Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers

Kwang-Jin Kim,1,2,3,4,5,* Tamer E. Fandy,6,* Vincent H. L. Lee,6,7 David K. Ann,1,3 Zea Borok,1,4,8 and Edward D. Crandall1,4,9

Departments of 1Medicine, 2Physiology and Biophysics, 3Molecular Pharmacology and Toxicology, 5Biomedical Engineering, 6Pharmaceutical Sciences, 7Ophthalmology, 8Biochemistry and Molecular Biology, and 9Pathology, and 4Will Rogers Institute Pulmonary Research Center, Keck School of Medicine and Schools of Pharmacy and Engineering, University of Southern California, Los Angeles, California 90033

Submitted 1 April 2004 ; accepted in final form 19 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We characterized immunoglobulin G (IgG) transport across rat alveolar epithelial cell monolayers cultured on permeable supports. Unidirectional fluxes of biotin-labeled rat IgG (biot-rIgG) were measured in the apical-to-basolateral (ab) and opposite (ba) directions as functions of [rIgG] in upstream fluids at 37 and 4°C. We explored specificity of IgG transport by measuring fluxes in the presence of excess Fc, Fab, F(ab')2, or chicken Ig (IgY). Expression of the IgG receptor FcRn and the effects of dexamethasone on FcRn expression and biot-rIgG fluxes were determined. Results show that ab flux of biot-rIgG is about fivefold greater than ba flux at an upstream concentration of 25 nM biot-rIgG at 37°C. Both ab and ba fluxes of rIgG saturate, resulting in net absorption with half-maximal concentration and maximal flow of 7.1 nM and 1.3 fmol·cm–2·h–1. At 4°C, both ab and ba fluxes significantly decrease, nearly collapsing net absorption. The presence of excess unlabeled Fc [but not Fab, F(ab')2, or IgY] significantly reduces biot-rIgG fluxes. RT-PCR demonstrates expression of {alpha}- and {beta}-subunits of rat FcRn. Northern analysis further confirms the presence of {alpha}-subunit of rat FcRn mRNA of ~1.6 kb. Dexamethasone exposure for 72 h decreases the steady-state level of mRNA for rat FcRn {alpha}-subunit and the ab (but not ba) flux of biot-rIgG. These data indicate that IgG transport across alveolar epithelium takes place via regulable FcRn-mediated transcytosis, which may play an important role in alveolar homeostasis in health and disease.

receptor mediated; saturable transcytosis; net IgG absorption; lung defense; pulmonary immune system


ALVEOLAR EPITHELIUM LINES the distal air spaces of the lung and provides high resistance to the leak of solutes and fluid from the surrounding interstitial and vascular spaces (18). Various serum proteins (e.g., albumin, transferrin, and IgG) are known to be present in alveolar fluid lining distal air spaces, although the underlying transport mechanisms that account for their presence are not well delineated (10, 12, 19). Alveolar protein clearance is essential for resolution of both hydrostatic and (especially) high permeability pulmonary edema. Understanding the mechanisms of alveolar protein clearance may be useful in the management of patients with alveolar pulmonary edema and in providing new insights into transpulmonary delivery of exogenous protein drugs.

Proteins in the alveolar space may be cleared by endocytosis and degradation inside alveolar epithelial cells, by transcytosis across the alveolar epithelium, or by restricted diffusion through the epithelium (10, 12, 19, 20). The relative contributions of each of these three pathways to total clearance of proteins from the air spaces are not known. Previous reports suggest the possibility of transcellular mechanism(s) [e.g., receptor-mediated or adsorptive transcytosis (24)] for transport of macromolecules across alveolar epithelium.

Protein transport studies utilizing intact lung are not ideal for inferring mechanistic information because of the anatomical complexity and inherent problems (e.g., series and parallel arrangement of biological barriers, presence of unstirred layers, and unknown distribution volumes and surface areas for solute transport) associated with interpretation of experimental data. In this regard, a simplified model of the alveolar epithelial barrier [primary cultured rat alveolar epithelial cell monolayers (RAECM) (16, 21)] has been utilized widely to study transport mechanisms. The monolayers exhibit morphologic and phenotypic characteristics of in vivo type I pneumocytes (4), develop high barrier resistance (>2,000 {Omega}cm2), and actively reabsorb Na+ (~0.2 µeq·cm–2·h–1) from apical fluid (17). Using this in vitro model, we showed transport of dextrans (22) to primarily occur paracellularly with little contribution by pinocytosis. Horseradish peroxidase (HRP) was shown to be transported transcellularly via nonspecific fluid phase endocytosis (23), whereas enkephalin transport mediated via simple diffusion has been reported (35). Transport of HRP (conjugated to transferrin) was enhanced due to receptor-mediated transcytosis of transferrin (6, 36).

