Immunoreactive epidermal growth factor receptors are present in gastrointestinal epithelial cells of preterm infants with necrotising enterocolitis

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Abstract

Introduction: Epidermal growth factor (EGF) affects epithelial cell proliferation, differentiation and migration in the gastrointestinal tract of experimental animals, and increases proliferation when given intravenously to children with congenital microvillous atrophy or necrotising enteritis. The aim of the present study is to determine whether EGF receptors (EGFR) are present in the gut epithelium of preterm infants, and to discover whether neonatal necrotising enterocolitis (NEC) is associated with the absence of EGFR from mucosal cells. Methods: Tissues were taken from involved colon and small intestine of four preterm infants with NEC, and control tissues were taken from four other neonates who had laparatomies for congenital malformations. Sections of the tissues were examined histopathologically after treatment with a monoclonal antibody against the external domain of the EGFR (Zymed Laboratories, San Francisco, CA, USA). Results: Histopathological examination confirmed diagnosis of NEC in the involved bowel and controls showed appearance within normal limit. Immunoreactive EGFR were present on the epithelial cells of both the colon and small intestine, localised on the basolateral membrane of the cells of both subject and the controls. There was no apparent reduction in expression compared with controls. Conclusion: NEC in preterm infants is not associated with absence of EGFR. The presence of EGFR in gut epithelial cells raises the possibility of using EGF for prophylaxis or treatment of NEC.

Introduction

Epidermal growth factor (EGF) is a heat stable 53-amino acid peptide (MW 6400), which is secreted into the gut lumen by the Salivary glands, Brunner's glands of the duodenum, kidneys and also by the ulcer-associated cell lineage (UACL) [1], [2].

The human EGF receptor (EGFR) has been cloned and is a 170-kDa protein that consists of a cell surface ligand binding domain, a single hydrophobic transmembrane domain, and a cytoplasmic tyrosine kinase domain. EGF binding to EGFR initiates a complex program of activation of intrinsic kinase activity, increases in cytosolic calcium, and ultimately DNA synthesis and cellular growth [1], [2], [3]. This receptor also binds transforming growth factor α, amphiregulin, and heparin binding EGF.

EGF helps promote the growth and maturation of various organ systems, including the gastrointestinal tract and lungs [4], [5], [6]. The predominant effects of EGF on the gastrointestinal tract can be categorised into the following three areas: suppression of gastric acid secretion, gastric cytoprotection, and stimulation of DNA synthesis [1]. Immunoreactive EGF is found in most biological fluids, including plasma, although the circulating concentration of EGF is extremely low. EGF is bound to platelets and is released during platelet aggregation [7]. EGF and other growth factors regulates cellular function through autocrine, endocrine, paracrine and juxtacrine pathways [1].

Numerous studies have identified both EGF and EGFR in a variety of different tissues, including the kidney, bone marrow, the lactating mammary gland, the pancreas, the adult small intestine and the anterior pituitary gland [8], [9]. However, no data are available on the expression of EGF or EGFR in preterm human gastrointestinal cells.

Experimental evidence supports a role for EGF on gut maturation and protection during development; EGF leads to increased growth of the gastric mucosa in neonatal rats, and inactivation of the EGFR in knockout mice results in a haemorrhagic enteritis that is similar to necrotising enterocolitis [10], [11], [12], [13]. Helmrath et al. [14], in a small study, demonstrated a lower concentration of EGF in both saliva and serum in preterm infants with necrotising enterocolitis. These results led us to ask whether reduced EGF or EGFR expression might be associated with NEC in human preterm infants.

The aim of the present study was thus to determine whether EGFR were present in the gut epithelium of preterm infants, and to define whether NEC is associated with loss or absence of EGFR from the mucosa of the affected tissue.

Section snippets

Patients

The Hammersmith Hospital Research Ethics Committee gave approval for his study and informed parental consent was obtained in each case.

We studied three females and one male infant with necrotising enterocolitis who were treated in the neonatal intensive care unit of the Hammersmith Hospital.

The infants were born at a median gestational age of 28 (range 27–30) weeks with the median birth weight of 635 (range 550–1730) g.

The age at diagnosis was median of 7 (range 7–15) days.

The studied infants

Histology

Tissues from small or large bowel from infants with NEC and also small or large bowel from the controls taken at operation were fixed in buffered normal formalin and paraffin-embedded for histological evaluation.

Immunohistochemistry

Immunohistochemistry was performed on tissues from infants with NEC and those of the controls. This was initially performed by the streptavidin–biotin method.

Paraffin sections were dewaxed and taken down to water. Endogenous peroxidase was blocked with hydrogen peroxide for 15 min. EGFR immunostaining was performed by incubating sections for 35 min with a monoclonal antibody against the external domain of the EGF receptor (Zymed Laboratories).

Sections were washed and incubated with biotinylated

Necrotising enterocolitis

Macroscopic examination of the involved bowel showed necrosis, oedema, haemorrhagic areas and gas in the bowel wall. Microscopical examination showed haemorrhagic infarction of the mucosa and submucosa with a range of inflammatory responses. Fibrin thrombi were present in the small capillaries. These findings confirmed the diagnosis of necrotising enterocolitis (Fig. 1).

Controls

Macroscopic examination confirmed the diagnosis of bowel atresia (2) or sigmoid volvulus (1) or isolated intestinal

Discussion

The present study showed that EGFR were present in the gastrointestinal epithelial cells of preterm infants and that EGFR were located on the basolateral membrane of the cells. There were no differences in the location of the EGFR between the infants with or without NEC.

Various studies in the past have described the appearance of EGFR in the human digestive tract and pancreas between the 12th and 17th weeks of gestation [18], [19], [20], [21]. Using immunohistochemistry, EGFR immunostaining was

Acknowledgements

We are grateful to the staff of the Neonatal Unit at the Hammersmith Hospital, as well as to those of the Histopathology laboratory, for their help in the preparation of slides and staining. We also thank Mahrokh Nohadani for preparing the photographs.

References (37)

  • A Lucas et al.

    Breast milk and neonatal necrotising enterocolitis

    Lancet

    (1990)
  • R.J Playford et al.

    Why is epidermal growth factor present in the gut lumen

    Gut

    (1996)
  • H Marquart et al.

    Rat transforming growth factor type1: structure and relation to epidermal growth factor

    Science

    (1984)
  • R.A Goodlad et al.

    Proliferative effects of urogastrone–EGF on the intestinal epithelium

    Gut

    (1987)
  • H.W Sundell et al.

    Effects of epidermal growth factor on the lung maturation in the fetal lambs

    Am. J. Pathol.

    (1980)
  • P Skov Olsen et al.

    Oral administration of synthetic human urogastrone promotes healing of chronic duodenal ulcers in rats

    Gastroenterology

    (1986)
  • K Kajikawa et al.

    Expression of epidermal growth factor in human tissues: immunohistochemical and biochemical analysis

    Virchows Arch. A: Pathol. Anat.

    (1991)
  • A.G Kasselberg et al.

    Immunocytochemical localisation of human epidermal growth factor/urogastrone in several human tissues

    J. Histochem. Cytochem.

    (1985)
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