Evidence for the role of alveolar epithelial gp60 in active transalveolar albumin transport in the rat lung

Abstract

1. Transcytosis of albumin, involving the 60 kDa albumin-binding glycoprotein, gp60, was studied in cultured type II alveolar epithelial cells obtained from rat lungs. 2. Type II cells internalized the interfacial fluorescent dye RH 414, which marks for plasmalemma vesicles. Fluorescent forms of albumin and anti-gp60 antibody colocalized in the same plasmalemma vesicles. 3. Antibody (100 microg ml(-1)) cross-linking of gp60 for brief periods (15 min) markedly stimulated vesicular uptake of fluorescently tagged albumin. The caveolar disrupting agent, filipin (10 nM), abolished the stimulated internalization of albumin. 4. The vast majority of plasmalemmal vesicles carrying albumin also immunostained for caveolin-1; however, lysosomes did not stain for caveolin-1. Filipin depleted the epithelial cells of the caveolin-1-positive, albumin-transporting plasmalemma vesicles. 5. Prolonged (> 1 h) stimulation of type II cells with cross-linking anti-gp60 antibody produced loss of cell-surface gp60 and abolished endocytic albumin uptake. 6. Transalveolar transport of albumin was also studied in the isogravimetric rat lung preparation perfused at 37 degrees C. (125)I-labelled albumin was instilled into distal airspaces of lungs, and the resulting (125)I-labelled albumin efflux into the vascular perfusate was determined. 7. Unlabelled albumin (studied over a range of 0-10 g (100 instilled ml)(-1)) inhibited 40 % of the transport of labelled albumin ((5.7 +/- 0.4) x 10(5) counts (instilled ml)(-1)) with an IC(50) value of 0.34 g (100 ml)(-1). 8. Filipin blocked the displacement-sensitive component of (125)I-labelled albumin transport, but had no effect on the transport of the paracellular tracer (3)[H]mannitol. 9. Displacement-sensitive (125)I-labelled albumin transport had a significantly greater Q(10) (27-37 degrees C) than the non-displaceable component. 10. Cross-linking of gp60 by antibody instillation stimulated only the displacement-sensitive (125)I-labelled albumin transalveolar transport in intact rat lungs. 11. To estimate the transport capacity of the displacement-sensitive system, the percentage of instilled (125)I-labelled albumin counts remaining in lung tissue was compared in lungs treated with instillates containing either 0.05 g (100 ml)(-1) unlabelled albumin or 5 g (100 ml)(-1) unlabelled albumin. Approximately 25 % of instilled (125)I-labelled albumin was cleared from the lung preparations per hour by the displacement-sensitive transport pathway. This component was blocked by filipin.

Figure 1. Identification of rat alveolar type II epithelial cells in culture

Cells were isolated, placed in cell culture for 48 h, fixed, and stained for surfactant protein C (SP-C) using anti-SP-C antibody in conjunction with rhodamine-labelled secondary Ab (see Methods). The confocal images show SP-C-containing granules (red) characteristic of type II alveolar epithelial cells. Scale bar, 10 μm.

Figure 4. Gp60 internalization and temperature-sensitive induction of endocytosis after gp60 activation in isolated rat type II alveolar epithelial cells

A, internalization of activated gp60 after warm (37 °C) incubation for 15 min in HBSS containing 10 mg ml⁻¹ albumin plus cy3-labelled anti-gp60 Ab. Cell-surface fluorescence was removed by acid washing (see Methods). Note the punctate distribution of internalized cy3 fluorescence. B-C, endocytosis marked with interfacial dye FM 1-43 with control (B) and gp60 (C) cross-linking Ab (see Methods). Note the augmentation of FM 1-43 fluorescence after gp60 cross-linking in C. D, blocking of FM 1-43 uptake by cold (4 °C) incubation in albumin (10 mg ml⁻¹)-HBSS after gp60 cross-linking. Results are representative of 3 experiments. Scale bar in D, 30 μm.

Figure 3. Albumin endocytosis in rat type II cells

A, colocalization of the styryl pyridinium dye RH 414 and Alexa 488-albumin in rat type II alveolar epithelial cells. Cells grown on glass coverslips were incubated (30 min, 37 °C) in HBSS containing 5 μg ml⁻¹ RH 414, 50 μg ml⁻¹ Alexa 488-albumin, plus 5 mg ml⁻¹ unlabelled albumin (the gp60 ligand). Cells were washed 3 times with ice-cold HBSS and the confocal images were collected 5 min after rewarming cells (37 °C). Albumin was found colocalized (yellow areas) with RH 414, which labels endocytic vesicles. B, evidence that albumin does not enter the lysosomal compartment. Cells were treated with Alexa-488-albumin, the lysosomal marker LysoTracker (50 nm), plus unlabelled albumin (5 mg ml⁻¹). Note the lack of coincidence of the two tracers in the composite. Results are representative of 3 experiments each in A and B. Scale bars, 20 μm throughout.

Figure 6. Desensitization of albumin uptake by depletion of cell-associated gp60

Rat type II alveolar epithelial cells, adhering to glass coverslips, were pretreated (2 h, 37 °C) with Hanks' balanced salt solution (HBSS) containing, as indicated, no additive, control antibody (20 μg ml⁻¹), or cross-linking anti-gp60 antibody (20 μg ml⁻¹). Cells were incubated with Alexa 488-labelled albumin (50 μg ml⁻¹) plus cy3-labelled anti-gp60 antibody (3.5 μg ml⁻¹) for 30 min and washed 3 times with cold (4 °C) HBSS to remove tracer from extracellular fluid. Live-cell images of both albumin (green) and gp60 labelling (red) were obtained with a confocal microscope (× 63 objective). Note the marked reduction in internalization of fluorescent forms of albumin and anti-gp60 (lower row) in cells that have lost gp60 staining (upper row). Results typical of 4 experiments in different cultures.