In this study, we explored the mechanisms of IgG transport across primary cultured rat alveolar epithelial cell monolayers. To test the hypothesis that IgG is transported via receptor-mediated transcytosis across alveolar epithelium, we investigated the effects of IgG concentration and temperature on fluxes of biotin-labeled IgG in the apical-to-basolateral (ab) and opposite (ba) directions. In addition we also investigated the effects of day in culture, apical fluid pH, and dexamethasone to provide further insights into IgG transport across alveolar epithelium. Our results support the hypothesis that IgG translocation across the alveolar epithelial barrier occurs predominantly via transcellular saturable processes mediated by the neonatal Fc receptor, FcRn.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Primary culture of RAECM. Type II pneumocytes isolated enzymatically from male Sprague-Dawley rats were further enriched by IgG panning and plated onto tissue culture-treated polycarbonate filters (0.45-µm pores, 12-mm-diameter Transwells; Costar-Corning, Cambridge, MA) at 1.2 x 106 cells/cm2 (2, 21). Cells were maintained for 48 h at 37°C in a humidified atmosphere of 5% CO2/95% air, with culture medium containing 10% newborn bovine serum in minimal defined serum-free medium (MDSF), a 1:1 mixture of DME/F-12 medium (Sigma, St. Louis, MO) supplemented with 1% nonessential amino acids, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1% bovine serum albumin. Medium was changed at 48 h, and monolayers were maintained thereafter in MDSF unless otherwise noted. From day 3 onward, confluent monolayers are populated with cells that have undergone transdifferentiation to exhibit morphologic [e.g., bulging nuclei with thin cytoplasmic extensions (4)] and phenotypic [e.g., reactivity towards an antibody recognizing a rat type I cell epitope (5)] features similar to those found in type I pneumocytes in vivo. Monolayers (n = 96) develop transepithelial electrical resistance (Rt) = 2.52 ± 0.03 k{Omega}cm2 and transmonolayer potential difference (PD) = 11.3 ± 0.1 mV (apical negative) on days 3–4 in primary culture.

Measurement of unidirectional fluxes of biotinylated rat IgG. For flux studies, both apical (0.5 ml) and basolateral (1.5 ml) fluids of RAECM cultivated on 12-mm Transwells were washed once with prewarmed MDSF and allowed to equilibrate in a humidified incubator (5% CO2 in air, 37°C) for 1 h. After equilibration and measurement of Rt and PD with a MilliCell ERS device (Millipore, Malborough, MA), unidirectional fluxes of IgG in the ab direction were initiated by removing 0.05 ml of the upstream fluid and immediately replacing it with 0.05 ml of 250 nM biotinylated rat IgG (biot-rIgG; Jackson Immunoresearch, West Grove, PA) at time 0, achieving a final concentration of 25 nM biot-rIgG in apical upstream fluid. At 3, 6, and 18 h, samples (0.2 ml) were taken from basolateral downstream fluid. These samples were stored at –20°C until performance of enzyme-linked immunosorbent assay (ELISA) for biot-rIgG (see below). Transport in the ba direction was similarly assessed, except that 0.15 ml of basolateral upstream fluid was replaced with 0.15 ml of 250 nM biot-rIgG, and downstream samples (0.05 ml) were taken from apical fluid. Steady-state fluxes of biot-rIgG were estimated from the linear slope of the relationship between the amount (fmol) of biot-rIgG appearing in downstream fluid and time (h). We determined apparent permeability by normalizing flux against the initial concentration gradient of biot-rIgG (25 nM) and the monolayer surface area (1.13 cm2). Monolayer PD and Rt were measured at the end of flux studies to monitor the integrity of the barrier, which showed no appreciable changes over the time periods studied.

