Cell differentiation is a key determinant of cathelicidin LL-37/human cationic antimicrobial protein 18 expression by human colon epithelium

Abstract

Antimicrobial peptides are highly conserved evolutionarily and are thought to play an important role in innate immunity at intestinal mucosal surfaces. To better understand the role of the antimicrobial peptide human cathelicidin LL-37/human cationic antimicrobial protein 18 (hCAP18) in intestinal mucosal defense, we characterized the regulated expression and production of this peptide by human intestinal epithelium. LL-37/hCAP18 is shown to be expressed within epithelial cells located at the surface and upper crypts of normal human colon. Little or no expression was seen within the deeper colon crypts or within epithelial cells of the small intestine. Paralleling its expression in more differentiated epithelial cells in vivo, LL-37/hCAP18 mRNA and protein expression was upregulated in spontaneously differentiating Caco-2 human colon epithelial cells and in HCA-7 human colon epithelial cells treated with the cell differentiation-inducing agent sodium butyrate. LL-37/hCAP18 expression by colon epithelium does not require commensal bacteria, since LL-37/hCAP18 is produced with a similar expression pattern by epithelial cells in human colon xenografts that lack a luminal microflora. LL-37/hCAP18 mRNA was not upregulated in response to tumor necrosis factor alpha, interleukin 1alpha (IL-1alpha), gamma interferon, lipopolysaccharide, or IL-6, nor did the expression patterns and levels of LL-37/hCAP18 in the epithelium of the normal and inflamed colon differ. On the other hand, infection of HCA-7 cells with Salmonella enterica serovar Dublin or enteroinvasive Escherichia coli modestly upregulated LL-37/hCAP18 mRNA expression. We conclude that differentiated human colon epithelium expresses LL-37/hCAP18 as part of its repertoire of innate defense molecules and that the distribution and regulated expression of LL-37/hCAP18 in the colon differs markedly from that of other enteric antimicrobial peptides, such as defensins.

FIG. 1.

Immunohistochemical detection of LL-37/hCAP18 in normal human colon and small intestine. Biopsy specimens from normal human colon (A and B) and proximal small intestine (C to F) were analyzed by indirect immunoperoxidase staining for LL-37/hCAP18 expression. The sections were immunostained with an LL-37/hCAP18-specific antibody (A, C, and E) or control rabbit IgG (B, D, and F). Magnification, ×400 (A to D) and ×100 (E and F).

FIG. 2.

Immunohistochemical detection of LL-37/hCAP18 in human colon cancer tissue. Colon tissue obtained at surgery from a patient with adenocarcinoma of the colon was analyzed by immunostaining for LL-37/hCAP18 expression. Sections were stained with LL-37/hCAP18-specific antibody (A and B), control rabbit IgG (C and D), or hematoxylin and eosin (E and F). The sections in panels A, C, and E are from the tumor, whereas the sections in panels B, D, and F are from normal mucosa from the same surgical specimen. Magnification, ×400.

FIG. 3.

LL-37/hCAP18 mRNA expression is upregulated by butyrate treatment of HCA-7 cells. HCA-7 cells were left unstimulated or were stimulated with 2 mM butyrate (+ butyrate) for up to 7 days, and total cellular RNA and cell lysates were prepared at the indicated time points. (A) Total cellular RNA was amplified by RT-PCR for LL-37/hCAP18 and β-actin, and the products were analyzed on a 1% agarose gel. As a negative control, RNA was omitted from RT and PCR amplification (No RNA). (B) LL-37/hCAP18 mRNA expression was analyzed by real-time PCR. The levels were normalized to those of β-actin, which were constant in both groups throughout the experiment. The data are expressed as the fold changes (± SD) in mRNA levels relative to the levels at day zero for unstimulated (○) and butyrate-treated (•) cells. (C) AP activity in cell lysates is expressed as changes (± SD) relative to levels at day zero.

FIG. 4.

LL-37/hCAP18 mRNA expression increases in spontaneously differentiating Caco-2 cells. Caco-2 cells were incubated for up to 21 days after reaching confluence to induce spontaneous cell differentiation. LL-37/hCAP18 mRNA levels were analyzed by qualitative RT-PCR (A) and real-time PCR (B, •) as described in the legend to Fig. 3. AP activity (B, ○) was analyzed by enzymatic assay.

FIG. 5.

Immunostaining of LL-37/hCAP18 in HCA-7 cells. Confluent HCA-7 cells were treated with butyrate for 5 days (C, D, G, and H) or were left unstimulated as controls (A, B, E, and F). LL-37/hCAP18 expression was analyzed by indirect immunofluorescence using an LL-37/hCAP18-specific antibody (A and C) or a control rabbit IgG (E and G). (B, D, F, and H) Nuclear counterstaining with Hoechst 33258 dye for each of the adjacent panels. Magnification, ×400.

FIG. 6.

Immunohistochemical detection of LL-37/hCAP18 in human colon xenografts. (A) Total DNA isolated from luminal contents of two different human fetal colon xenografts (XG1 and XG2) was amplified by PCR using universal primers targeting a highly conserved region of the bacterial 16S rRNA gene. In the lanes designated “XG1 + E. coli 10³” and “XG2 + S. bovis 10³,” the indicated bacteria (10³ CFU) were mixed with xenograft luminal content before DNA extraction. DNA samples from mouse feces and human colon lavage fluid were analyzed as positive controls. (B and C) Human colon xenografts were immunostained with LL-37/hCAP18-specific antibody (B) or control rabbit IgG (C). Magnification for both panels, ×400.

FIG. 7.

Epithelial LL-37/hCAP18 expression by cultured human colonic epithelial cells stimulated with inflammatory mediators. HCA-7 cells were left unstimulated (Control) or were stimulated with the indicated agonists. Total cellular RNA was isolated 6 h later and analyzed for LL-37/hCAP18, hBD-2, CCL9/Mig and β-actin mRNA expression by RT-PCR. As a negative control, RNA was omitted from RT and PCR amplification (No RNA).

FIG. 8.

Immunohistochemical detection of LL-37/hCAP18 in inflamed human colon. Biopsy specimens from inflamed human colon were analyzed by indirect immunoperoxidase staining for LL-37/hCAP18 expression. The sections were immunostained with an LL-37/hCAP18-specific antibody (A) or control rabbit IgG (B). Magnification, ×400.

FIG. 9.

LL-37/hCAP18 mRNA expression by human colonic epithelial cell lines infected with enteroinvasive bacteria. HCA-7 cells were left uninfected (Control) or were infected with the indicated bacteria as described in Materials and Methods. (A) LL-37/hCAP18 mRNA levels were analyzed by real-time RT-PCR at 6 (open bars) and 24 (solid bars) h after infection and are expressed as fold increases over controls (+ SD). (B) hBD-2 and β-actin mRNA expression was analyzed by qualitative RT-PCR 6 h after infection.