Potential role of epithelial cell-derived histone H1 proteins in innate antimicrobial defense in the human gastrointestinal tract

Abstract

In the human gastrointestinal tract, microorganisms are present in large numbers in the colon but are sparse in the proximal small intestine. In this study, we have shown that acid extracts of fresh human terminal ileal mucosal samples mediate antimicrobial activity. Following cation-exchange chromatography, one of the eluted fractions demonstrated antibacterial activity against bacteria normally resident in the human colonic lumen. This activity was further fractionated by reverse-phase high-performance liquid chromatography and identified as histone H1 and its fragments. We have also shown that in tissue sections, immunoreactive histone H1 is present in the cytoplasm of villus epithelial cells. In vitro culturing of detached (from the basement membrane) villus epithelial cells led to the release of antimicrobial histone H1 proteins, while the cells demonstrated ultrastructural features of programmed cell death. Our studies suggest that cytoplasmic histone H1 may provide protection against penetration by microorganisms into villus epithelial cells. Moreover, intestinal epithelial cells released into the lumen may mediate antimicrobial activity by releasing histone H1 proteins and their fragments.

FIG. 1

Activity against S. typhimurium CS015 by acid extracts of human terminal ileal mucosa. The mucosal extract (in 0.1% acetic acid) (▪) and the control (0.1% acetic acid only) (•) were applied, together with bacteria, to 96-well microtiter plates, and bacterial growth was assessed hourly. The data represent one of 29 experiments performed.

FIG. 2

(a) RP-HPLC fractionation of semipurified ileal mucosal extract. Antimicrobial fraction 15 eluted from the cation-exchange column (1 M NaCl) was applied to an Aquapore C₄ column, and of the eluted fractions, F, G, and H demonstrated antimicrobial activity. (b) AU-PAGE of fraction 15 (lane 1), fraction H (lane 3), and synthetic magainin (lane 2). Of the many positively charged peptides and proteins present in fraction 15, one was purified by RP-HPLC (fraction H in panel a).

FIG. 3

C₄ RP-HPLC separation of fraction H (Fig. 2) following digestion with Lys-C. Eluted peaks (1, 2, and 3) were collected and sequenced. Peak 1 (retention time, 9.45 min) gave a single unequivocal sequence. Peak 2 (retention time, 11.86 min) gave two signals for some cycles of sequence analysis, indicating a mixture of two peptides. However, these two signals were of sufficiently different intensities to allow the two sequences to be assigned (2a and 2b). Peak 3 gave no sequence, suggesting that it contained the undigested N terminally blocked protein. The amino acid sequence (amino acids 31 to 80) of human histone H1d is shown. Amino acid sequences of peptides 1, 2a, and 2b showed 100% homology to histone H1d. In addition, these peptides also showed 100% sequence homology to histones H1b and H1c.

FIG. 4

AU-PAGE of an antimicrobial fraction of human ileal mucosal extract. This fraction was obtained by cation-exchange chromatography, followed by RP-HPLC. AU-PAGE analysis demonstrated the presence of four distinct protein bands (stained by Coomassie blue). Following transfer to a PVDF membrane, amino acid sequence analyses of these four proteins revealed 100% homology to histone H1 proteins.

FIG. 5

Expression of histone H1 in human terminal ileal mucosa. Immunohistochemical analysis was performed with an anti-histone H1 monoclonal antibody, but a nuclear stain was not used. (a) Histone H1 immunoreactivity was seen in the cytoplasm of epithelial cells, at the tip and on the lateral aspect of villi (arrows), as well as in nuclei. (b) Villus (from the left margin of panel a) at a high magnification. Arrows indicate immunoreactivity. Approximate magnifications: a, ×224; b, ×389.

FIG. 6

Immunoelectron microscopy of a section of normal human small intestinal mucosa labelled with an anti-human histone H1 monoclonal antibody by the peroxidase technique. Two epithelial cells near the villus tip are shown. (a) In one, immunoreactive histone H1 (arrows) was present in the cytoplasm below the microvilli. (b) The cytoplasm of an adjacent epithelial cell did not contain immunoreactive histone H1.

FIG. 7

Expression of immunoreactive histone H1 in detached villus epithelial cells of human terminal ileal mucosa. Cytospin preparations of epithelial cells were made following detachment with EDTA (and subsequent washing with PBS). Photomicrographs of the same cytospin preparation were taken. Strong histone H1 immunoreactivity was seen in nuclei. Strong histone H1 immunoreactivity was seen in the cytoplasm (arrows) of some epithelial cells (b). Magnification, ×590.

FIG. 8

Acid extracts (in 0.1% acetic acid) of supernatants of detached and cultured (for 3 h [▪] or for 18 h [▴]) villus epithelial cells mediate antimicrobial activity. Microtiter plate antimicrobial assays were performed with S. typhimurium CS015, and 0.1% acetic acid only (•) was used as a control. The data represent one of three experiments performed.

FIG. 9

Dot blot analysis demonstrating the presence of immunoreactive histone H1 in supernatants of detached and cultured (for 18 h) terminal ileal villus epithelial cells. Acid extracts were applied to PVDF membranes, which were subsequently incubated with an anti-human histone H1 monoclonal antibody (b) or control buffer (a). Strong histone H1 immunoreactivity was seen with the monoclonal antibody.

FIG. 10

Transmission electron micrograph of detached villus epithelial cells cultured for 3 h. Apoptotic bodies (small arrows) and an apoptotic epithelial cell (large arrow) are shown.