastrointestinal SMCs.9, 11, 15, 16, 21, 22, 23 The major source of endogenous CO is through heme breakdown.9, 10, 11, 12, 13 Colocalization of HO-2 IR in ICCs was reported in the opossum annorectum,9 rat small intestine,11 mouse small intestine,13, 15 and in the human stomach.21 In the current study, strong HO-2 IR was shown in the ganglion cells in the myenteric plexus and nerve fibres in the muscularis propria in the human colon suggesting that enteric neurones have a capacity to synthesize CO and thus indicate a possible role of CO as a neurotransmitter. The lack of HO-2 IR in the intramuscular nerve fibres and in the hypertrophic nerve trunks suggests impaired CO synthesis in the aganglionic bowel.

The most striking finding in this study was the colocalization of HO-2 IR in the ICCs in the normal colon, indicating that CO is produced by ICCs, a finding not reported previously in the human colon. Double labelling with c-kit and HO-2 using confocal laser microscopy showed HO-2 IR in the stellatelike ICCs surrounding the myenteric plexus and also in the bipolar ICCs in the smooth muscle layers. The presence of HO-2 IR in the ICCs of normal colon implies that CO released from ICCs may act on the enteric neurones and modulate inhibitory neurotransmission. The lack or deficiency of ICCs in the aganglionic bowel may impair endogenous production of CO.

Carbon monoxide that is produced by enteric neurones and ICCs is an important neurotransmitter that mediates relaxation of the smooth muscle of GIT. The findings of the current study indicate that there is impaired CO synthesis by the neurones and ICCs in the aganglionic bowel, which may be responsible for the motility dysfunction in HD patients.

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References 

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☆ Address reprint requests to Professor Prem Puri, MS, FRCS, FRCS (Ed), FACS, Director of Research, Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin 12, Ireland.

PII: S0022-3468(02)63022-2

doi:10.1053/jpsu.2003.50014

Journal of Pediatric Surgery
Volume 38, Issue 1 , Pages 73-77, January 2003

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