The ability of endotoxin-stimulated enterocytes to produce bactericidal factors.
OBJECTIVE
Bactericidal peptides, specifically defensins, are produced by polymorphonuclear cells. Intestinal epithelial cells also produce bactericidal peptides, perhaps as part of their barrier function, to the greatest load of endogenous bacteria present in the body. We sought to determine whether and under what conditions intestinal cell lines could produce bactericidal compounds.


METHODS
Laboratory investigation.


METHODS
Children's burn hospital.


METHODS
Caco-2, IEC-6, and HT-29 cell lines.


METHODS
Three different enterocyte lines were cultured for 1 day +/- lipopolysaccharide (1 or 10 microg/mL), and their supernatants were tested for bactericidal activity. Also, reverse transcription-polymerase chain reaction of Caco-2 cells was performed to assess the expression of defensin-6 mRNA.


RESULTS
After culture, enterocytes all were found to release one or more soluble factors with bactericidal activity (as measured fluorometrically by using a metabolizable dye) when stimulated by lipopolysaccharide (1 microg/mL). The bactericidal activity of these culture supernatants was saturated by increased bacterial load, additive to the effects of normal human peripheral blood polymorphonuclear cells, and was reduced by serial supernatant dilution. Enterocyte stimulation with larger amounts of lipopolysaccharide (10 microg/mL) resulted in greater bactericidal activity. After supernatant fractionation based on molecular weight, the bactericidal effect was best retained in the <10-kDa fraction. In addition, the expression of mRNA for defensin-6, a bactericidal peptide produced by neutrophils, was seen in Caco-2 cells.


CONCLUSIONS
Enterocytes are shown to produce a soluble, low molecular weight, bactericidal compound in response to endotoxin stimulation. The expression of defensin-6 mRNA in Caco-2 cells suggests that intestinal cells may release defensins as bactericidal peptides. This experimental system provides an in vitro model to study the activity and production of bactericidal factors by enterocytes.
Ogle
Cora K
CK
Shriners Burns Hospital for Children and the Department of Surgery, University of Cincinnati, Cincinnati, OH 45229-3095, USA. coraogle@hotmail.com
Noel
J Gregory
JG
Guo
Xialing
X
Wells
Denise A
DA
Valente
John F
JF
Ogle
James D
JD
Alexander
J Wesley
JW
D000704:Analysis of Variance; D000818:Animals; D018938:Caco-2 Cells / Q000378:metabolism; D002460:Cell Line; D023082:Defensins / Q000378:metabolism*; D004306:Dose-Response Relationship, Immunologic; D020895:Enterocytes / Q000378:metabolism* / Q000382:microbiology*; D019073:HT29 Cells / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008070:Lipopolysaccharides; D015999:Multivariate Analysis; D009504:Neutrophils / Q000378:metabolism / Q000382:microbiology; D011498:Protein Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
D023082:Defensins; D008070:Lipopolysaccharides; D011498:Protein Precursors; D012333:RNA, Messenger; C059569:cryptdin