Interstitial Cells Of Cajal Phenotype Research Articles

Intestinal proinflammatory macrophages induce a phenotypic switch in interstitial cells of Cajal.

Interstitial cells of Cajal (ICCs) are pacemaker cells in the intestine, and their function can be compromised by loss of C-KIT expression. Macrophage activation has been identified in intestine affected by Hirschsprung disease-associated enterocolitis (HAEC). In this study, we examined proinflammatory macrophage activation and explored the mechanisms by which it downregulates C-KIT expression in ICCs in colon affected by HAEC. We found that macrophage activation and TNF-α production were dramatically increased in the proximal dilated colon of HAEC patients and 3-week-old Ednrb-/- mice. Moreover, ICCs lost their C-KIT+ phenotype in the dilated colon, resulting in damaged pacemaker function and intestinal dysmotility. However, macrophage depletion or TNF-α neutralization led to recovery of ICC phenotype and restored their pacemaker function. In isolated ICCs, TNF-α-mediated phosphorylation of p65 induced overexpression of microRNA-221 (miR-221), resulting in suppression of C-KIT expression and pacemaker currents. We also identified a TNF-α/NF-κB/miR-221 pathway that downregulated C-KIT expression in ICCs in the colon affected by HAEC. These findings suggest the important roles of proinflammatory macrophage activation in a phenotypic switch of ICCs, representing a promising therapeutic target for HAEC.

Understanding the Biology of Human Interstitial Cells of Cajal in Gastrointestinal Motility

Millions of patients worldwide suffer from gastrointestinal (GI) motility disorders such as gastroparesis. These disorders typically include debilitating symptoms, such as chronic nausea and vomiting. As no cures are currently available, clinical care is limited to symptom management, while the underlying causes of impaired GI motility remain unaddressed. The efficient movement of contents through the GI tract is facilitated by peristalsis. These rhythmic slow waves of GI muscle contraction are mediated by several cell types, including smooth muscle cells, enteric neurons, telocytes, and specialised gut pacemaker cells called interstitial cells of Cajal (ICC). As ICC dysfunction or loss has been implicated in several GI motility disorders, ICC represent a potentially valuable therapeutic target. Due to their availability, murine ICC have been extensively studied at the molecular level using both normal and diseased GI tissue. In contrast, relatively little is known about the biology of human ICC or their involvement in GI disease pathogenesis. Here, we demonstrate human gastric tissue as a source of primary human cells with ICC phenotype. Further characterisation of these cells will provide new insights into human GI biology, with the potential for developing novel therapies to address the fundamental causes of GI dysmotility.

Effects of lipopolysaccharide-induced inflammation on the interstitial cells of Cajal

Interstitial cells of Cajal (ICC) have recently been found to display phenotypic changes. The present study is designed to determine whether phenotypic changes occur in ICC associated with an inflammatory microenvironment and whether the ICC phenotype could be recovered after the discontinuation of inflammatory stimuli. Immunohistochemistry studies revealed that the functional ICC marker, c-kit, was markedly reduced in patients with Hirschsprung's disease (n = 34) compared with controls (n = 12), whereas another marker of ICC, CD34, was not altered significantly. Compared with the vehicle group (n = 6), intraperitoneal injection of lipopolysaccharide (LPS; 1.5 mg/kg) in mice (n = 6) significantly induced plasma tumor necrosis factor-alpha (TNF-α) levels as determined by enzyme-linked immunosorbent assay. Western blot and real-time polymerase chain reaction assessment further showed that LPS injection markedly suppressed intestinal c-kit protein and mRNA expression, which could be blocked by Toll-like receptor 4 (TLR4) deficiency (n = 6) rather than TLR2 deficiency (n = 6) and had no effects on CD34. Compared with the vehicle group (n = 6), intraperitoneal TNF-α (30 μg/kg) administration (n = 6) also significantly reduced intestinal c-kit protein and mRNA levels but not CD34 levels. However, the reduction of c-kit induced by TNF-α injection was not suppressed by TLR4 deficiency (n = 6). Intestinal c-kit protein and mRNA levels were markedly restored after the discontinuation of TNF-α administration for 7 days. Moreover, immunofluorescence analysis of primary ICC further confirmed that exposure to TNF-α for 24 h suppressed c-kit expression, which could be restored after discontinuation of TNF-α exposure. CD34 expression was not altered upon exposure to TNF-α. Thus, phenotypic changes in ICC occur in an inflammatory microenvironment in the gut and LPS, TLR4 and TNFα are crucial to this process.

