CD14 is expressed and released as soluble CD14 by human intestinal epithelial cells in vitro: lipopolysaccharide activation of epithelial cells revisited

Abstract

Human endothelial as well as epithelial cells were shown to respond to lipopolysaccharides (LPSs). However, the expression and release of CD14 by these so-called CD14-negative cells have not been studied in detail. We investigated three human intestinal epithelial cell lines (ECLs), SW-480, HT-29, and Caco-2, for their expression of CD14 and CD11c/CD18 as well as their responsiveness to endotoxins. Fluorescence-activated cell sorter analysis revealed no expression of CD11c/CD18, but there was low expression of membrane-bound CD14 on HT-29, Caco-2, and SW-480 ECLs. Both Western blotting and reverse transcription-PCR confirmed the CD14 positivity of all three intestinal ECLs. No substantial modulation of CD14 expression was achieved after 6, 8, 18, 24, and 48 h of cultivation with 10-fold serial dilutions of LPS ranging from 0.01 ng/ml to 100 microg/ml. Interestingly, soluble CD14 was found in the tissue culture supernatants of all three ECLs. Finally, only HT-29 and SW-480, and not Caco-2, cells responded to LPS exposure (range, 0.01 ng/ml to 100 microg/ml) by interleukin 8 release. Thus, we show that HT-29, SW-480, and Caco-2 human intestinal ECLs express membrane-bound CD14. As Caco-2 cells did not respond to LPS, these cell lines might be an interesting model for studying the receptor complex for LPS. The fact that human intestinal epithelial cells are capable not only of expression but also of release of soluble CD14 may have important implications in vivo, e.g., in shaping the interaction between the mucosal immune system and bacteria in the gut and/or in the pathogenesis of endotoxin shock.

FIG. 1

Flow cytometry analysis of CD14 and CD18 surface expression on HT-29, SW-480, and Caco-2 intestinal ECLs. Expression of CD14 was assessed by indirect immunofluorescence using anti-CD14, MEM-15, and MEM-18 MAbs (IgG1) at a 1:500 dilution followed by FITC-conjugated, goat anti-mouse IgG F(ab′)₂ (black profile). CD18 expression was assessed by direct immunoflourescence using PE-conjugated mouse anti-human CD18 (IgG1) MAb (black profile). FITC- and PE-conjugated isotype-matched (IgG1) Abs and staining without primary MAb were used as negative controls (gray profile). The data are expressed as cell number versus log fluorescence and are representative of two to five independent experiments. The highest mCD14 positivity is documented by MFI values.

FIG. 2

SDS-PAGE and Western blot detection of mCD14 on HT-29, SW-480, and Caco-2 intestinal ECLs. Cells (10⁷) were incubated with 2 U of PI-PLC per ml at 37°C for 1 h, and 20 μl of the GPI protein-enriched fraction was run under nonreducing conditions on SDS–10% PAGE, transferred to a nitrocellulose membrane, and stained with MEM-15 (IgG1) mouse anti-human CD14 MAb at a dilution of 1:500, followed by biotinylated goat anti-mouse IgG F(ab′)₂ Ab and peroxidase-conjugated streptavidin. Bound proteins were visualized by the enhanced-chemiluminescence detection system, and PBMNC were used as a positive control. The figure shows the results of one experiment representative of four.

FIG. 3

SDS-PAGE and Western blot detection of mCD14 on the HT-29 intestinal ECL with multiple anti-CD14 MAbs. Using the same experimental design used to obtain the results shown in Fig. 2, mCD14 was detected by MEM-15, MEM-18, MoP9, MoP15, and MoS39 anti-CD14 MAbs (IgG1) at a dilution of 1:500. IN-05 mouse anti-insulin (IgG1) MAb at a dilution of 1:200 was used as an isotype-matched negative control. The figure presents data representative of two separate experiments.

FIG. 4

Expression of CD14 mRNA in intestinal ECLs. Total RNA from the HT-29 SW-480, and Caco-2 ECLs, as well as HT-29 Glc⁻ cells differentiated by cultivation in glucose-free medium as described in Materials and Methods, was reverse transcribed with Superscript II and amplified with CD14-specific primers. The expected sizes of PCR products from DNA and mRNA were 356 and 284 bp, respectively. The data are representative of results from three independent cell cultures and RT-PCR experiments.

FIG. 5

Effect of differentiation stage of the HT-29 intestinal ECL on CD14 expression. The HT-29 intestinal ECL was differentiated by cultivation in Glc⁻ DMEM supplemented with 10% fetal bovine serum as described in Materials and Methods and reference . (A) Flow cytometry analysis of the undifferentiated HT-29 and differentiated HT-29 Glc⁻ ECLs. Cells (5 × 10⁵ cells per sample) were stained with mouse anti-human CD14 MAb MEM-15 (IgG1) followed by secondary FITC-conjugated, goat anti-mouse IgG F(ab′)₂ (black profile). FITC-conjugated isotype-matched (IgG1) antibody and staining without primary MAb were used as negative controls (gray profile). The data are representative of three independent experiments. (B) SDS-PAGE and Western blot detection of mCD14 in undifferentiated HT-29 and differentiated HT-29 Glc⁻ ECLs using MEM-15 (IgG1) mouse anti-human CD14 MAb and the same experimental design as that described for Fig. 2. The data are representative of two separate experiments.

