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Assessment of transport rates of proteins and peptides across primary human alveolar epithelial cell monolayers
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 196–203
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Assessment of transport rates of proteins and peptides across primary human alveolar epithelial cell monolayers Michael Bur a , Hanno Huwer b , Claus-Michael Lehr a , Nina Hagen c , Mette Guldbrandt c , Kwang-Jin Kim d , Carsten Ehrhardt a,e,∗ a
¨ Saarland University, Biopharmaceutics and Pharmaceutical Technology, 66123 Saarbrucken, Germany ¨ ¨ Department of Cardiothoracic Surgery, Volklingen Heart Centre, 66333 Volklingen, Germany c Novo Nordisk A/S, Department of Drug Metabolism, 2760 Maløv, ˚ Denmark d Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, Departments of Medicine, Physiology and Biophysics, Biomedical Engineering, and Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA 90033, USA e School of Pharmacy and Pharmaceutical Sciences, University of Dublin, Trinity College, Dublin 2, Ireland b
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Article history:
In this study, we investigated bi-directional fluxes (i.e., in absorptive and secretive direc-
Received 4 October 2005
tions) of human serum proteins [albumin (HSA), transferrin (TF), and immunoglobulin
Received in revised form 30 January
G (IgG)] and peptides/proteins of potential therapeutic relevance [insulin (INS), glucagon-
2006
like peptide-1 (GLP-1), growth hormone (GH), and parathyroid hormone (PTH)] across tight
Accepted 4 February 2006
monolayers of human alveolar epithelial cells (hAEpC) in primary culture. Apparent perme-
Published on line 14 March 2006
ability coefficients (Papp ; ×10−7 cm/s, mean ± S.D.) for GLP-1 (6.13 ± 0.87 (absorptive) versus 1.91 ± 0.51 (secretive)), HSA (2.45 ± 1.02 versus 0.21 ± 0.31), TF (0.88 ± 0.15 versus 0.30 ± 0.03),
Keywords:
and IgG (0.36 ± 0.22 versus 0.15 ± 0.16) were all strongly direction-dependent, i.e., net absorp-
Pulmonary drug absorption
tive, while PTH (2.20 ± 0.30 versus 1.80 ± 0.77), GH (8.33 ± 1.24 versus 9.02 ± 3.43), and INS
Protein absorption
(0.77 ± 0.15 versus 0.72 ± 0.36) showed no directionality. Trichloroacetic acid precipitation
Serum proteins
analysis of tested molecules collected from donor and receiver fluids exhibited very little
Human pneumocytes
degradation. This is the first study on permeability data for a range of peptides and proteins across an in vitro model of the human alveolar epithelial barrier. These data indicate that there is no apparent size-dependent transport conforming to passive restricted diffusion for the tested substances across human alveolar barrier, in part confirming net absorptive transcytosis. The obtained data differ significantly from previously published reports utilising monolayers from different species. It can be concluded that the use of homologous tissue should be preferred to avoid species differences. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Although immunocytochemical and biochemical approaches have been used to demonstrate the presence of serum proteins (e.g., albumin, transferrin, cerulo-plasmin, and immunoglob-
∗
Corresponding author. Tel.: +353 1 608 2441; fax: +353 1 608 2783. E-mail address: [email protected] (C. Ehrhardt).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.02.002
ulin G) in bronchoalveolar lavage fluid and respiratory tract of lungs of various animal species, the rates and modes for traversing of proteins across the alveolar epithelium remain an issue of debate (Kim and Malik, 2003; Hastings et al., 2004). In a related subject matter, the lung is considered as a potential
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Table 1 – Molecular weight, isoelectric point (IEP), and apparent permeability coefficient (Papp ) of tested proteins and peptides across hAEpC monolayers
GLP-1(7–37) PTH(1–38) Insulin Growth hormone Albumin Transferrin IgG
Molecular weight (Da)
IEP
Papp , a-to-b
Papp , b-to-a
3355 4458 5800 22125 65000 76500 150000
4.9 8.6 5.4 5.0 4.9 5.9 5.8–7.3
6.13 ± 0.87 (n = 12) 2.20 ± 0.30 (n = 10) 0.77 ± 0.15 (n = 8) 8.33 ± 1.24 (n = 8) 2.45 ± 1.02 (n = 10) 0.88 ± 0.15 (n = 8) 0.36 ± 0.22 (n = 8)
1.91 ± 0.51 (n = 12) 1.80 ± 0.77 (n = 11) 0.72 ± 0.36 (n = 8) 9.02 ± 3.43 (n = 12) 0.21 ± 0.31 (n = 8) 0.30 ± 0.03 (n = 9) 0.15 ± 0.16 (n = 8)
Papp , a-to-b, rat AEpC na na 0.21 ± 0.02 (n = 3) 0.041 ± 0.002 (n = 3–6) 0.77 ± 0.32 (n = 3) 1.10 ± 0.35 (n = 3) 0.91 ± 0.06 (n = 3)
Papp , b-to-a, rat AEpC na na 0.12 ± 0.02 (n = 3) 0.074 ± 0.013 (n = 3–6) 0.39 ± 0.01 (n = 3) 0.47 ± 0.02 (n = 3) 0.17 ± 0.09 (n = 3)