Down-Regulation of Hydrogen Sulfide Biosynthesis Accompanies Murine Interstitial Cells of Cajal Dysfunction in Partial Ileal Obstruction

PurposeTo investigate the role of endogenous hydrogen sulfide (H2S) in partial obstruction-induced dysfunction of the interstitial cells of Cajal (ICC) in mice ileum.Materials and MethodsPartial intestinal obstruction was induced surgically in male imprinting control region (ICR) mice. ICC networks were studied by Immunohistochemistry. Electrical activity was recorded by intracellular recording techniques. The expression of ICC phenotype marker c-kit receptor tyrosine kinase (c-kit), membrane binding stem cell factor (mSCF), the endogenous H2S biosynthesis enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) was studied by Western blotting. The expression of tumor necrosis factor-α (TNF-α) mRNA was observed by using real-time polymerase chain reaction.ResultsPartial intestinal obstruction resulted in ICC networks were disrupted above obstruction 14 days after the operation. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed and their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized. The expression of c-kit and mSCF was significantly decreased, also suggesting the disruption of the ICC network. The expression of TNF-α was significantly increased in the tunica muscularis of the obstructed intestine. Treatment of cultured intestinal smooth muscle cells with TNF-α caused dramatic down regulation of mSCF. The expression of CBS and CSE was significantly decreased in the tunica muscularis of the obstructed intestine. Intraperitoneal injection (i.p) of DL-propargylglycine, an irreversible inhibitor of CSE, and aminooxyacetic acid, an inhibitor of CBS, elevated the expression of TNF-α mRNA in the tunica muscularis of the ileum. Obstruction-induced over expression of TNF-α was significantly improved by supplementation of NaHS, but not the expressions of mSCF and c-kit.ConclusionsThe down regulation of endogenous H2S biosynthesis is related to over expression of TNF-α in obstructed small intestine. TNF-α-mediated mSCF down-regulation is not the only reason of partial intestinal obstruction-induced loss of ICC.

Interstitial cells in smooth muscles Review Series

Interstitial cells in smooth muscles Review Series

Downregulation of msh-like 2 (msx2) and neurotrophic tyrosine kinase receptor type 2 (ntrk2) in the developmental gut of KIT mutant mice

In the gastrointestinal tract, interstitial cells of Cajal (ICC) are the regulatory cells of gut movement. W/W mutant mice that have receptor tyrosine kinase KIT mutation lack ICC along the myenteric plexus layer of small intestine. The development and maintenance of the ICC phenotype have been related to KIT, but the other genes involved in ICC development during embryogenesis are not clear. Our aim was to identify ICC-specific genes in the embryonic stage. We examined genes that are expressed less in ICC-deficient W/W mice than in wild type (WT) at embryonic day 14 (E14) in order to clarify the genes associated with the ICC development using subtractive hybridization and microarray. Among them, we identified msh-like 2 (msx2) and neurotrophic tyrosine kinase receptor type 2 (ntrk2). Using real-time PCR, msx2 and ntrk2 were found to be expressed at significantly lower levels in W/W than in WT during embryogenesis. Msx2 immunoreactivity was high in the WT small intestine. These data suggest that the gene expressions of ntrk2 and msx2 were significantly suppressed in KIT mutant mouse embryo and neonate and that these genes are likely to regulate ICC development.

Myometrial interstitial cells and the coordination of myometrial contractility

A strict regulation of contractility in the uterus and fallopian tube is essential for various reproductive functions. The uterus contributes, through either increased contractility or periods of relative quiescence, to: (i) expulsion of menstrual debris, (ii) sperm transport, (iii) adequate embryo placement during implantation, (iv) enlarging its capacity during pregnancy and (v) parturition. The dominant cell population of the uterine wall consists of smooth muscle cells that contain the contractile apparatus responsible for the generation of contractile force. Recent interest has focused on a new population of cells located throughout the myometrium on the borders of smooth muscle bundles. These cells are similar to interstitial cells of Cajal (ICC) in the gut that are responsible for the generation of electrical slow waves that control peristalsis. A precise role for myometrial Cajal-like interstitial cells (m-ICLC) has not been identified. m-ICLC express the c-kit receptor, involved in creating and maintaining the ICC phenotype in the gastrointestinal tract. However, both acute and prolonged inhibition of this receptor with the c-kit antagonist imatinib mesylate does not appear to affect the spontaneous contractility of myometrium. Calcium imaging of live tissue slices suggests that contractile signalling starts on the borders of smooth muscle bundles where m-ICLC are located and recently the possible role of extracellular ATP signalling from m-ICLC has been studied. This manuscript reviews the evidence regarding tissue-level signalling in the myometrium with a particular emphasis on the anatomical and possible functional aspects of m-ICLC as new elements of the contractile mechanisms in the uterus.