ELISA. ELISA for IgG detection (7) was modified to measure the level of biot-rIgG in bathing fluids of RAECM used for flux studies. Streptavidin-coated 96-well plates (Boehringer Mannheim, Indianapolis, IN) were blocked for 1 h with gentle shaking in the presence of freshly made 0.2 ml of blocking solution [comprising 0.5% (vol/vol) fish gelatin (Sigma) in 0.05% (vol/vol) Tween 20, 1 mM ethylenediamine-tetraacetic acid disodium salt, and 0.05% (wt/vol) sodium azide]. After we removed the blocking solution by flicking a 96-well plate, we added the thawed fluid samples (0.2 ml, which, in the case of apical fluid samples, required dilution with fresh MDSF) to each well and incubated them for 1 h at room temperature with shaking to allow binding of biot-rIgG to streptavidin. We estimated background levels using bathing fluids of monolayers that were treated similarly but without exposure to biot-rIgG. Some of the microplate wells contained serial dilutions of fresh biot-rIgG (ranging from 0.125 to 100 pM) dissolved in MDSF for generation of a standard curve.

The 96-well plate was washed three times for 5 min each with shaking, using 0.2 ml of wash solution (comprising 0.1 M phosphate-buffered saline, pH 7.4, supplemented with 0.05% Tween 20) per washing step per well. The washed plate was blocked again for 1 h with shaking, followed by incubation at room temperature with 0.1 ml of 1 µg/ml primary antibody (goat anti-rat IgG, Jackson Immunoresearch) for 1 h. The plate was then washed as above, and each well was further incubated at room temperature with shaking in 0.1 ml of 2 µg/ml secondary antibody [donkey anti-goat IgG conjugated with HRP (Jackson Immunoresearch)] for 1 h. The plate was again washed as above, and each well was incubated for 15 min with shaking in 0.1 ml of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (KPL, Gaithersburg, MD) comprising equal parts of 0.4 TMB and 0.02% fresh H2O2 in citric acid buffer, which was mixed immediately before use. Absorbance of the blue color produced from the reaction of TMB and HRP was measured spectrophotometrically at 650 nm with a microplate reader (Labsystems, Franklin, MA).

Investigation of concentration dependency and competitive inhibition of IgG transport. To determine whether IgG flux saturates with the upstream concentration of IgG, we varied upstream concentrations up to 25 µM. To determine whether other immunoglobulin-related macromolecules compete with IgG transport, we added unlabeled molecules [e.g., Fc, Fab, and F(ab')2 or chicken immunoglobulin (IgY)] in 100x molar excess (i.e., 2,500 nM) to upstream fluid 30 min before the instillation of biot-rIgG (25 nM). After 3 h, downstream appearance of biot-rIgG in the presence and absence of competing molecules was measured. In a separate series of experiments, unidirectional fluxes of biot-rFc were measured (at 25 nM) for comparison with biot-rIgG fluxes at the same upstream concentration. The amount of biot-rFc was determined by ELISA, using the same procedure as for biot-rIgG estimation, except for use of goat anti-rFc and rabbit anti-goat IgG conjugated with HRP as primary and secondary antibodies, respectively.

Effects of temperature, apical fluid pH, and day in culture on IgG transport. To determine the degree of cellular energy expenditure required for IgG transport, we studied biot-rIgG fluxes (at 25 nM) at 4°C. In a separate series of experiments, the effects of apical fluid pH (e.g., 5.5, 6.0, 6.5, 7.0, and 7.4) on biot-rIgG fluxes (with upstream concentrations of 25 nM) were determined, while keeping basolateral fluid pH at 7.4. For maintaining apical pH at different values, we supplemented MDSF with 15 mM 2-(N-morpholino)ethanesulfonic acid for pH 5.5–6.5 or N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) for pH 7.0 or 7.4, respectively. In another series of experiments, the effects of day in culture on biot-rIgG transport rates (at 25 nM) were investigated with monolayers maintained for 3, 6, 14, and 25 days.

Effects of glucocorticoids on IgG transport. The effects of glucocorticoid exposure on IgG transport were investigated. Dexamethasone (100 nM) was added at various time points in culture to both apical and basolateral bathing fluids and allowed to incubate for different time intervals (e.g., 12, 24, 48, and 72 h). Unidirectional fluxes of biot-rIgG (at 25 nM) were measured as above.