Data appearing in the last two columns for rat AEpC monolayers are taken from Matsukawa et al. (2000), Yamahara et al. (1994), and Bosquillon et al. (2004b). All Papp values are expressed as mean ± S.D. (cm/s) × 10−7 . The abbreviations of a-to-b and b-to-a denote the apical-to-basolateral and basolateral-to-apical directions, respectively. na: not available.
alternative route for the systemic delivery of biopharmaceuticals (i.e., proteins and peptides) (Byron and Patton, 1994; Paul et al., 2005). In this study, we investigated proteins and peptides with molecular weights (MW ) ranging from 3300 to 150,000 Da (Table 1) in bi-directional transport studies across an in vitro model of the human alveolar barrier. The molecules tested in this study are briefly described hereafter. Glucagon and related peptides constitute a family included in the proglucagon molecule, which has the identical sequence in the pancreas, intestine and brain. In gut Lcells, the C-terminal portion of proglucagon is predominantly processed to glucagon-like peptide-1 (GLP-1). Further processing produces the truncated and amidated forms of the peptide: GLP-1(1–36) amide (MW 4111 Da), GLP-1(7–36) amide (MW 3297 Da), and GLP-1(7–37) (MW 3355 Da), which all retain biological activity. Upon binding to its receptor, GLP-1 stimulates insulin secretion in a glucose-dependent manner (Richter et al., 1990). Parathyroid hormone (PTH) is secreted by the parathyroid glands and is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. It appears to be a protein containing 84 amino-acid residues, a sequence of which about 33–35 are necessary for biological activity. PTH(1–34) was reported with an absolute bioavailability of ∼34% in in vivo rat lung studies, making it a promising candidate for pulmonary drug delivery (Codrons et al., 2003, 2004). Insulin (INS) is a twochain polypeptide hormone of 5800 Da produced by the -cells of pancreatic islets. It regulates the cellular uptake, utilisation, and storage of glucose, amino acids, and fatty acids and inhibits the breakdown of glycogen, protein, and fat (Morishige et al., 1977). The human growth hormone (GH) is a member of the somatotropin/prolactin family of hormones which play an important role in growth control. The isoform I has 191 amino acid residues and a molecular weight of 22,125 Da. Action of GH is regulated upon binding to the growth hormone receptor (GHR) expressed at cytoplasmatic membranes of various cells and/or the soluble growth hormone binding protein (GHBP) (Allen et al., 2000). Albumin (HSA) is a soluble, monomeric protein which comprises about one-half of the blood serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones and plays a role in stabilising extracellular fluid volume (Kim et al., 2003). It is a globular unglycosylated serum protein of a molecular weight 65,000 Da. Albumin is synthesised in the
liver as preproalbumin which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted form of albumin. Transferrin (TF) is a glycoprotein of an approximate molecular weight of 76,500 Da. It transports iron from the intestine, reticuloendothelial system, and liver parenchymal cells to all proliferating cells in the body. In addition to its function in iron transport, this protein may also have a physiologic role as granulocyte/pollen-binding protein (GPBP) involved in the removal of certain organic matter/allergens from serum (Widera et al., 2003a). Immunoglobulin G (IgG) antibody molecules have biological properties conferred by transport across the maternal–foetal membranes, interaction with the classical complement system, and fixation to heterologous tissues via the Fc fragment of IgG. IgG molecules have a molecular weight of ∼150,000 Da (Spiekermann et al., 2002). Although we know peptides and proteins get transported across the rodent alveolar epithelium via various mechanisms, information specific to human alveolar epithelium is lacking to date (Kim and Malik, 2003). Thus, in this study, we measured in vitro permeability characteristics of a series of compounds across monolayers of primary cultured human alveolar epithelial cells (hAEpC) grown on tissue culturetreated filter inserts. These monolayers comprise alveolar epithelial type I-like cells and develop a high barrier resistance (>1000 cm2 ) (Elbert et al., 1999; Ehrhardt et al., 2005). We found that there is no apparent size-dependent transport conforming to passive restricted diffusion for serum proteins (HSA, TF, and IgG) across human alveolar barrier, in part confirming the net absorptive transcytosis of these macromolecules. Some of the investigated therapeutic proteins and peptides (PTH, GH, and INS) appear to be passively transported, while GLP-1 exhibited a significant net absorption.
