Intestinal heat shock protein 110 regulates expression of CD1d on intestinal epithelial cells - PubMed (original) (raw)

Intestinal heat shock protein 110 regulates expression of CD1d on intestinal epithelial cells

Sean P Colgan et al. J Clin Invest. 2003 Sep.

Abstract

CD1d is expressed on the surface of professional and nonprofessional APCs, including intestinal epithelial cells (IECs), for a role in the presentation of glycolipid-based antigens to subsets of T cells. The mechanisms that regulate CD1d expression in any cell type are unknown. To investigate the possibility that expression of CD1d is influenced by exogenous factors present within the intestinal lumen, CD1d expression was analyzed in several IEC lines after culturing in the presence of lumenal contents (LC) of the normal human intestine. Exposure of the colon-derived cell lines T84, HT-29, and Caco-2 to soluble LC resulted in a marked induction of CD1d expression as determined by RT-PCR, confocal microscopy, cell surface ELISA, and Western blot analysis. Similarly, exposure of human IECs to LC isolated from mice bred in both specific pathogen-free and germfree conditions also resulted in the induction of CD1d expression, with the maximum CD1d-inducing activity observed in the small intestine. Biochemical and biophysical characterization of the human CD1d-inducing activity identified heat shock protein 110 (Hsp110) as a major functional component of the LC that contributes to CD1d surface regulation, and immunolocalization studies revealed Hsp110 expression in subsets of human IECs in vivo. These data support the presence of a novel autocrine pathway of CD1d regulation by Hsp110.

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Figures

Figure 1

Figure 1

Soluble components present within human LC selectively enhance CD1d expression on the surface of IECs. (a) Cell surface ELISA of T84 monolayers following incubation with either 6 μg/ml (black bars) or 3 μg/ml (gray bars) of LC sample for varying periods of time. (b) Relative levels of CD1d, MHC class II, and ICAM-1 expression were compared by ELISA on T84 cells incubated alone (CNT) or in the presence of 6 μg/ml LC sample (LC) for a period of 48 hours. As a positive control for the detection of all inducible epithelial markers, monolayers were exposed to IFN-γ for 48 hours at a final concentration of 1,000 IU/ml. ODs at 405 nm ± SEM, generated by ELISA, were blanked against isotype-matched control antibodies.

Figure 2

Figure 2

LC-induced upregulation of cell surface CD1d expression is accompanied by an elevation in the levels of CD1d-encoded mRNA transcripts and protein. (a) RT-PCR of total RNA isolated from T84 cells treated for varying lengths of time with media containing 6 μg/ml human LC sample. Primers specific to CD1d and the housekeeping gene GAPDH were used as described. (b) Immunoprecipitation (IP) of whole-cell lysates following treatment with LC material, using the CD1d-specific antibodies D5 and 51.1.3. Immunoprecipitates were resolved by 12% SDS-PAGE under nonreducing conditions. Proteins were transferred to nitrocellulose, and CD1d was detected by immunoblotting with the D5 antibody. Locations of 48-kDa (filled arrowhead) and 37-kDa (open arrowhead) forms of CD1d are indicated. The molecular weight is indicated on the left. (c) Confocal microscopy of T84 cells following incubation for 48 hours alone or in the presence of LC sample (6 μg/ml) or IFN-γ (1,000 IU/ml). CD1d (green) and the tight-junction marker ZO-1 (red) were stained with FITC- and Texas red–conjugated antibodies, respectively.

