Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line

Abstract

The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.

Figure 1

FcRn expression in normal adult human small intestine and human intestinal epithelial cell lines. Western blots of total cellular protein (13 μg protein per lane, a; 10 μg protein per lane, b) isolated from the indicated source using affinity-purified rabbit antisera raised against amino acids 112–125 (a) or amino acids 174–188 (b). (c) RT-PCR detection of FcγRI transcripts. Total RNA (2 μg) from T84 (lanes 3 and 4), MOLT-4 (lanes 5 and 6; negative control), and U937 (lanes 1 and 2; positive control) cell lines was incubated with an oligo-dT primer with (odd-numbered lanes) or without (even-numbered lanes) avian myeloblastosis virus–RT (AMV-RT), and a nested PCR was performed with primers specific for FcγRI cDNA (top) or for β-actin (bottom).

Figure 2

Immunolocalization of FcRn in polarized T84 monolayers (a–c) and normal adult human small intestinal mucosa (d–g). (a) Whole-mount T84 monolayers show a diffuse, punctate staining pattern. The Z0-1 image was captured slightly above the focal plane of FcRn. (b) FcRn staining of whole-mount T84 monolayers viewed as confocal vertical sections. (c) FcRn staining was absent in the presence of an isotype-matched, irrelevant antiserum. (f) Villous enterocytes of normal adult human small intestine show delicate linear staining in the region of the apical cytoplasmic membrane. (g) Crypt enterocytes show an apical and punctate staining pattern visible not only at the level of the apical cytoplasmic membrane, but also in the apical cytoplasm below the level of the apical membrane. (d and e) FcRn staining was absent in the presence of an irrelevant antiserum or with secondary antibody alone (not shown).

Figure 3

Functional expression of FcRn in polarized T84 cells. T84 cells grown on collagen-coated Transwells were cell surface–biotinylated and solubilized at either pH 6.0 or pH 8.0 lysates were incubated with Sepharose beads (N) or beads coupled to IgG (IgG). Immunoprecipitated proteins were analyzed by SDS-PAGE and avidin blot. A 12-kDa band consistent with the mobility of β₂M (small arrow) and a 45-kDa band consistent with the mobility of human FcRn α chain (large arrow) were immunoprecipitated by IgG-coupled beads at pH 6.0 but not at pH 8.0.

Figure 4

Binding of rhodamine-labeled IgG to T84 cell surfaces. T84 cells grown on glass coverslips were incubated at 4°C with rhodamine-labeled IgG at pH 6.0 or pH 8.0 in the presence or absence of excess competing nonlabeled IgG. Ligand binding was assessed by epifluorescence and Nomarski bright-field microscopy.

Figure 5

FcRn-dependent bidirectional transcytosis of IgG across T84 cell monolayers. (a) Transcytosis of IgG-biotin occurs at 37°C (lanes 1 and 3) but not at 4°C (lane 2). (b) Specificity of IgG transport for FcRn. T84 cells transport human IgG-biotin at 37°C (lane 3) but not at 4°C (lane 4). IgG heavy and light chains are indicated by the bars to the left of the IgG standard. In contrast, chicken IgY-biotin does not cross T84 cell monolayers at 37°C in either direction (lanes 2 and 5). IgY heavy and light chains are indicated by asterisks to the right of the IgY standard. (c) Quantitative ELISA of IgG and IgY transport at 37°C and 4°C.

Figure 6

Specificity of IgG transport and functional dependence on the vacuolar H⁺ ATPase. Where transport occured, the 25-kDa biotinylated light chain of IgG is shown. (a) Apical-to-basolateral transcytosis of IgG-biotin (60 nM) in the absence or presence of a 500-fold molar excess of nonlabeled chicken IgY (30 μM) or human IgG (30 μM) (lanes 4 and 5, respectively). Lanes 2 and 3 show incubation in the absence of competitive inhibitor at 37°C and 4°C as positive and negative controls, respectively. (b) Basolateral-to-apical transcytosis of IgG-biotin (60 nM) in the absence or presence of a 500-fold molar excess of nonlabeled chicken IgY (30 μM) (lane 3) or human IgG (30 μM) (lane 4) or excess fragment B of Staphylococcal protein A (0.1 mg/mL; lane 5). Lane 2 shows incubation at 37°C as a positive control. (c) T84 cells were incubated in the presence (lane 2) or absence (lanes 3 and 4) of bafilomycin A1 (0.1 μM), and basolaterally directed transport of IgG-biotin (60 nM) was measured after 1 hour of incubation in symmetrical HBSS+ buffered to pH 8.0.