The SRC family tyrosine kinase HCK and the ETS family transcription factors SPIB and EHF regulate transcytosis across a human follicle-associated epithelium model

Abstract

A critical step in the induction of adaptive mucosal immunity is antigen transcytosis, in which luminal antigens are transported to organized lymphoid tissues across the follicle-associated epithelium (FAE) of Peyer's patches. However, virtually nothing is known about intracellular signaling proteins and transcription factors that regulate apical-to-basolateral transcytosis. The FAE can transcytose a variety of luminal contents, including inert particles, in the absence of specific opsonins. Furthermore, it expresses receptors for secretory immunoglobulin A (SIgA), the main antibody in mucosal secretions, and uses them to efficiently transcytose SIgA-opsonized particles present in the lumen. Using a human FAE model, we show that the tyrosine kinase HCK regulates apical-to-basolateral transcytosis of non-opsonized and SIgA-opsonized particles. We also show that, in cultured intestinal epithelial cells, ectopic expression of the transcription factor SPIB or EHF is sufficient to activate HCK-dependent apical-to-basolateral transcytosis of these particles. Our results provide the first molecular insights into the intracellular regulation of antigen sampling at mucosal surfaces.

FIGURE 1.

Cocultured TC7 monolayers transcytose particles in opsonin-independent and SIgA-dependent manners. A, non-opsonized particles (1 × 10¹⁰ particles/ml) were added to the apical surface of monocultures (Raji −) and cocultures (Raji +). The number of particles transcytosed at the indicated temperatures was determined as described under “Experimental Procedures.” Each point represents a value from a single monoculture or coculture. The mean value (a longer horizontal line) and S.D. value (error bar) are also shown. B, TC7 cocultures were incubated at 37 °C for 60 min in the absence (blue line) or presence (red line) of non-opsonized fluorescent particles (1 × 10⁸ particles/ml) added to the apical surface. TC7 monocultures were also incubated in the absence (orange line) or presence (green line) of the particles and used as controls. After incubation, monolayers were trypsinized, and isolated cells were analyzed by a flow cytometer as described under “Experimental Procedures.” The number in the histogram indicates the percentage of the cells that display enhanced fluorescence in cocultured monolayers incubated with particles (red line). The result shown is a representative of three independent experiments. C and D, the apical surface of monocultures (Raji −) and cocultures (Raji +) was incubated with SIgA or IgG, and cell surface-bound antibodies were detected as described under “Experimental Procedures” using an anti-κ light chain antibody conjugated to HRP and a chemiluminescence substrate. Generated light was captured by a CCD camera, and representative images are shown in C. The amounts of cell surface-bound antibodies are expressed as relative luminescence intensities and shown in D. Values are the mean ± S.D. of three independent experiments. AU, arbitrary units. E, non-opsonized particles (N) or the same particles opsonized with IgG (G) or SIgA (A) were added to the apical surface of monocultures (Raji −) and cocultures (Raji +). The number of particles transcytosed at the indicated temperatures was determined and presented as in A.

FIGURE 2.

HCK is expressed in TC7 cocultures. RT-PCR was carried out as described under “Experimental Procedures.” The sizes (bp) of the expected PCR products and DNA molecular weight markers are shown. Left, an HCK-specific fragment was amplified from total RNA isolated from TC7 monocultures (Raji −) and cocultures (Raji +). As a positive control, total RNA was also isolated from dimethyl sulfoxide-treated HL-60 human promyelocytic leukemia cells that express HCK (55). Middle, as a control for potential contamination of cocultured TC7 cells with Raji cells, a DNA fragment specific for CD20, which is strongly expressed in Raji cells (56), was amplified from total RNA isolated from TC7 cocultures (Raji +) and Raji cells. Right, as a control for RNA quality and quantity, a DNA fragment specific for GAPDH was amplified from total RNA isolated from TC7 monocultures (Raji −) and cocultures (Raji +).

