Production of proinflammatory cytokines and inflammatory mediators in human intestinal epithelial cells after invasion by Trichinella spiralis

Abstract

Epithelial cells are the first point of host contact for invasive intestinal pathogens and may initiate mucosal inflammatory responses via production of proinflammatory cytokines and mediators. The aim of the present study was to investigate in vitro the initial invasion of a parasitic nematode (Trichinella spiralis), to measure the early production of specific epithelial cytokines and inflammatory mediators after invasion, and to compare these responses with those to invasive bacteria. Monolayers of human colonic epithelial cell lines (HT29, T84, and Caco-2) were infected by T. spiralis or Listeria monocytogenes. Bile-activated infective larvae of T. spiralis invaded and migrated into the epithelial cell monolayers, leaving trails of dead cells. Transmission electron microscopy studies of damaged cells along the trail showed a progressive increase in size, disruption of cell membranes, loss or dilution of cytoplasmic proteins, and swelling of mitochondria and nuclei. However, no nuclear fragmentation was observed. With reverse transcription-PCR and an enzyme-linked oligonucleotide chemiluminescent assay, mRNA transcripts of interleukin-1beta (IL-1beta), IL-8, and epithelial neutrophil-activating peptide 78 were shown to increase in epithelial cells invaded by T. spiralis or L. monocytogenes, but only L. monocytogenes elicited increased inducible nitric oxide synthase (iNOS) mRNA. No increase in tumor necrosis factor alpha or transforming growth factor beta mRNA was seen after T. spiralis invasion. Increased levels of IL-8 were also released from the basolateral surfaces of infected monolayers as detected by sandwich enzyme-linked immunosorbent assay. Induction and secretion of proinflammatory cytokines in epithelial cells after nematode or bacterial invasion may initiate the acute inflammatory response of the small intestine. The upregulation of iNOS in bacterial infections may contribute to mucosal defense and may also be associated with subsequent cell death, whereas different mechanisms appear to operate after nematode invasion.

FIG. 1

Light microscopy of human colonic epithelial monolayers infected by T. spiralis. (A) With trypan blue staining, the infective larva (arrow) can be seen to have left a trail of dead cells after invasion and migration in Caco-2 epithelial monolayers. (B) Trails of damaged T84 epithelial cells consist of many dead cells (arrow), which are larger than adjacent cells and have enlarged nuclei.

FIG. 2

TEM of human colonic epithelial cells infected by T. spiralis. (A) Infective larva (L) penetrating an HT29 epithelial monolayer. The cytoplasm and nuclei of adjacent cells (AC) are less electron dense than intact cells. (B) Invasion of infective larva (L) into HT29 cells. Note the disruption (arrows) of the membranes of adjacent cells. (C) Damage of cells along the path of the larva. Note the disruption of the membrane (large arrow) and the loss of cytoplasmic proteins of the cell (AC1). Only the cell membrane in direct contact with the cuticle (C) of the larva is disrupted, whereas the lateral membrane (small arrow) of the same cell remains intact. (D) The mitochondrion (arrow) and nucleus (N) of the infected HT29 cell (AC) are swollen compared to the adjacent normal cell (NC) and its mitochondrion (M). (E) The nuclear membrane (arrow) of a Caco-2 cell in direct contact with the infective larva (L) is disrupted.

FIG. 3

Ratios of cytokine PCR products of intestinal epithelial cell lines infected with T. spiralis and L. monocytogenes obtained by RT-PCR and ELOCA. Monolayers of HT29 (A), T84, (B), and Caco-2 (C) cells were infected with 1 × 10³, 3 × 10³, and 5 × 10³ infective larvae of T. spiralis or 10⁷ CFU of L. monocytogenes per ml. After 5 h of infection, total RNA extracted from each sample was used for semiquantitative RT-PCR and ELOCA to examine the levels of expression of mRNA of IL-1β, IL-8, ENA-78, and iNOS. The experiments were repeated twice, and results are presented as ratios of infected to control cell lines, as described in Materials and Methods. *, statistically significant compared with uninfected medium control (P < 0.05).