RT-PCR to identify cDNA fragments of {alpha}- and {beta}-subunits of rat FcRn gene. Total RNA from day 6 RAECM (grown on 24-mm tissue culture-treated Transwells) was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA). The integrity of the extracted RNA was further evaluated by denaturing-agarose (1%) gel electrophoresis. The primer pair 5'-CGGAGCTCAAGTTTCGATTC-3' and 5'-GAAGCAGGCCACAAAAGAAG-3', encompassing a 542-bp segment of the {alpha}-subunit of rat FcRn, was utilized for RT-PCR analysis. The Superscript One-Step RT-PCR kit with platinum Taq (GIBCO-BRL, Rockville, MD) and the Mastercycler Gradient 5331 Thermal Cycler (Eppendorf-Brinkmann, Westbury, NY) were used. The RT-PCR procedure comprises one cycle of 50°C for 30 min followed by heating at 94°C for 2 min and subsequent 35 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and one cycle of 72°C for 7 min. The volume of all the components added together was 50 µl, and the final concentration of the primers was 0.4 µM. The same RT-PCR procedure was used to amplify a 194-bp segment of rat {beta}2-microglobulin (the {beta}-subunit of rat FcRn), except for using a primer pair of 5'-GTCTCAGTTCCACCCACCTC-3' and 5'-TTTTGGGCTCCTTCAGAGTG-3'. We conducted electrophoresis using the NuSieve 3:1 agarose (3%; BioWhittaker, Rockland, ME) to analyze the PCR amplification product. In one of the lanes, a DNA molecular weight ladder (GIBCO-BRL) was loaded to estimate the relative molecular size of RT-PCR products. GelStar stain (BioWhittaker) was used for DNA detection in a UV Transilluminator (Fotodyne, Hartland, WI) and photographed with a GelStar filter (BioWhittaker).

To further confirm the molecular sequence of the RT-PCR products obtained above, we excised DNA bands of interest from the agarose gel and the cDNA fragments extracted using the Ultrafree DA centrifugal filter device (Millipore, Bedford, MA). The extracted cDNA fragments were cloned with chemically competent Escherichia coli that was supplied with the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA), and plasmid DNA was isolated with the HiSpeed plasmid purification kit (Qiagen). We analyzed the plasmid by restriction analysis using EcoRI digestion based on the vector map provided by the supplier. Molecular sequence of the DNA was identified by standard DNA sequencing methods.

Northern analysis. Total RNA was extracted from day 6 cell monolayers that were either exposed to dexamethasone (100 nM) for 72 h as above or grown in MDSF (control). Five micrograms each of these RNA samples were fractionated on 1% denaturing-agarose gel and transferred to Nytran Supercharge membrane (Schleicher & Schuell, Keene, NH) by the rapid downward transfer method using the Turboblotter Kit (Schleicher & Schuell) by capillary action of the transfer solution of 3 M NaCl in 0.3 M sodium citrate (20x SSC, pH 7.0). The membrane was prehybridized for 2 h at 55°C, followed by 4 h of hybridization at 55°C with the labeled cDNA ({alpha}-subunit of rat FcRn or rat GAPDH). The {alpha}-subunit of rat FcRn cDNA fragment obtained above was purified with the Microcon PCR filter units (Millipore) and labeled with the North2South HRP labeling and detection kit (Pierce, Rockford, IL). We generated rat GAPDH cDNA fragment using two primers [forward (F): 5'-GCCAAAAGGGTCATCATCTC-3' and reverse (R): 5'-CTCAGTGTAGCCCAGGATGC-3'] with the same RT-PCR procedures used for {alpha}- and {beta}-subunits of rat FcRn gene (except for the primers) and labeled with HRP.

Hybridized membranes were washed three times with 2x SSC at 55°C for 20 min each, followed by another three washes at room temperature with 1x SSC, and finally with 0.5x SSC, all for 20 min each. These wash solutions all contain 0.1% (wt/vol) SDS. Washed membranes were then incubated with a 1:1 mixture of the peroxidase substrate and luminol enhancer for 5 min. The membrane was exposed to X-ray film (Kodak, Rochester, NY) for up to 8 h and processed for further analysis by densitometry (see below). For estimation of GAPDH level, the membrane was stripped in boiling 0.1% (wt/vol) SDS for 5 min with gentle shaking, followed by another wash in boiling 0.1% (wt/vol) SDS with shaking. The membrane was cooled to room temperature and further rinsed in 3 M NaCl in 200 mM NaH2PO4 and 20 mM EDTA (pH 7.4) for 5 min. Reprobing of the membrane with HRP-labeled GAPDH cDNA fragment was then performed for the determination of the difference in RNA loading. Densitometric analysis was performed with NIH Image Analysis software.