FIGURE 3.

p59HCK regulates opsonin-independent and SIgA-dependent transcytosis across the FAE model. A, monolayers were generated using TC7 cells stably transfected with an empty vector (V) or the same vector constitutively expressing p59HCK^CA (59) or p61HCK^CA (61). As a control, monolayers were also generated using untransfected (U) TC7 cells. Monolayers were then cultured in the presence (+) or absence (−) of Raji cells and used to analyze transcytosis of non-opsonized particles. The number of particles transcytosed at the indicated temperatures is presented as in Fig. 1A. Expression levels of p59HCK^CA and p61HCK^CA were determined by immunoblotting and are shown to the right. B, monolayers were generated using TC7 cells stably transfected with an empty vector (Vector) or the same vector constitutively expressing p59HCK^DN (p59^DN) or p61HCK^DN (p61^DN). Monolayers were then cocultured (Raji +) and used to analyze, at 37 °C, transcytosis of non-opsonized (N), IgG-opsonized (G), and SIgA-opsonized (A) particles. The number of particles transcytosed is presented as in Fig. 1A. None of these particles were significantly transcytosed by cocultures at 4 °C or by monocultures (data not shown). Expression levels of p59HCK^DN and p61HCK^DN were determined by immunoblotting and are shown to the right. C, untransfected and p59HCK^DN-expressing monolayers were cultured in the presence (+) or absence (−) of Raji cells. Apical AP activities were determined as described under “Experimental Procedures” and expressed as the absorbance at 405 nm (A405). Each point represents a value from a single monoculture or coculture. The mean values (longer horizontal lines) and S.D. (error bars) are also shown. D and E, differentiated TC7 monolayers stably expressing p59HCK^DN (p59^DN) from a Dox-inducible promoter were cocultured (Raji +) for 2 days without Dox (−Dox). At day 2 of coculture, some of the monolayers were used to analyze, at 37 °C, transcytosis of non-opsonized (N) and SIgA-opsonized (A) particles. The rest of the monolayers were cocultured for an additional 2 days in the absence or presence (+Dox) of Dox and used for the same transcytosis analysis at day 4. The number of particles transcytosed was determined and presented as in Fig. 1A.

FIGURE 4.

Raji cells can regulate the expression of HCK through the induction of SPIB and EHF in the FAE model. A, a putative SPIB binding site (27) (boxed) in the human HCK promoter. Nucleotide +1 is the C of the CTG translation initiation codon for p61HCK. B, the HCK promoter-firefly LUC reporter plasmid (HCK-LUC) was constructed by inserting the HCK promoter fragment (−1790 to −100) into a LUC reporter vector (LUC vector) as described in the supplemental Experimental Procedures. TC7 cells were transiently transfected with the LUC vector or the HCK-LUC plasmid, together with expression vectors encoding SPIB and SPIB^DBD, as indicated at the bottom. LUC reporter assays were carried out as described under “Experimental Procedures,” and the results were expressed as the mean ± S.D. (error bars) of triplicate assays. The mean value obtained with the LUC vector in the absence of SPIB and SPIB^DBD was set to 1. Expression levels of SPIB and SPIB^DBD in cells transfected with HCK-LUC were confirmed by immunoblotting and are shown above the corresponding columns. C, the firefly LUC reporter plasmids SPIB-LUC and EHF-LUC were constructed as described in the supplemental Experimental Procedures by inserting the SPIB (−722 to +2) and EHF (−21,849 to −21,472) promoter fragments into a LUC reporter vector (LUC vector), respectively. (Nucleotide +1 is the A of the ATG initiation codon of each gene.) TC7 cells grown on Transwell permeable membranes were transiently transfected with the LUC vector, SPIB-LUC, or EHF-LUC, and treated with Raji cell-conditioned medium (CM +) or fresh Raji cell medium (CM −) as described under “Experimental Procedures”. The mean value obtained with the LUC vector in the absence of CM was set to 1. D, TC7 cells were transiently transfected with the LUC vector or the HCK-LUC plasmid, together with expression vectors encoding EHF as indicated. LUC reporter assays were carried out as described in B. The mean value obtained with the LUC vector in the absence of EHF was set to 1.

