The human cationic antimicrobial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6

Abstract

Peptide antibiotics are widespread in nature and, by providing a rapid first line of defense, may be key players in the innate immune system. Although epithelia are the main barriers shielding the internal environment from microorganisms, the role for peptide antibiotics in epithelial protection is unclear. We recently reported that the human cationic antimicrobial protein hCAP18, the precursor of the antimicrobial peptide called LL-37, is not expressed by normal human keratinocytes but is induced in various inflammatory skin disorders. In the present study we demonstrate that hCAP18 is consistently expressed at both mRNA and protein levels in squamous epithelia of the mouth, tongue, esophagus, cervix, and vagina in humans. The gene for hCAP18 contains promoter elements that are potentially regulated by interleukin-6, and our data further show a colocalization between interleukin-6 and hCAP18 expression in these tissues. Our finding that hCAP18 is widely produced in squamous epithelia suggests a role for this peptide in epithelial antimicrobial defense. Furthermore, colocalization with interleukin-6 indicates a potential local mechanism for the upregulation of hCAP18 at the epithelial surfaces.

FIG. 1

Autoradiogram of a multiple tissue Northern blot hybridized with a 0.4-kb CAP18 cDNA fragment. The sizes of the RNA markers are indicated on the left. A single transcript of less than 1 kb was detected specifically in bone marrow, in agreement with published data.

FIG. 2

hCAP18 is expressed in the squamous epithelium of the tongue and colocalizes with immunoreactivity for IL-6. (A) Section of tongue demonstrating positive immunostaining for hCAP18 in the epithelium. Immunoreactivity (red precipitate) is most pronounced in the basal cell layers, but scattered immunopositive cells can also be seen in the suprabasal layers. (B) Section from the same tissue hybridized with antisense ³⁵S-labeled cRNA probe for hCAP18 mRNA. Intense autoradiographic signal for hCAP18 mRNA (appears as white grains under dark-field illumination) is seen in the lower portion of tongue epithelium. (C) Immunoreactivity for IL-6 is seen in tongue epithelium with the same pattern as for hCAP18 and in submucosal blood vessels. (D) Hybridization with the control sense probe for hCAP18 lacks the autoradiographic signal for hCAP18 mRNA. The shiny appearance is due to the autofluorescence of the tissue. Bars, 100 μm.

FIG. 3

hCAP18 and IL-6 colocalize in esophagus epithelia. (A) Section of esophagus demonstrates hCAP18-immunoreactive epithelial cells. (B) No immunoreactivity is detected in the epithelium when rabbit serum is used instead of hCAP18 antibody. (C) Immunoadsorption with hCAP18 recombinant peptide almost completely abolished hCAP18 immunoreactivity in esophagus epithelium. (D) Immunostaining for IL-6 demonstrates immunoreactive cells in the same areas as for hCAP18. Bars, 100 μm.

FIG. 4

In the dysplastic and inflammatory cervix, hCAP18 is expressed in a bandlike pattern in the superficial epithelial layers. (A) Noninflammatory cervix demonstrates positive immunoreactivity for hCAP18 predominantly found in the basal epithelium. (B) In situ hybridization shows matching signal for hCAP18 mRNA in the same tissue. The shiny appearance of the surface layer is due to the autofluorescence of the tissue and does not represent an autoradiographic signal. (C) Sample of a dysplastic and inflammatory cervix demonstrates a striking band of hCAP18 immunoreactivity in the superficial epithelium. (D) In situ hybridization shows that hCAP18 is actively expressed in the outermost layers of the epithelium. (E) High-power view of superficial hCAP18-immunoreactive epithelial cells. (F) hCAP18-immunoreactive fibroblast-like cell in the cervix stroma (same tissue sample as in panel A). (G) hCAP18-immunoreactive mucous glands in the cervix (same tissue sample as in panel C). Bars: A to D and G, 100 μm; E and F, 25 μm.