Statistical analysis. Data are presented as means ± SE. Unpaired Student’s t-tests were used to compare differences between two group means. For comparisons of multiple group means, one-way analysis of variance followed by Dunn’s multiple comparisons was used to contrast the difference(s). P < 0.05 is considered to be statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Figure 1 illustrates the linear relationships between amount of biot-rIgG transported into respective downstream fluids after 3, 6, and 18 h when 25 nM biot-rIgG was present in upstream apical (ab) or basolateral (ba) fluid. Unidirectional fluxes of 1.90 ± 0.20 and 0.43 ± 0.07 fmol·cm–2·h–1 can be estimated in the ab and ba directions, respectively, from steady-state rates of biot-rIgG appearing in downstream fluids. Asymmetric biot-rIgG fluxes indicate that biot-rIgG is preferentially absorbed across RAECM in the ab direction.



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Fig. 1. Cumulative biotinylated rat IgG (biot-rIgG) transported across day 6 rat alveolar epithelial cell monolayers (RAECM) as a function of time. The upstream concentration of biot-rIgG is 25 nM. ab, Apical-to-basolateral direction; ba, basolateral-to-apical direction. Data represent means ± SE (n = 6).

 
Both ab and ba transport of biot-rIgG saturate, with apparent half-maximal concentrations (Kt) of 16.0 ± 0.3 and 32.1 ± 0.4 nM in the ab and ba directions, respectively (Fig. 2). Corresponding maximal fluxes (Jmax) of 2.0 ± 0.1 and 0.7 ± 0.1 fmol·cm–2·h–1 can be noted for the ab and ba fluxes, respectively, indicating involvement of receptor-mediated processes for transport of IgG across alveolar epithelial barrier. Net absorption of IgG in the ab direction across RAECM also saturates, with corresponding Kt and Jmax of 7.1 nM and 1.3 fmol·cm–2·h–1.



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Fig. 2. Unidirectional fluxes of biot-rIgG across day 6 RAECM as a function of rIgG concentration ([rIgG]) in upstream fluid. Apparent half-maximal concentration (Kt) and maximal flux (Jmax) of 16 nM and 2.0 fmol·cm–2·h–1 for the ab (Jab) direction and 32 nM and 0.7 fmol·cm–2·h–1 for the ba (Jba) direction can be noted for the respective saturable processes responsible for IgG transport. As a result, net IgG transport (i.e., the difference in unidirectional fluxes at each concentration studied between ab and ba directions) in the ab direction also saturates with Kt of 7 nM and Jmax of 1.3 fmol·cm–2·h–1. Data represent means ± SE (n = 6).

 
We investigated the effects of various IgG-related macromolecules on receptor-mediated transport of biot-rIgG, using 100x molar excess unlabeled macromolecules (i.e., 2,500 nM) in upstream fluid that contained 25 nM biot-rIgG. As seen in Fig. 3, the presence of unlabeled rFc, but not rFab, rF(ab')2, or IgY, led to significant decreases (by ~80%) in unidirectional fluxes of biot-rIgG in both ab and ba directions. These data indicate that the Fc portion of the IgG molecule plays an important role in receptor-mediated IgG transport by competing with biot-rIgG binding to its cognate receptor. In support of this competition afforded by Fc in IgG transport, no significant differences were observed for biot-rIgG vs. biot-rFc fluxes in the ab or ba direction (Fig. 4).



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Fig. 3. Effects of excess unlabeled macromolecules on biot-rIgG fluxes across day 6 RAECM in the apical-to-basolateral and in the basolateral-to-apical directions. Biot-rIgG concentration in upstream fluid was 25 nM, and respective unlabeled macromolecules were present at 100x molar excess (i.e., 2,500 nM). * and # Significant differences compared with control in the ab and ba directions, respectively. Data represent means ± SE (n = 6).