Kit mutants and gastrointestinal physiology

There has been considerable speculation about the function of interstitial cells of Cajal (ICC) since their discovery more than 100 years ago. It has been difficult to study these cells under native conditions, but great insights about the function of ICC have come from studies of genetic models with loss-of function mutations in the Kit signalling pathway. First it was discovered that signalling via Kit (a receptor tyrosine kinase) was vital for the development and maintenance of the ICC phenotype in gastrointestinal (GI) muscles. In compound heterozygotes (W/W(V) and Sl/Sl(d) animals), where there are partial loss-of-function mutations in Kit receptors or Kit ligand (stem cell factor), ICC failed to develop in various regions of the GI tract, but no major changes in the smooth muscle layers or enteric nervous system occurred in the absence of these cells. Animals with these mutations provided an unprecedented opportunity to understand the role of ICC in GI motor function, and it is now clear from these studies that ICC serve as: (i) pacemaker cells, generating the spontaneous electrical rhythms of the gut known as slow waves; (ii) a propagation pathway for slow waves so that large areas of the musculature can be entrained to a dominant pacemaker frequency; (iii) mediators of excitatory cholinergic and inhibitory nitrergic neural inputs from the enteric nervous system, and (iv) stretch receptors that modulate membrane potential and electrical slow wave frequency. This review describes the use of genetic models to understand the important physiological role of ICC in the GI tract.

Interstitial cells of Cajal at the clinical and scientific interface

Considerable work over the past two decades has determined that interstitial cells of Cajal (ICC) serve as pacemaker cells, conduits for active transmission of electrical slow waves, sites of innervation by peripheral motor neurons, and mechanotransducers. While most of the physiology of ICC has been learned from studies of the cells within the gastrointestinal tract, ICC are found in a variety of smooth muscle tissues and may have analogous or novel physiological functions in those organs. Clinical investigations of muscles from patients with a variety of gastrointestinal motility disorders have raised the exciting possibility that loss of ICC may be responsible for the development of motor dysfunction. This review discusses the development of ICC, the kinds of human disorders in which ICC loss may be important, what factors regulate the ICC phenotype, and what therapeutic approaches might be utilized to restore or regenerate ICC. This field is primed for translational discoveries. ICC are responsible for critical physiological functions in smooth muscle tissues, they are lost in pathophysiological conditions, and it will be important now to decipher the conditions that are responsible for ICC loss and develop new therapies to relieve patients of this problem. Success in this endeavour might improve the quality of life for millions of patients.

Kit signaling is essential for development and maintenance of interstitial cells of Cajal and electrical rhythmicity in the embryonic gastrointestinal tract

Interstitial cells of Cajal (ICC) are specialized cells in smooth muscle organs that generate and propagate pacemaker activity, receive inputs from motor neurons, and serve as mechanosensors. In the gastrointestinal tract, development and maintenance of the ICC phenotype have been linked to intracellular signaling via Kit, but its role in development of ICC during embryogenesis is controversial. Here we have studied the development of functional ICC-MY during the late gestational period in mice. Blocking Kit with a neutralizing antibody before and after development of spontaneous electrical activity (E17 to P0) caused loss of ICC-MY networks and pacemaker activity. ICC-MY and pacemaker activity developed normally in W/+ and W(V)/+ heterozygotes, but failed to develop between E17 to P0 in W/W(V) embryos with compromised Kit function. Muscles treated with Kit neutralizing antibody or the tyrosine kinase inhibitor, imatinib mesylate (STI571), from E17-P0 for 3 days caused loss of functionally developed ICC-MY networks, but ICC-MY and pacemaker activity recovered within 9 days after discontinuing treatment with neutralizing antibody or imatinib mesylate. These data suggest that Kit signaling is an important factor in lineage decision and in the development of functional ICC in late gestation. ICC-MY demonstrate significant plasticity in gastrointestinal tissues. Manipulation of the ICC phenotype might provide useful therapies in gastrointestinal disease where the Kit-positive cell population is either lost or amplified.

Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction.

1. Partial obstruction of the murine ileum led to changes in the gross morphology and ultrastructure of the tunica muscularis. Populations of interstitial cells of Cajal (ICC) decreased oral, but not aboral, to the site of obstruction. Since ICC generate and propagate electrical slow waves in gastrointestinal muscles, we investigated whether the loss of ICC leads to loss of function in partial bowel obstruction. 2. Changes in ICC networks and electrical activity were monitored in the obstructed murine intestine using immunohistochemistry, electron microscopy and intracellular electrophysiological techniques. 3. Two weeks following the onset of a partial obstruction, the bowel increased in diameter and hypertrophy of the tunica muscularis was observed oral to the obstruction site. ICC networks were disrupted oral to the obstruction, and this disruption was accompanied by the loss of electrical slow waves and responses to enteric nerve stimulation. These defects were not observed aboral to the obstruction. 4. Ultrastructural analysis revealed no evidence of cell death in regions where the lesion in ICC networks was developing. Cells with a morphology intermediate between smooth muscle cells and fibroblasts were found in locations that are typically populated by ICC. These cells may have been the redifferentiated remnants of ICC networks. 5. Removal of the obstruction led to the redevelopment of ICC networks and recovery of slow wave activity within 30 days. Neural responses were partially restored in 30 days. 6. These data describe the plasticity of ICC networks in response to partial obstruction. After obstruction the ICC phenotype was lost, but these cells regenerated when the obstruction was removed. This model may be an important tool for evaluating the cellular/molecular factors responsible for the regulation and maintenance of the ICC phenotype.

Physiology and pathophysiology of the interstitial cell of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks.

Interstitial cells of Cajal (ICC) are the pacemaker cells in gastrointestinal (GI) muscles. They also mediate or transduce inputs from enteric motor nerves to the smooth muscle syncytium. What is known about functional roles of ICC comes from developmental studies based on the discovery that ICC express c-kit. Functional development of ICC networks depends on signaling via the Kit receptor pathway. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells within the tunica muscularis, and the loss of ICC in some motility disorders. Manipulating Kit signaling with reagents to block the receptor or downstream signaling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies of the development of these cells within the tunica muscularis and also allowed the study of specific functions of different classes of ICC in several regions of the GI tract. This article examines the role of ICC in GI motility, focusing on the functional development and maintenance of ICC networks in the GI tract and the phenotypic changes that can occur when the Kit signaling pathway is disrupted.

Expression of the Intermediate Filament Nestin in Gastrointestinal Stromal Tumors and Interstitial Cells of Cajal

It has recently been proposed that gastrointestinal stromal tumors (GISTs) originate from stem cells that differentiate toward a phenotype of interstitial cells of Cajal (ICCs). Nestin is a newly identified intermediate filament protein, and is predominantly expressed in immature cells, such as neuroectodermal stem cells and skeletal muscle progenitor cells, and tumors originating from these cells. In this study, we examined, using immunohistochemistry, the nestin expression in GISTs and ICCs to clarify the origin of GISTs. Strong immunoreactivity for nestin was observed in all 18 GISTs, and its expression was confirmed by Western blot and Northern blot analyses. In contrast, three leiomyomas and a schwannoma that developed in the gastrointestinal tract showed no apparent immunoreactivity for nestin. Among 17 mesenchymal tumors (seven leiomyosarcomas, five malignant peripheral nerve sheath tumors, and five fibrosarcomas) that occurred in sites other than the gastrointestinal tract, only two malignant peripheral nerve sheath tumors were moderately immunoreactive for nestin. Furthermore, with fluorescence double immunostaining of the normal small intestine, nestin expression was demonstrated in ICCs. These results show that nestin may be a useful marker for diagnosis of GISTs, and support the current hypothesis that GISTs are tumors of stem cells that differentiate toward an ICC phenotype.

Cellular origin of gastrointestinal stromal tumors: a study of 27 cases.

