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. 2004 Dec;78(24):13755-68.
doi: 10.1128/JVI.78.24.13755-13768.2004.

Glycocalyx restricts adenoviral vector access to apical receptors expressed on respiratory epithelium in vitro and in vivo: role for tethered mucins as barriers to lumenal infection

Jaclyn R Stonebraker  1 , Danielle Wagner, Robert W Lefensty, Kimberlie Burns, Sandra J Gendler, Jeffrey M Bergelson, Richard C Boucher, Wanda K O'Neal, Raymond J Pickles
Affiliations

Glycocalyx restricts adenoviral vector access to apical receptors expressed on respiratory epithelium in vitro and in vivo: role for tethered mucins as barriers to lumenal infection

Jaclyn R Stonebraker et al. J Virol. 2004 Dec.

Abstract

Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.

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Figures

FIG. 1.
FIG. 1.
AdV-mediated expression of GPI-hCAR in HAE and localization of GPI-hCAR to ciliated cell shafts. (A) Representative en face images of HAE inoculated with AdVGFP (i and ii) or AdVgpi-hCAR (iv and v) without (i and iv) or with (ii and v) disruption of epithelial tight junctions by sodium caprate, with gene expression being assessed 48 h later. Original magnification, ×10. Confocal XZ optical sectioning of HAE expressing GFP (green) and probed with anti-β-tubulin IV conjugated to Texas Red (red) indicated that predominately ciliated columnar epithelial cells were transduced by AdVGFP (iii), and GPI-hCAR expressed in HAE was localized to the apical surface, as detected by anti-hCAR (RmcB) conjugated to Texas Red (red) (vi). Original magnification, ×63. (B) Transmission electron micrographs of HAE inoculated with AdV-gpi-hCAR and probed with RmcB conjugated to immunogold particles (12-nm diameter) revealed that GPI-hCAR was predominately localized to the cilial shafts of ciliated cells (i, arrows), with less localization to the microvillus structures on ciliated cells (ii, arrowheads). HAE inoculated with AdVGFP or a vehicle control alone and probed with RmcB showed no immunogold labeling. Bar = 1 μm.
FIG. 2.
FIG. 2.
Inefficient AdV-mediated gene transfer to HAE in the presence of apical GPI-hCAR. (A) Representative en face views of control HAE (i and iii) and GPI-hCAR HAE (ii and iv) either probed with anti-hCAR conjugated to Texas Red (i and ii) or monitored for GFP expression 48 h after inoculation with AdVGFP (iii and iv). Original magnification, ×10. (B) Quantitative LacZ enzymatic analyses of control HAE (hatched bars) or GPI-hCAR HAE (solid bars) 48 h after inoculation of the apical surfaces of the HAE cultures with AdVLacZ at 2 × 1010 or 2 × 1011 particles/ml. The data represent 14 samples for each bar from cultures derived from four different patient samples and are means ± standard errors (SE).
FIG. 3.
FIG. 3.
Immunodetection of glycoconjugates on the apical surfaces of HAE cultures. Cultures were exposed to probes for the following: (i) α2-6-linked sialic acid residues, (ii) α2-3-linked sialic acid residues, (iii) MUC1 glycoprotein, (iv) keratan sulfate, and (v) heparan sulfate or chondroitin sulfate. The probes were then detected with secondary reagents conjugated to Cy3 or Texas Red. Secondary reagents in the absence of specific probes showed no fluorescence on HAE (vi). Original magnification, ×10.
FIG. 4.
FIG. 4.
Enhancement of AdV-mediated gene transfer to GPI-hCAR HAE after elimination of apical surface glycocalyx. (A) Quantitative LacZ enzyme analyses of AdV-mediated gene transfer 48 h after inoculation of HAE (hatched bars) or GPI-hCAR HAE (solid bars) either without treatment (CTL; n = 5 for each group) or after treatment of the apical surface with PXIV (0.01%; n = 6), PYR (5 mM; n = 8), or a combination of both reagents (PYR + PXIV; n = 5). Data represent means ± SE for two different patient samples. (B) Representative en face images of HAE (i and iii) and GPI-hCAR HAE (ii and iv) with no treatment (i and ii) and after apical surface treatment with a combination of PYR and PXIV (iii and iv). Original magnification, ×10. (C) Confocal XZ optical sectioning of GPI-hCAR HAE pretreated with PYR and PXIV, inoculated with AdVGFP, and assessed for GFP expression 48 h later. Colocalization with anti-hCAR conjugated to Texas Red (i) or anti-β-tubulin IV conjugated to Texas Red (ii) revealed that GFP-expressing cells also expressed GPI-hCAR and were predominately ciliated. The rare occurrence of GPI-hCAR-negative cells that were positive for GFP most likely reflected the absence of GPI-hCAR detection within the optical plane analyzed. Nonciliated cells were also occasionally positive for GFP expression (ii, arrows). Original magnification, ×63. (D) Effect of PYR and PXIV treatment on the TERs of HAE (hatched bars) and GPI-hCAR HAE (solid bars). Data represent means ± SE for at least five cultures from two different patient samples.