 


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Fig. 4. Comparison of biot-rIgG flux with biot-rFc flux across day 6 RAECM. Upstream concentrations of biot-rIgG and biot-rFc were the same at 25 nM. No significant differences between respective unidirectional fluxes of IgG and Fc in the ab or ba directions were found. Data represent means ± SE (n = 6).

 
When IgG fluxes were studied as a function of day in culture, no significant changes over time were observed in either the ab or ba direction, maintaining the asymmetrical unidirectional fluxes of IgG and relatively constant net absorption (Fig. 5). In contrast to this observation, Rt of RAECM declines steadily over time, decreasing by ~50% on day 25 compared with that on day 3. These data further indicate that restricted paracellular passive diffusion of IgG does not contribute appreciably to the observed unidirectional fluxes.



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Fig. 5. Effects of days in culture on biot-rIgG flux across RAECM. Upstream concentration of biot-rIgG was the same at 25 nM for each day studied. No significant differences were noted in fluxes in ab or ba directions as functions of culture day. Data represent means ± SE (n = 6–7).

 
When experimental temperature was lowered from 37 to 4°C, significant decreases in both ab and ba fluxes of biot-rIgG were observed (Fig. 6). These drastic decreases in IgG fluxes occur in the presence of relatively small decreases in Rt (by ~16%) observed at the lower temperature. Apparent Q10 for ab and ba transport of IgG can be estimated as 1.5 and 1.8, respectively. When pH in apical fluid was lowered to <7.4 (with constant pH in basolateral fluid), no significant changes in any of the unidirectional fluxes were observed (data not shown).



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Fig. 6. Effects of temperature on biot-rIgG fluxes across day 6 RAECM. Upstream concentration of biot-rIgG was 25 nM. Significant decrements in fluxes in both ab and ba directions were noted when experimental temperature was lowered from 37 to 4°C. * and # Significant differences compared with the flux at 37°C in the ab and ba directions, respectively. Data represent means ± SE (n = 6).

 
We determined whether RAECM express the gene for an IgG receptor, FcRn, reported to be present in various epithelial/endothelial tissues using RT-PCR. As seen in Figs. 7 and 8, 542-bp and 194-bp bands expected from the F- and R-primer sets for {alpha}- and {beta}-subunits of rat FcRn gene, respectively, were detected with RNA obtained from day 6 RAECM. When the gel-purified {alpha}-subunit fragment of rat FcRn gene was ligated into the pCR II-TOPO vector and subsequently analyzed using EcoRI enzyme, an expected size of 560 bp for the fragment of the {alpha}-subunit of rat FcRn gene was found. Moreover, sequencing of the purified plasmid with the FcRn forward primer yielded 100% identical sequence to that reported for the {alpha}-subunit of rat FcRn gene. By similar approaches, we also confirmed the sequence of the {beta}-subunit of rat FcRn gene (data not shown). These data suggest that RAECM express both {alpha}-and {beta}-subunits of rat FcRn gene.



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Fig. 7. RT-PCR products for {alpha}-subunit of rat FcRn gene in day 6 RAECM. Lane 1: 1,000-bp DNA ladder; lane 2: negative control (Taq polymerase only, no reverse transcription); lane 3: RT-PCR product showing a signal at 542 bp.

 


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Fig. 8. RT-PCR products for {beta}-subunit of rat FcRn gene, {beta}2-microglobulin, in day 6 RAECM. Lane 1: 1,000-bp DNA ladder; lane 2: RT-PCR product showing a signal at 194 bp.

 
We determined the effects of dexamethasone (100 nM) on IgG transport across RAECM by measuring unidirectional fluxes and mRNA levels for FcRn. As seen in Fig. 9, for up to 24 h following exposure to dexamethasone, biot-rIgG flux in the ab direction measured with 25 nM biot-rIgG in apical upstream fluid did not change. After 48 and 72 h of exposure, ~50 and 62% decreases in the ab flux of IgG were observed. By contrast, ba flux of IgG was not affected by dexamethasone for up to 72 h of exposure. These data indicate that dexamethasone exposure of RAECM differentially regulates IgG transport in a time-dependent manner.



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Fig. 9. Effects of dexamethasone (100 nM) on IgG fluxes. Dexamethasone inhibits ab flux of biot-rIgG after 48 and 72 h only, while not affecting ba flux across RAECM. Upstream concentration of biot-rIgG was 25 nM. *Significant differences from IgG flux estimated in the ab direction at 0 h of dexamethasone exposure. Data represent means ± SE (n = 6–7).