Context Interstitial cells of Cajal (ICCs), also known as pacemaker cells, are cells in the gastrointestinal tract that play a role in the control of gut motility. The ICCs express the c-kit proto-oncogene encoding a type III tyrosine kinase (KIT) receptor, a ligand that is known as stem cell factor (SCF). The maturation of ICCs is dependent on SCF-KIT interaction. The cellular origin, differentiation, nomenclature, and prognosis of gastrointestinal stromal tumors (GISTs) are controversial. Objective To test the hypothesis that GISTs originate from CD34-positive stem cells and differentiate toward an ICC phenotype. Materials and methods We studied 27 cases of smooth muscle differentiated GISTs collected for 14 years (1985-1999), including 8 benign (leiomyoma), 15 malignant primary (leiomyosarcoma), and 4 metastatic to the liver. Immunohistochemical studies of selected lineage-directed monoclonal antibodies of c-kit (CD117), CD34, vimentin, desmin, alpha-actin, S100, and MIB-1 were performed on both normal and tumor tissues. Results Immunoperoxidase stains of normal gastrointestinal tract showed both c-kit and CD34-positive cells surrounding the Auerbach ganglia plexus in the gastrointestinal tract. Twenty-seven of 27 tumors strongly expressed c-kit. Fourteen of 27 tumors were positive for CD34. Of the malignant GISTs, 14 of 19 were positive for CD34; of the benign tumors, 0 of 8 were positive for CD34. Thus, CD34 was the best indicator of malignant phenotype. Conclusion This is the first description of benign smooth muscle GISTs negative for CD34. The results of this study suggest that GISTs originate from CD34-positive stem cells and differentiate toward pacemaker cell phenotype. The lack of expression of CD34 in the benign GIST may indicate that benign GISTs are composed of more mature ICCs, whereas malignant GISTs are composed of dedifferentiated ICCs that express CD34-positive stem cells.

Cellular origin of gastrointestinal stromal tumors: a study of 27 cases.

Interstitial cells of Cajal (ICCs), also known as pacemaker cells, are cells in the gastrointestinal tract that play a role in the control of gut motility. The ICCs express the c-kit proto-oncogene encoding a type III tyrosine kinase (KIT) receptor, a ligand that is known as stem cell factor (SCF). The maturation of ICCs is dependent on SCF-KIT interaction. The cellular origin, differentiation, nomenclature, and prognosis of gastrointestinal stromal tumors (GISTs) are controversial. To test the hypothesis that GISTs originate from CD34-positive stem cells and differentiate toward an ICC phenotype. We studied 27 cases of smooth muscle differentiated GISTs collected for 14 years (1985-1999), including 8 benign (leiomyoma), 15 malignant primary (leiomyosarcoma), and 4 metastatic to the liver. Immunohistochemical studies of selected lineage-directed monoclonal antibodies of c-kit (CD117), CD34, vimentin, desmin, alpha-actin, S100, and MIB-1 were performed on both normal and tumor tissues. Immunoperoxidase stains of normal gastrointestinal tract showed both c-kit and CD34-positive cells surrounding the Auerbach ganglia plexus in the gastrointestinal tract. Twenty-seven of 27 tumors strongly expressed c-kit. Fourteen of 27 tumors were positive for CD34. Of the malignant GISTs, 14 of 19 were positive for CD34; of the benign tumors, 0 of 8 were positive for CD34. Thus, CD34 was the best indicator of malignant phenotype. This is the first description of benign smooth muscle GISTs negative for CD34. The results of this study suggest that GISTs originate from CD34-positive stem cells and differentiate toward pacemaker cell phenotype. The lack of expression of CD34 in the benign GIST may indicate that benign GISTs are composed of more mature ICCs, whereas malignant GISTs are composed of dedifferentiated ICCs that express CD34-positive stem cells.

Development and plasticity of interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal (GI) muscles, and these cells also mediate or transduce inputs from the enteric nervous system. Different classes of ICC are involved in pacemaking and neurotransmission. ICC express specific ionic conductances that make them unique in their ability to generate and propagate slow waves in GI muscles or transduce neural inputs. Much of what we know about the function of ICC comes from developmental studies that were made possible by the discoveries that ICC express c-kit and proper development of ICC depends upon signalling via the Kit receptor pathway. Manipulating Kit signalling with reagents to block the receptor or downstream signalling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies into the specific functions of the different classes of ICC in several regions of the GI tract. Kit is also a surface antigen that can be used to conveniently label ICC in GI muscles. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells in the gut wall, and the loss of ICC in some clinical disorders. Preparations made devoid of ICC have also allowed analysis of the consequences of losing specific classes of ICC on GI motility. This review describes recent advances in our knowledge about the development and plasticity of ICC and how developmental studies have contributed to our understanding of the functions of ICC. We have reviewed the clinical literature and discussed how loss or defects in ICC affect GI motor function.