FIG. 5

Effect of differentiation stage of the HT-29 intestinal ECL on CD14 expression. The HT-29 intestinal ECL was differentiated by cultivation in Glc⁻ DMEM supplemented with 10% fetal bovine serum as described in Materials and Methods and reference . (A) Flow cytometry analysis of the undifferentiated HT-29 and differentiated HT-29 Glc⁻ ECLs. Cells (5 × 10⁵ cells per sample) were stained with mouse anti-human CD14 MAb MEM-15 (IgG1) followed by secondary FITC-conjugated, goat anti-mouse IgG F(ab′)₂ (black profile). FITC-conjugated isotype-matched (IgG1) antibody and staining without primary MAb were used as negative controls (gray profile). The data are representative of three independent experiments. (B) SDS-PAGE and Western blot detection of mCD14 in undifferentiated HT-29 and differentiated HT-29 Glc⁻ ECLs using MEM-15 (IgG1) mouse anti-human CD14 MAb and the same experimental design as that described for Fig. 2. The data are representative of two separate experiments.

FIG. 6

Modulation of mCD14 expression on HT-29, SW-480, and Caco-2 intestinal ECLs. (A) Effect of LPS on CD14 mRNA expression in the HT-29 intestinal ECL. CD14 mRNA was determined by RT-PCR using CD14-specific primers with an expected product size of 535 bp. RT-PCR was performed on total RNA from the HT-29 intestinal ECL cultivated in DMEM with 10% fetal bovine serum and with 0.01, 0.1, 1, and 10 μg of LPS per ml (serovar Minnesota) for 6 h. RT without Superscript II and PCR without template cDNA were carried out as negative controls. (B) Flow cytometry analysis of LPS's effect on modulation of mCD14 expression in HT-29, SW-480, and Caco2 intestinal ECLs. The cell lines were cultivated in complete medium supplemented with 10% fetal bovine serum and in the presence of 0.1 μg of LPS per ml (E. coli serotype O55:LB5) for 6, 18, and 48 h. The cells (5 × 10⁵ cells per sample) were then used for indirect immunofluorescence staining as described for Fig. 1. The data are representative of two to four independent experiments.

FIG. 6

Modulation of mCD14 expression on HT-29, SW-480, and Caco-2 intestinal ECLs. (A) Effect of LPS on CD14 mRNA expression in the HT-29 intestinal ECL. CD14 mRNA was determined by RT-PCR using CD14-specific primers with an expected product size of 535 bp. RT-PCR was performed on total RNA from the HT-29 intestinal ECL cultivated in DMEM with 10% fetal bovine serum and with 0.01, 0.1, 1, and 10 μg of LPS per ml (serovar Minnesota) for 6 h. RT without Superscript II and PCR without template cDNA were carried out as negative controls. (B) Flow cytometry analysis of LPS's effect on modulation of mCD14 expression in HT-29, SW-480, and Caco2 intestinal ECLs. The cell lines were cultivated in complete medium supplemented with 10% fetal bovine serum and in the presence of 0.1 μg of LPS per ml (E. coli serotype O55:LB5) for 6, 18, and 48 h. The cells (5 × 10⁵ cells per sample) were then used for indirect immunofluorescence staining as described for Fig. 1. The data are representative of two to four independent experiments.

FIG. 7

Detection of sCD14 in cell-free cell culture supernatants of HT-29, SW-480, Caco-2, and differentiated HT-29 Glc⁻ intestinal ECLs. Cell-free supernatants were collected after 48 h of cell culture, concentrated three times using Centricon-10 tubes (cutoff, 10 kDa), and assessed by SDS–10% PAGE under nonreducing conditions followed by Western blotting and immunodetection as described for Fig. 2. Three times-concentrated complete DMEM with 10% fetal bovine serum and three times-concentrated 48 h supernatant of the HT-29 cell line (with the primary anti-CD14 MAb MEM-15 omitted) were used as control I and control II, respectively. The figure shows the results of one experiment representative of three.

FIG. 8

IL-8 secretion by HT-29 (filled circles), SW-480 (open circles), and Caco-2 (filled triangles) intestinal ECLs stimulated with LPS (E. coli serotype O55:LB5) for 8 (A) and 24 (B) h. LPS concentrations ranged from 0.01 ng/ml to 100 μg/ml. Cell-free supernatants were collected after 8 (A) or 24 (B) h of cell culture, and levels of IL-8 were determined by ELISA. Data are presented as means ± standard errors of values from three parallel cell cultures and are representative of three independent experiments.