 
Using the cDNA fragments for {alpha}-subunit of rat FcRn gene obtained from RT-PCR as above, we performed Northern analysis to determine the mRNA expression level of rat FcRn. As seen in Fig. 10, the HRP-labeled cDNA fragment of the {alpha}-subunit of the rat FcRn gene detected a signal at ~1.6-kb for RNA purified from day 6 RAECM. Another signal was also detected at ~3.1 kb (data not shown), similar to the pattern of FcRn mRNA expression previously reported in other tissues. These data confirm expression of the FcRn gene in RAECM.



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Fig. 10. Northern analysis of total RNA (5 µg) extracted from day 6 RAECM using a 542-bp fragment of {alpha}-subunit of rat FcRn cDNA as a probe. Lane 1: dexamethasone-treated (from day 3 through day 6) monolayers; lane 2: untreated (control) day 6 monolayers.

 
We also determined the mRNA levels for RAECM exposed to 100 nM dexamethasone for 72 h starting from day 3. As seen in Fig. 10, Northern analysis of RNA extracted from these monolayers showed that dexamethasone exposure led to ~41% decrease in mRNA level for the {alpha}-subunit of rat FcRn gene compared with control. On the other hand, mRNA levels for an internal control, GAPDH, did not appreciably change (data not shown). These data indicate that dexamethasone downregulates the mRNA level of the rat FcRn gene.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we demonstrate that IgG transport across primary cultured rat alveolar epithelial cell monolayers occurs via a regulable saturable (i.e., receptor-mediated) process, yielding net absorption. The saturable process appears to favor interaction of the Fc region with the cognate IgG receptor, showing no influence by other regions of IgG [including Fab or F(ab')2]. Glucocorticoids downregulate IgG transport in the ab direction only, significantly decreasing expression of the FcRn mRNA. These data are consistent with the hypothesis that net absorption of IgG across alveolar epithelium occurs via regulable transcytosis mediated by FcRn.

Saturation of IgG flux with increasing upstream [IgG] occurs via IgG binding to its cognate receptors (i.e., a carrier-mediated process). Evidence for the presence of such receptors includes inhibition by excess unlabeled Fc fragment but not by other molecules [Fab, F(ab')2, and IgY] of IgG transport in both the ab and ba directions, the presence of both {alpha}- and {beta}-subunits of rat FcRn gene transcripts by RT-PCR analyses, and confirmation by Northern analyses of the expression of mRNA for {alpha}-subunit of rat FcRn. It has been shown that FcRn lacks binding affinity for IgY (7). Moreover, other Fc receptors (e.g., Fc{gamma}RI or FcR{gamma}II) have not been reported to be expressed in any epithelium to date. The similarity of the transport rates of biot-rIgG and biot-rFc, in both the ab and ba directions, provides further evidence for FcRn mediating saturable IgG transport across alveolar epithelium.

The invariant fluxes of rIgG across monolayers in either direction at different days in culture (up to 25 days) indicate that rates of IgG transport do not change as days in culture increase (suggesting maintenance of FcRn levels) and that passive paracellular leak of IgG does not play a major role in IgG transport (since Rt of day 25 cell monolayers is only ~50% of that for cell monolayers at day 3). Because the Stokes radius of IgG is ~5.5 nm, close to the equivalent pore radius of RAECM (22), passive paracellular diffusion of IgG would be expected to be small compared with transcellular transport. We also found that alveolar epithelial IgG transport is strongly dependent on ambient temperature, with Q10 values comparable to those we reported for alveolar epithelial albumin transcytosis (20), providing further evidence for nondiffusional IgG transport.