2.
Materials and methods
2.1.
Peptides and proteins
Unlabelled human recombinant insulin, fluorescein isothiocyanate (FITC)-labelled human serum albumin (HSA) and FITC-labelled human immunoglobulin G were all obtained
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from Sigma (Deisenhofen, Germany). 125 I-labelled human parathyroid hormone (1–38) (PTH) and glucagon-like peptide-1 (7–37) (GLP-1) were purchased from Phoenix Pharmaceuticals (Karlsruhe, Germany) and 125 I-labelled human growth hormone (GH) and transferrin (TF) from Perkin-Elmer (Rodgau, Germany).
2.2.
Cell culture
Fresh human type II alveolar epithelial cells (hAEpC) were isolated from non-tumour lung tissue which was obtained from patients undergoing lung resection. The use of human material for isolation of primary cells was reviewed and approved by the local ethical committees (Saarland State Medical Board, Germany). Isolation of primary human type II pneumocytes was performed according to a protocol modified from those of Elbert et al. (1999) and Ehrhardt et al. (2005). Briefly, finely minced lung tissues were digested for 40 min at 37 ◦ C using a combination of 150 mg trypsin type I (Sigma, Seelze, Germany) and 0.641 mg elastase (CellSystems, St. Katharinen, Germany) in 30 ml HEPES-buffered balanced salt solution (BSS; 137 mM NaCl, 5.0 mM KCl, 0.7 mM Na2 HPO4 ·7H2 O, 10 mM HEPES (N-[2hydroxy-ethyl]piperazine-N -[2-ethanesulfonic acid]), 5.5 mM glucose, and preservatives (penicillin (100 units/ml) and streptomycin (100 g/ml)), pH 7.4). The human alveolar epithelial type II cell (ATII) population was purified from the crude cell mixture, using a combination of differential cell attachment, centrifugation with a percoll density gradient, and cell sorting with magnetic beads (anti-HEA (EpCAM) MicroBeads, Miltenyi Biotec, Bergisch Gladbach, Germany). The average yield of ATII cells was 0.8 × 106 cells/g tissue (n = 19) with a purity of >90% determined by staining cells for alkaline phosphatase. Purified ATII cells were then seeded at a density of 600,000 cells/cm2 on collagen/fibronectin coated polyester filter inserts (Transwell Clear, 6.5 mm in diameter, 0.4 m pore size, Corning, Wiesbaden, Germany) using small airways growth medium (Cambrex Bio Science, Verviers, Belgium) supplemented with penicillin (100 units/ml), streptomycin (100 g/ml), and 1% foetal calf serum. Under the chosen culture conditions, the type II pneumocytes transdifferentiate into monolayers of type I-like phenotype (Fuchs et al., 2003; Demling et al., 2006). Integrity of cell monolayers was routinely determined by measuring transepithelial electrical resistance (TEER) using an epithelial voltohmmeter (EVOM, WPI, Berlin, Germany). We used hAEpC monolayers of >1000 cm2 on days 7–9 post plating. Formation of tight junctions was also monitored by immunolabelling for a tight junctional protein, occludin.
2.3.
