The ability of endotoxin-stimulated enterocytes to produce bactericidal factors Cora K. Ogle, PhD; J. Gregory Noel, MS; Xialing Guo, MS; Denise A. Wells, MS; John F. Valente, MD; James D. Ogle, PhD; J. Wesley Alexander, MD, ScD T he gut mucosal barrier pos- sesses a variety of mechanisms to defend against microbial in- filtration. These include ana- tomical and chemical barriers as well as numerous cell-mediated and humoral re- sponses. Peptide-based antimicrobial de- fenses are conserved components of host defense and are found in both the animal and plant kingdoms (1). The sequence and structure of these antimicrobial pep- tides show significant diversity, but in general they are membrane-active, am- phipathic molecules with a net positive charge at physiologic (neutral) pH (2). Defensins are one family of well-charac- terized antimicrobial peptides that are produced by myeloid-derived cells and have been isolated from a number of mammalian species (3). This family of relatively short peptides (30 –34 amino acids in length) has potent bactericidal properties and significantly contributes to myeloid cell-mediated bacterial killing (3). Recently, a number of intestinal epi- thelial cell peptides, which are analogs to these myeloid-derived peptides, have been characterized. They possess similar, if not more potent, bactericidal proper- ties and may be produced by intestinal crypt cells, thus termed the “cryptdins” (4). Cryptdins represent a potentially im- portant component of the gut barrier to microbial invasion. Study of the inducing stimuli and subsequent control of crypt- din production is hampered by the inher- ent difficulties in the isolation of viable enterocytes. We investigated the ability of several intestinal cell lines to release compounds consistent with these bacte- ricidal peptides. We also investigated en- dotoxin as an important stimulus to which enterocytes might respond. To de- termine whether our peptide was similar to defensins, we investigated expression of defensin-6, a known bactericidal pep- tide produced by neutrophils, in entero- cytes. MATERIALS AND METHODS Cell Lines and Culture Methods. For these studies, we initially attempted to use entero- cytes isolated freshly from rats. These cells, however, showed poor viability for the length of our studies. Instead, we decided to use three common intestinal cell lines. We selected Caco-2, HT-29, and IEC-6 cells for this study. All of these cell lines share many common characteristics with enterocytes. Caco-2 cells differentiate and resemble enterocyte mor- phology. Likewise, IEC-6 cells differentiate and function like rat intestinal epithelial cells. From Shriners Burns Hospital for Children (CKO, JGN, XG, DAW, JDO), Cincinnati, OH, and the Depart- ment of Surgery (CKO, JFV, JWA), University of Cin- cinnati, Cincinnati, OH. Supported, in part, by Shriners Hospital for Chil- dren and by grant AI12936-19 from the National In- stitutes of Health. Address requests for reprints to: Cora K. Ogle, PhD, Shriners Burns Hospital for Children, 3229 Burnet Avenue, Cincinnati, OH 45229-3095. E-mail: coraogle@hotmail.com Copyright © 2002 by Lippincott Williams & Wilkins Objective: Bactericidal peptides, specifically defensins, are produced by polymorphonuclear cells. Intestinal epithelial cells also produce bactericidal peptides, perhaps as part of their bar- rier 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 com- pounds. Design: Laboratory investigation. Setting: Children’s burn hospital. Subjects: Caco-2, IEC-6, and HT-29 cell lines. Interventions: Three different enterocyte lines were cultured for 1 day  lipopolysaccharide (1 or 10 g/mL), and their super- natants were tested for bactericidal activity. Also, reverse tran- scription-polymerase chain reaction of Caco-2 cells was per- formed to assess the expression of defensin-6 mRNA. Measurements and Main Results: After culture, enterocytes all were found to release one or more soluble factors with bacteri- cidal activity (as measured fluorometrically by using a metabo- lizable dye) when stimulated by lipopolysaccharide (1 g/mL). The bactericidal activity of these culture supernatants was satu- rated by increased bacterial load, additive to the effects of normal human peripheral blood polymorphonuclear cells, and was re- duced by serial supernatant dilution. Enterocyte stimulation with larger amounts of lipopolysaccharide (10 g/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 neutro- phils, was seen in Caco-2 cells. Conclusion: Enterocytes are shown to produce a soluble, low molecular weight, bactericidal compound in response to endo- toxin 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. (Crit Care Med 2002; 30:428 –434) KEY WORDS: enterocyte; host defense; cryptdins; defensins; bac- tericidal peptides; Paneth cells; gut; sepsis; bacterial transloca- tion; endotoxin 428 Crit Care Med 2002 Vol. 30, No. 2