Figure 3

Figure 3

Soluble LC material elevates CD1d expression in the colonic epithelium-derived cell lines HT-29 and Caco-2. (a) CD1d-specific cell surface ELISA of confluent monolayers of HT-29 cells following incubation with LC samples. As a positive control, cells were exposed to IFN-γ at a final concentration of 1,000 IU/ml for a period of 48 hours. (b) Western blot of whole-cell lysates isolated from HT-29 and Caco-2 cells following treatment with soluble LC material at a concentration of 6 μg/ml. Proteins contained within the lysates were resolved by 12% SDS-PAGE and transferred to nitrocellulose. The presence of CD1d was subsequently assessed by probing with the anti-human CD1d-specific antibody D5. CD1d was observed as dominant bands of 48-kDa (filled arrowhead) and 37-kDa (open arrowhead) forms. No bands were detected when nitrocellulose membranes were probed with normal mouse serum (data not shown). The blots were reprobed for the presence of β-actin (bottom panels).

Figure 4

Figure 4

Non–microbe-derived factors are present within the lumen of the murine small and large intestine that upregulate CD1d expression on human T84 cells. Cell surface ELISA was performed on confluent T84 monolayers using the CD1d-specific antibody 51.1.3. (a) BALB/c mice housed in either germfree (GF) or SPF environments were sacrificed and the LC of the colon isolated. T84 cells were cultured alone (–) or exposed to the soluble lumenal preparations for 48 hours at a final concentration of 6 μg/ml. (b) Lumenal material from the stomach (St), small intestine (SI), or large intestine (LI) of SPF-housed mice was tested for its ability to enhance epithelial cell surface CD1d expression. The CD1d-inducing activity of both soluble lumenal components (black bars) and mucosal scrapings (gray bars) was assessed relative to untreated controls (–).

Figure 5

Figure 5

Preliminary biochemical characterization of the active CD1d-inducing factor present within soluble LC material. ELISA was performed on T84 cells treated for a period of 48 hours with various preparations of LC isolates. (a) Monolayers were cultured alone (CNT), in the presence of unmodified whole LC samples (–), or with LC samples that had been preincubated at 80°C for 10 minutes. (b) LC material was separated into aqueous (AQ) or hydrophobic (HP) phase by chloroform extraction and C18 column chromatography, respectively, as indicated. Individual fractions were subsequently assessed for their ability to induce CD1d expression. (c) LC samples were passed through a Centricon filter (Millipore Corp., Bedford, Massachusetts, USA)of 100-kDa molecular weight cut-off. The 100-kDa filtrate was then passed consecutively through 30-kDa and 10-kDa Centricon filters. The filtrates (white bars) and retentates (gray bars) from each filtration step were subsequently resuspended in PBS, tested for their CD1d-inducing activity (black bars), and compared with those from the no-treatment (CNT) and crude-fraction (LC) groups. (d) Coomassie-stained SDS-PAGE gel of partially purified material, indicating the major bands selected for microsequencing (arrows), and the migration position of the band with sequence homology to Hsp110. A control (CNT) derived from a100-kDa filtrate fraction is also shown.

Figure 6

Figure 6

Role of Hsp110 in induction of epithelial CD1d. (a) Western blot analysis of LC for Hsp110. LC or purified recombinant Hsp110 (0.3 μg) was resolved by SDS-PAGE under nonreducing conditions, and blots were probed with polyclonal rabbit anti-Hsp110. Also shown is the control with primary antibody omitted from the reaction mixture. A nonspecific band (NS) of approximately 70 kDa was observed in both LC and control samples. (b) Cell surface ELISA of T84 monolayers following incubation with 6 μg/ml LC in the presence of indicated concentrations of anti-Hsp110 or control anti-Hsp30. (c) Cell surface ELISA of T84 monolayers following incubation with indicated concentrations of purified recombinant Hsp110 in the presence of anti-Hsp110 or control anti-Hsp30. OD values generated by ELISA were blanked against isotype-matched control antibodies. (d) Flow cytometric analysis of Hsp110 on native small intestinal enterocytes (black line) compared with equivalent concentrations of an isotype-matched control (gray line). (e and f) Immunohistochemical localization of Hsp110 in normal small intestinal tissue (e) and in normal colon (f). Positive staining with anti-Hsp110 but not with control Ig (Control) is seen in epithelial cells and diffusely in the lamina propria. Magnification, ×400.

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