Transport studies
Transport experiments were conducted using hAEpC monolayers from days 7 to 9 obtained from two different isolations, when TEER values peaked. Both sides of monolayers were washed twice with pre-equilibrated bicarbonated Krebs–Ringer’s solution (KRB; 15 mM HEPES, 116.4 mM NaCl, 5.4 mM KCl, 0.78 mM NaH2 PO4 , 25 mM NaHCO3 , 1.8 mM CaCl2 , 0.81 mM MgSO4 , and 5.55 mM glucose, pH 7.4). Monolayers were then placed in new 24-well cluster plates containing 800 l per well of KRB, pre-warmed to 37 ◦ C. After 60 min of equilibration, transport experiments were initiated (i.e., t = 0)
by replacing the donor fluid with 220 l (apical) or 820 l (basolateral) of KRB containing the respective proteins or peptides to be tested for transport. The initial concentrations of test molecules in the donor fluid were assayed by drawing 20 l samples immediately after t = 0. Receiver samples (100 l) were drawn serially from the respective downstream fluids at t = 30, 60, 120, 180, and 240 min. After each sampling, fresh transport buffer of an equal volume was returned to the receiver side to maintain a constant volume. At the end of the transport experiments, 20 l samples were drawn from the donor fluids for assay. Each transport experiment was performed using four to six monolayers (obtained from two cell preparations) for flux measurements in either the apical-to-basolateral (i.e., absorptive, a-to-b) or basolateral-to-apical (i.e., secretive, bto-a) direction. In order to assess the integrity of monolayers during the flux studies, TEER was measured before and after each transport experiment. Unidirectional fluxes (J) were determined from steady-state appearance rates of each compound accumulating in the receiver fluid. The apparent permeability coefficient, Papp , is calculated according to the equation Papp =
J A × Ci
where Ci is the initial concentration of the substance under investigation in the donor fluid and A is the nominal surface area of cell monolayers (0.33 cm2 ) utilised in this study.
2.4.
Sample analysis
Samples of 125 I-labelled proteins (GLP-1, PTH, GH, and TF) were collected in scintillation vials which were mixed with 2 ml each of Ultima Gold scintillation cocktail (Perkin-Elmer). Activity of these samples was assessed with a Tri-Carb liquid scintillation counter (Perkin-Elmer). Fluorescence of FITC-labelled HSA and IgG samples was analysed in 96-well plates using a fluorescence plate reader (Cytofluor II, PerSeptive Biosystems, Wiesbaden, Germany) at excitation and emission wavelengths of 485 and 530 nm, respectively. Samples from insulin flux studies were analysed by an enzyme-linked immunosorbent assay (ELISA, Active Insulin, Diagnostic Systems Laboratories, Sinsheim, Germany) according to the manufacturer’s instructions.
2.5.
Trichloroacetic acid precipitation assay
The fraction of intact proteins labelled with either 125 I or FITC was determined by precipitation assay. Briefly, an equal volume of ice cold 20% trichloroacetic acid (TCA) was added to the respective protein samples and incubated for 30 min on ice. Subsequent to centrifugation at 14,000 × g for 15 min at 4 ◦ C, the supernatant was carefully removed, 300 l cold acetone was added to the pellet, and centrifuged again with the same settings. Supernatants were then assessed for either radioactivity or fluorescence.
2.6.
Data analyses
Data are presented as mean ± standard deviation (n), where n is the number of observations. Differences among group
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means were determined by one-way analysis of variance followed by post hoc Newman–Keuls–Student procedures and p < 0.05 taken as the level of significance.
ues were 2976 ± 1257 cm2 (absorptive) and 3336 ± 661 cm2 (secretive).
3.1.7.
3.
Results
3.1.
Transport studies
3.1.1.
Glucagon-like peptide 1
Unidirectional flux studies of GLP-1(7–37) using hAEpC monolayers from two cell preparations are shown in Fig. 1 and Table 1. Mean TEER values during flux studies were 1965 ± 589 and 1966 ± 411 cm2 for monolayers in absorptive (n = 12) and secretive (n = 12) directions, respectively. GLP1(7–37) showed a significant (p < 0.05) net absorption across the monolayers.
3.1.2.