Consistent with the functional data for receptor-mediated transport of IgG, we demonstrated that both {alpha}- and {beta}-subunits of FcRn (epithelial type-specific receptors that recognize IgG) are expressed in primary cultured pneumocytes. The mRNA (~1.6-kb) transcript we found for the {alpha}-subunit of rat FcRn is in concordance with that previously reported for rat FcRn mRNA of ~1.6 kb (29, 30) and human FcRn transcript of ~1.5 kb (7, 31, 32). A larger transcript of ~3.1 kb was also detected by Northern analysis (data not shown), which may be due to hybridization with an unidentified primary mRNA transcript of FcRn or with a partially spliced transcript. A similarly sized transcript for rat FcRn was also noted by Simister and colleagues (29, 30) in addition to the regular 1.6-kb transcript. {beta}2-Microglobulin (the {beta}-subunit of FcRn not covalently bound to {alpha}-subunit) is also expressed in primary cultured alveolar epithelial cells, providing additional evidence for the functional presence of both subunits of rat FcRn, since it was reported that IgG transport in endothelium is disrupted when {beta}2-microglobulin is knocked out (15).

The delayed effect of dexamethasone (100 nM) on IgG transport in the ab direction only after 48 and 72 h may be attributed to transcriptional or posttranscriptional effects on FcRn gene expression. We do not currently know how the differential effects of dexamethasone on IgG transport occur. Dexamethasone-induced reduction in the steady-state level for FcRn mRNA (by ~40%) is expected to significantly decrease total cellular FcRn protein, which may decrease the more rapid ab transport of IgG without affecting the slower ba transport of IgG. In other tissues, it has also been reported that mechanisms of ab transport of IgG are different from those in the ba direction (25, 26). Decreased absorption of IgG and FcRn expression in the presence of dexamethasone is in accord with a previous report in newborn rats showing disappearance of Fc receptors from enterocytes of the proximal small intestine after dexamethasone injection (11). Moreover, when young rats are exposed to corticosterone (5 mg ip for 3 days), IgG transport from the lumen of the small intestine to blood decreased markedly (27).

Studies of IgG transport in other tissues have suggested involvement of the FcRn. Small intestine of neonatal rat (30), adult human intestine (14), human placenta (34), fetal yolk sac of rats and mice (28), adult rat hepatocytes (1), adult human kidney (13), mouse endothelial cells (3), and mouse lung epithelial cells (33) all exhibit FcRn expression. Asymmetric transport of IgG across alveolar epithelium found in this study (about threefold greater ab flux over ba flux) at saturating concentrations of IgG >50 nM is remarkably pronounced. For comparison, an in vitro model of rat inner medullary collecting duct cell line transfected with rat FcRn cDNA has been reported to exhibit net absorption of iodinated human Fc (26), and absorptive IgG transport has been noted for a trophoblast cell model, the BeWo cell line, although the net absorption rates are lower than that found in alveolar epithelial cell monolayers (8, 9). IgG transport in other tissues, however, is not always absorptive. For example, an in vitro intestinal crypt T84 epithelial cell model exhibits ~3.6-fold greater ba transport of IgG compared with ab transport when studied with an upstream IgG concentration of 60 nM (7). These data suggest that rates, direction dependence, and associated mechanisms of IgG transport are tissue and/or cell specific.

In summary, we have demonstrated that IgG transport across primary cultured rat alveolar epithelial cell monolayers takes place via a saturable (i.e., receptor-mediated) process, yielding net absorption. Interaction of IgG with its cognate receptor (FcRn) is mediated by the Fc region only. Alveolar epithelial IgG transport is regulated by glucocorticoids, where only ab transport of IgG is decreased along with diminished expression of FcRn. We conclude that net absorption of IgG across alveolar epithelium occurs via regulable transcytosis mediated by FcRn. We suggest that net IgG absorption across alveolar epithelium may play important roles in alveolar homeostasis and mucosal defense of the distal respiratory epithelial tract.


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 MATERIALS AND METHODS
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This work was supported in part by National Institutes of Health Grants DE-14183, GM-12356, HL-38578, HL-38621, HL-38658, HL-62569, HL-64365, and HL-72231 and by the Hastings Foundation.


    ACKNOWLEDGMENTS
 
The authors appreciate the technical assistance of Zerlinde Balverde and Raymond Alvarez. V. H. L. Lee is Herbert Gavin Professor of Pharmaceutical Sciences. E. D. Crandall is Hastings Professor and Kenneth T. Norris Jr. Chair of Medicine.

Present address for T. E. Fandy: Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K.-J. Kim, Rm. HMR 914, Dept. of Medicine, USC Keck School of Medicine, 2011 Zonal Ave., Los Angeles, CA 90033 (E-mail: kjkim{at}usc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* K.-J. Kim and T. E. Fandy contributed equally to this work. Back


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