Figure 5. Filipin depletes rat type II alveolar epithelial cells of caveolin-1-positive, albumin-containing vesicles, but not lysosomes

Control or filipin-treated (10 nm, 30 min) cells were stained (37 °C, 30 min) with LysoTracker (50 nm) plus Alexa 488-labelled albumin (50 μg ml⁻¹), fixed and immunostained with anti-caveolin-1 mAb plus goat-anti-mouse Alexa 350-conjugated secondary Ab. Control cells received only the filipin vehicle DMSO. Results are representative of 3 experiments. Scale bar, 5 μm in all panels.

Figure 2. Gp60-dependent uptake of albumin in rat type II alveolar epithelial cells

A-C, effect of gp60 activation and caveolar disruption on uptake of cy3-labelled anti-gp60 antibody into rat type II alveolar epithelial cells. A, 30 min preincubation with vehicle (0.005 % DMSO). B, gp60 cross-linking as described in Methods. C, preincubation with filipin (10 nm, 30 min, 37 °C) followed by gp60 cross-linking. Note that filipin pretreatment blocked the uptake of cy3-labelled anti-gp60 antibody induced by gp60 cross-linking. D, colocalization of fluorescent forms of anti-gp60 Ab and albumin in alveolar type II epithelial cells. The cells were incubated with Alexa 488-albumin plus cy3-labelled anti-gp60 Ab (30 min, 37 °C), fixed with 4 % paraformaldehyde, treated with DAPI (1 μg ml⁻¹) for nuclear staining (blue), and mounted on glass slides. Yellow is indicative of colocalized Alexa 488 fluorescence (green) and cy3 fluorescence (red). Results are representative of 4 experiments. Scale bar, 10 μm in all panels.

Figure 8. Unidirectional transalveolar flux of instilled ¹²⁵I-labelled albumin tracer into vascular perfusate of isolated rat lung preparations

Slope of regression line (r²= 0.95) through the data points between 0 and 30 min was calculated to be 7.4 s⁻¹. Values shown are means ± 1 s.e.m. (n = 3 lung preparations).

Figure 13. Capacity of gp60-regulated albumin transport system in intact rat lung

Isogravimetric preparations received, for indicated period, the instillate containing ¹²⁵I-labelled albumin plus unlabelled albumin at 5 g (100 ml)⁻¹ (•) or 0.05 g (100 ml)⁻¹ with (▵) or without filipin (▪). Each plotted point represents an individual lung preparation. Fitted functions are regression lines. Difference between regression lines provides estimate for displaceable (i.e. gp60-regulated and filipin-sensitive) albumin transport capacity. See text for further details.

Figure 7. Stability of lung preparation undergoing double instillation

A, lung wet weight changes on instillation of 5 ml of albumin (5 g (100 ml)⁻¹)-Krebs solution (first arrow) and retained volume (≈1 ml) after draining excess instillate. On re-instillation (second arrow) and draining of remaining ≈4 ml of instillate, an additional 0.4 ml of liquid was retained. Instillates contained Evans Blue dye to mark distribution of retained liquid. B, pulmonary artery pressure (P_pa) during the experiment.

Figure 9. Effect of unlabelled albumin concentration (0-10 g (100 ml)⁻¹) in instillates on ¹²⁵I-labelled albumin permeability surface area (PS) product

*Significant difference with respect to zero unlabelled albumin (P < 0.05). Bars, ± 1 s.e.m.; n = 4-6 lung preparations. The best-fitting theoretical function was drawn through the points with an IC₅₀ value of 50 μm (maximal inhibitory effect of 40 % and Hill coefficient of 1.0).

Figure 10. The inhibitory effect of filipin (instillate concentration, 100 nm) on displaceable transalveolar ¹²⁵I-labelled albumin transport

A, absence of filipin effect on PS product of paracellular tracer ³[H]mannitol. B, filipin's inhibitory action on ¹²⁵I-labelled albumin PS at low (0.05 g (100 ml)⁻¹,) but not elevated (5 g (100 ml)⁻¹, ▪) unlabelled albumin concentration. Note that filipin affected only displaceable ¹²⁵I-labelled albumin transport. Error bars, 1 s.e.m.; n = 4-6 lung preparations. *Significant compared to basal value (P < 0.05).

Figure 11. Temperature dependency of ¹²⁵I-labelled albumin PS product

Each bar represents mean ±s.e.m. for 3 perfused lung preparations. Abscissa gives unlabelled albumin concentration in the instillate. Lowered temperature produced statistically significant reductions (*P < 0.05) for low and high unlabelled albumin concentrations. Note more marked inhibition found at 0.05 g (100 ml)⁻¹ unlabelled albumin concentration.

Figure 12. Effects of gp60 cross-linking on ¹²⁵I-labelled albumin PS product in isolated-perfused rat lungs

Note the enhancement of ¹²⁵I-labelled albumin transport at low (0.05 g (100 ml)⁻¹) unlabelled albumin concentration following gp60 cross-linking. Control instillates (□) contained control Ab plus secondary (goat anti-rabbit IgG) Abs, each at 100 μg ml⁻¹. Experimental instillates (▪) contained anti-gp60 Ab plus secondary Ab, each at 100 μg ml⁻¹. *Statistical significance vs. control (P < 0.05).