Insulin
As seen in Fig. 1 and Table 1, insulin did not exhibit a significant (p < 0.05) asymmetry in permeability. The observed insulin Papp values were in the same order of magnitude as those reported for FITC-labelled dextran (MW 4000) across hAEpC monolayers by Elbert et al. (1999), indicating that no active transport process is involved. Cells from two different isolations were used. Mean TEER values were 1430 ± 310 cm2 for absorptive and 1220 ± 190 cm2 for secretive direction. A similar magnitude of insulin Papp with apparent degradation was reported for rat alveolar epithelial cell monolayers (Yamahara et al., 1994).
3.1.4.
Growth hormone
In our unidirectional transport studies with growth hormone, 8 monolayers of hAEpC were used for absorptive transport experiments and 12 monolayers for secretive transport experiments (Fig. 1 and Table 1). The cells were cultured from two cell isolations. Respective mean TEER values were 1702 ± 598 cm2 (absorptive) and 2243 ± 460 cm2 (secretive). GH showed no significant directionality.
3.1.5.
Albumin
Unidirectional fluxes of human serum albumin across hAEpC monolayers are shown for absorptive (n = 10) and secretive (n = 8) directions in Fig. 1 and Table 1. The monolayers were cultured from two isolations. Respective mean TEER values were 4278 ± 1779 cm2 (absorptive) and 2056 ± 535 cm2 (secretive). HSA is net absorbed (p < 0.05) at the concentration tested.
3.1.6.
Immunoglobulin G
The transport of human IgG was conducted using eight hAEpC monolayers each for absorptive and secretive directions. The monolayers were cultured from two isolations. Respective mean TEER values were 1523 ± 274 cm2 (absorptive) and 1767 ± 463 cm2 (secretive). In Fig. 1 and Table 1, Papp values for IgG transport are shown. IgG showed net absorption, albeit small, across hAEpC monolayers.
3.2.
Trichloroacetic acid precipitation assay
None of the supernatant fractions showed any appreciable activity for 125 I or FITC, indicating that the observed fluxes for these macromolecules represent intact solutes (data not shown).
Parathyroid hormone
Apparent permeability coefficients (Papp ) of PTH(1–38) using 10 hAEpC monolayers in absorptive direction and 11 monolayers in secretive direction obtained from two cell preparations are shown in Fig. 1 and Table 1. Respective mean TEER values were 2982 ± 1310 cm2 (absorptive) and 2938 ± 800 cm2 (secretive). PTH(1–38) showed no significant directionality.
3.1.3.
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Transferrin
In Fig. 1 and Table 1, net absorption (p < 0.05) of TF across hAEpC monolayers is shown. Eight monolayers for absorptive and nine for secretive transport experiments were used from two different cell preparations. Respective mean TEER val-
4.
Discussion
In this study, transport characteristics of a series of proteins and peptides across monolayers of polarised primary human alveolar epithelial cells (hAEpC) have been assessed. Permeability data of these molecules across hAEpC monolayers have not been reported to date, however, some of the compounds have been used in transport experiments in other in vitro and/or in situ models (Kim and Malik, 2003; Hastings et al., 2004; Matsukawa et al., 2000). For glucagon-like peptide-1 (7–37) and parathyroid hormone (1–38) no permeability studies are available at all, while reports on the permeability of insulin, growth hormone, serum albumin (HSA), transferrin, and immunoglobulin G are available to a certain extent. Of the investigated compounds, GLP-1, HSA, TF, and IgG showed net absorptive transport behaviour, while PTH, GH, and INS exhibited no distinct directionality. None of the compounds revealed net secretion or any significant breakdown during the flux studies. Due to the high potential of inhalational application as opposed to injections required, permeability characteristics of insulin have been widely investigated in several respiratory in vitro models. The results of these investigations, however, are not always very consistent. Pezron et al. (2002) reported net insulin secretion across Calu-3 bronchial epithelial cells, while other groups, using 16HBE14o-cell layers, observed symmetric transport (Ahsan et al., 2003; Ehrhardt, unpublished data), suggesting paracellular diffusion of insulin. As for GH transport, receptors for GH (GHR) have been reported in Caco-2 cells (Bogazzi et al., 2004), where previously an asymmetric, but P-glycoprotein inhibitor-dependent, absorption of GH was shown (Wu and Robinson, 1999). Over the last 15 years, the absorption of GH via the pulmonary route has been reported with relatively good bioavailability (8–45%) (Folkesson et al., 1992). Regional deposition as well as formulation appears to make significant impact on the absolute bioavailability of GH (Colthorpe et al., 1995; Bosquillon et al., 2004a). Non-passive (i.e., transcytotic) transport of albumin across monolayers of human intestinal epithelial Caco-2 cells
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Fig. 1 – Time courses of cumulative amount of 125 I-GLP-1(7–37), 125 I-PTH(1–38), insulin (INS), 125 I-growth hormone (GH), FITC-albumin (HSA), 125 I-transferrin (TF), and FITC-IgG in apical () or basolateral (䊉) receiver fluids of primary human alveolar epithelial cell monolayers. Each data point represents the mean ± S.D. for n = 8–12.