FIGURE 5.

SPIB^DBD prevents the conversion of differentiated TC7 cells into M-like cells. A and B, TC7 monolayers stably expressing SPIB^DBD from a Dox-inducible promoter were cultured for 21 days and then cocultured (Raji +) in the absence of Dox (−Dox). At day 2 of coculture, Dox was added to half of the monolayers (+Dox) to induce SPIB^DBD. At day 4, transcytosis of non-opsonized (N) and SIgA-opsonized (A) particles was analyzed at the indicated temperatures. The number of particles transcytosed was determined and presented as in Fig. 1A. Induction of SPIB^DBD was confirmed by immunoblotting using GAPDH as a control and is shown in B. C–E, TC7 monolayers stably expressing SPIB^DBD or mSPIB^DBD from a Dox-inducible promoter were cultured for 19 days. Dox was then added to half of the monolayers (+Dox) to induce SPIB^DBD or mSPIB^DBD. Two days after Dox addition, coculture was started by adding Raji cells (Raji +) to both Dox-treated and untreated (−Dox) monolayers. At day 4 of coculture, transcytosis of non-opsonized (N) and SIgA-opsonized (A) particles was analyzed at 37 °C. The number of particles transcytosed was determined and presented as in Fig. 1A. Induction of SPIB^DBD and mSPIB^DBD was confirmed by immunoblotting as in B. F, TC7 monolayers stably expressing SPIB^DBD or mSPIB^DBD from a Dox-inducible promoter were cultured for 19 days. Dox was then added to all of the monolayers to induce SPIB^DBD or mSPIB^DBD. Two days after Dox addition, Raji cells were added to half of the monolayers, and cocultures were incubated for 4 days (Raji +). The rest of the monolayers were incubated without Raji cells for the same period of time (Raji −). Finally, apical AP activities were determined and presented as described in the legend to Fig. 3C. Untransfected (U) TC7 monolayers were treated identically and served as controls. Error bars, S.D.

FIGURE 6.

SPIB and EHF can induce p59HCK-dependent transcytosis of non-opsonized and SIgA-opsonized particles in TC7 monocultures. A and B, TC7 monolayers stably expressing full-length SPIB from a Dox-inducible promoter were differentiated and incubated for 4 days in the absence (−) or presence (+) of Dox. Non-opsonized (N) or SIgA-opsonized (A) particles (1 × 10¹⁰ particles/ml) were then added to the apical surface of the monolayers, and transcytosis was analyzed at the indicated temperatures. The number of particles transcytosed is presented as in Fig. 1A. Induction of SPIB was confirmed by immunoblotting as in Fig. 5B. C, TC7 cells stably expressing full-length SPIB from a Dox-inducible promoter were stably transfected with an empty vector (V) or the same vector expressing p59HCK^DN (59^DN) or p61HCK^DN (61^DN) from a Dox-inducible promoter. Monolayers of transfected cells were differentiated and incubated in the presence of Dox. Four days after the addition of Dox, particle transcytosis was analyzed at 37 °C as described above. Induction of SPIB, p59HCK^DN, and p61HCK^DN was confirmed by immunoblotting. D, TC7 cells were stably transfected with a Dox-inducible expression vector encoding full-length EHF, SPDEF, or ETS2. TC7 cells were also stably co-transfected with two Dox-inducible expression vectors encoding full-length EHF and p59HCK^DN, respectively (EHF + p59^DN). Monolayers were generated from these transfected cells and, after differentiation, treated with Dox for 4 days. Transcytosis of non-opsonized (N) and SIgA-opsonized (A) particles (1 × 10¹⁰ particles/ml) were analyzed as described above. Induction of EHF, SPDEF, and ETS2 was confirmed by immunoblotting. Their activities were separately confirmed by LUC reporter assays using a promoter containing five consensus ETS binding sites (57) (data not shown). Error bars, S.D.