FIG. 5.
FIG. 5.
RT-PCR analyses of human tethered mucin genes (MUC) in human placental (Plac), colon (Col), bronchial epithelial (HBE), and nasal epithelial (HNE) cells. + and −, RT-PCRs performed in the presence and absence, respectively, of reverse transcriptase.
FIG. 6.
FIG. 6.
Assessment of glycocalyx abundance on the surfaces of human airway epithelia in vitro and ex vivo by freeze substitution and transmission electron microscopy of HAE cultures (i and iii) and freshly excised human airway epithelium (ii and iv). Representative regions of the apical surfaces of ciliated cells (i and ii) and nonciliated cells that possess microvillus structures but not cilia are shown (iii and iv). Bar = 1 μm.
FIG. 7.
FIG. 7.
RT-PCR analyses of murine tethered mucin genes (Muc) in the murine colon (Col), murine trachea (Trach), or murine whole lung (Lung). + and −, RT-PCRs performed in the presence and absence, respectively, of reverse transcriptase.
FIG. 8.
FIG. 8.
Localization of RmcB immunoreactivity in the murine airway epithelium of K18-GPI-hCAR transgenic mice. Representative histological sections from murine nasal epithelia (i and ii) or small airway epithelia (iii and iv) derived from transgenics (i and iii) or littermate controls (ii and iv) and probed with RmcB and secondary antibodies conjugated to fluorescein isothiocyanate (green) are shown. Note that RmcB immunoreactivity below the surface of the nasal epithelium occurred in regions associated with glandular structures. Original magnification, ×100.
FIG. 9.
FIG. 9.
Expression of GPI-hCAR and Muc1 glycoprotein at the apical surfaces of murine tracheal epithelia derived from GPI-hCAR transgenic mice. (A) Immunolocalization of GPI-hCAR (green) to the apical surfaces of murine tracheas derived from K18-GPI-hCAR transgenics (i) but not littermate controls (ii). The colocalization of GPI-hCAR (iii, green) and Muc1 (iv, red) in GPI-hCAR/Muc1+/+ transgenic animals and the presence of GPI-hCAR (v, green) but not Muc1 (vi, red) at the apical surfaces of murine tracheal epithelia from GPI-hCAR/Muc1−/− animals are shown. Bar = 20 μm. (B) Electron micrographs of GPI-hCAR-expressing murine tracheal epithelium probed with anti-hCAR and detected with anti-mouse IgG conjugated to immunogold particles (12-nm diameter). Immunogold was detected on the cilial shafts of ciliated cells (arrows) and the apical surfaces of Clara cells (arrowheads). Similar procedures performed with a littermate control tracheal epithelium revealed no immunogold localization. Bar = 500 nm.
FIG. 10.
FIG. 10.
AdV-mediated gene transfer to murine tracheal epithelium is enhanced by GPI-hCAR only in the absence of Muc1 glycoprotein from the lumenal surface. (A) Representative photomicrographs of excised murine tracheas displaying LacZ expression in the surface epithelium. Tracheal epithelia from control (i), GPI-hCAR transgenic (ii), Muc1−/− knockout (iii), and GPI-hCAR/Muc1−/− (iv) animals were inoculated with AdVLacZ (20 μl at 1011 particles/ml) and assessed 48 h later for gene transfer. Histological sections of murine tracheal epithelia from GPI-hCAR/Muc1−/− animals revealed that airway surface epithelial cells expressed the transgene, with both ciliated cells (v, arrows) and Clara cells (vi, arrows) being transduced by AdVLacZ. (B) Quantitative morphological analyses of the percentages of epithelial surface area exposed to AdVLacZ that expressed LacZ after 48 h for tracheas derived from Muc1+/+ (n = 30), GPI-hCAR/Muc1+/+ (n = 30), Muc1−/− (n = 7), and GPI-hCAR/Muc1−/− (n = 22) animals. For comparison, the percentage of epithelial surface area expressing LacZ after sodium caprate treatment (25 mM) prior to AdVLacZ inoculation is shown (n = 12). Data represent means ± SE. * and **, statistically significant differences, with P < 0.0002 and P < 0.02, respectively.
FIG. 11.
FIG. 11.
Assessment of glycocalyx depth on the apical surface of murine tracheal epithelium in the presence and absence of Muc1 glycoprotein. (A) Representative transmission electron micrograph of murine tracheal epithelium stained with ruthenium red to assess the abundance of glycoconjugates. Ruthenium red staining on the apical surfaces of ciliated cells (CC) and nonciliated cells (Clara cell; NCC) is shown. Bar = 2 μm. (B) Quantitative morphological analyses of the depths of ruthenium red staining on the surfaces of ciliated cells (CC), nonciliated cells (NCC), or all cells (AC) of trachea epithelia derived from Muc1+/+ (solid bars) and Muc1−/− (open bars) mice. The data shown represent means ± SE. *, statistically significant difference, with P < 0.05.
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