FIGURE 7.

CD300LF is a SPIB-, EHF-, and Raji cell-inducible receptor for SIgA. A, TC7 cells stably transfected with an empty vector (−) or the same vector constitutively expressing CD300LF (+) were grown to confluent monolayers and incubated with SIgA or IgG. The amount of cell surface-bound antibody was determined and presented as in Fig. 1, C and D. Apical expression of CD300LF was separately confirmed as described below. B, comparison of the human, mouse, and rat CD300LF promoter sequences identified a putative SPIB binding site (27) (boxed) in a conserved region. Dashes indicate the nucleotides in the mouse and rat sequences that are identical to those in the human sequence. The numbers represent the positions in the human sequence relative to the ATG translation initiation codon (the A is +1). C, reporter plasmids were constructed by fusing different lengths of the human CD300LF promoter sequence to the firefly LUC coding sequence as described in the supplemental Experimental Procedures. The conserved SPIB site in the promoter is shown. Each of the plasmids was introduced into TC7 cells together with an empty vector (open bars) or the same vector constitutively expressing SPIB (filled bars) or EHF (hatched bars). LUC reporter assays were carried out as described under “Experimental Procedures,” and the results are expressed as the mean ± S.D. (error bars) of triplicate assays. The mean value obtained from the longest promoter fragment cotransfected with the empty vector was set to 1. D, TC7 monolayers stably expressing full-length SPIB or EHF from a Dox-inducible promoter were differentiated and then incubated for 4 days in the absence (−) or presence (+) of Dox. The apical surface of the monolayers was then incubated with anti-CD300LF (rat IgG) or an isotype-matched control antibody (Cont. Ab), and the amount of cell surface-bound antibody was determined using anti-rat IgG conjugated to HRP and presented as in Fig. 1, C and D. E, apical expression of CD300LF was analyzed as above using TC7 monocultures (Raji −) and cocultures (Raji +). Raji cells do not express CD300LF on their surface (47) (T. Asai and S. L. Morrison, unpublished data).

FIGURE 8.

CD300LF is a transcytotic receptor that can interact with p59HCK. A, TC7 cells were transiently transfected with expression vectors encoding CD300LF, FLAG-tagged p59HCK (HCK-FLAG), and FLAG-tagged YES (YES-FLAG), as indicated. Cells were then incubated with anti-CD300LF (rat monoclonal IgG) and, after washing, treated with polyclonal anti-rat IgG to cross-link CD300LF. Control cells (No crosslink) were incubated with an isotype-matched control antibody instead of anti-CD300LF. CD300LF was also cross-linked by incubating with SIgA-opsonized particles (SIgA). Cells were lysed and subjected to immunoprecipitation (IP) with goat polyclonal anti-CD300LF. The precipitates were immunoblotted with rabbit polyclonal anti-CD300LF and anti-FLAG. Cell lysates were also immunoblotted with anti-FLAG to verify the expression of p59HCK and YES. B, TC7 monolayers constitutively expressing CD300LF were differentiated, and transcytosis of IgG-opsonized (G) and SIgA-opsonized (A) particles was analyzed at the indicated temperatures as in Fig. 6B. C, TC7 cells were stably transfected with a bicistronic vector constitutively coexpressing CD300LF and the green fluorescent protein (GFP), p59HCK^CA (59^CA), or p61HCK^CA (61^CA). Monolayers of transfected cells were differentiated, and transcytosis of SIgA-opsonized particles was analyzed at 37 °C as in Fig. 6B. Expression of CD300LF, p59HCK^CA, and p61HCK^CA was confirmed by immunoblotting. Error bars, S.D.