(Caillard and Tome, 1995), rat alveolar epithelial cells (Kim et al., 2003), and endothelial cells (John et al., 2003) has been reported. A concentration-dependent absorption of albumin was also shown for the intact rat lung (Hastings et al., 2004). Although the underlying mechanisms are slow to emerge, it appears that albumin transport is most likely dominated by the transcytosis pathway, in that albumin transport saturates
with physiological concentrations and that binding of albumin to a limited number of high-affinity sites at the caveolae on the epithelial and endothelial cell may constitute albumin transport across lung air–blood barrier (John et al., 2001, 2003; Ghinea et al., 1988). It should be noted that most experiments were conducted using serum albumin of bovine origin, and not homologous type as in our studies.
european journal of pharmaceutical sciences
Receptors for TF have been found in Caco-2 cells (Shah and Shen, 1994), human bronchial epithelial cells (Franklin et al., 1996), and rat alveolar type II epithelial cells (Widera et al., 2003b). In all cases, the receptor is mostly expressed on the basal aspect of the cells. In Calu-3 cells, TF was found to be net secreted into apical fluid (Foster et al., 2000), while significant net absorption was observed in rat alveolar epithelial cell monolayers (Matsukawa et al., 2000). Intriguingly, an enhanced absorption of TF-conjugates has been demonstrated, despite the basal localisation of the transferrin receptor in Caco-2 and rat pneumocytes (Foster et al., 2000; Widera et al., 2003c, 2004). Exactly how IgG crosses epithelial barriers to function in host defence and mucosal immunity remains unclear, although MHC class I-related Fc receptors (FcRn) are reported to be expressed at the epithelial barriers of the intestine (Shah et al., 2003), bronchi (Spiekermann et al., 2002), and alveoli (Kim et al., 2004). Transcytosis of IgG mediated by FcRn across rat alveolar epithelial cell monolayers and other barriers has been published recently (Kim et al., 2004; Sakagami et al., 2006). It should be noted that almost all proteins were transported at rates which were faster than values reported earlier from our laboratory for FITC-labelled dextrans of similar molecular weights (Elbert et al., 1999). The only exception to this general observation was insulin, whose permeability values were similarly low as those of comparably sized dextrans. It is entirely possible that enzymatic degradation, as previously reported for rat pneumocyte monolayers (Yamahara et al., 1994), might not have been detected by the ELISA method used in our study. This suggests that biologically active intact insulin is transported across the human alveolar barrier by passive sizedependent diffusion only. When permeability data were contrasted with molecular weight values, no clear cut “inverse” relation can be found
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Fig. 3 – The relation between apparent permeability coefficients (Papp ) measured across human (current study) and rat (taken from Matsukawa et al., 2000; Bosquillon et al., 2004b and Yamahara et al., 1994) alveolar epithelial cell (AEpC) monolayers in absorptive (a-to-b, 䊉) or secretive direction (b-to-a, ). The pronounced difference observed for some compounds, especially in the a-to-b direction may be attributable to species difference.
using data generated in our studies (Fig. 2). This may be related to involvement of non-passive transport processes, at least for some of the macromolecules. In general, the observed permeability values of the peptides and proteins followed the same trend and were in the same order of magnitude as those observed in an in vitro model of rat pneumocyte monolayers (Table 1; Fig. 3). Apparent differences in the absorption profile (i.e., poor correlation between human versus rat monolayer studies) might be attributable to the use of homologous proteins used in the present study, as opposed to non-homologous proteins (e.g., bovine serum albumin and human growth hormone) used in the rat monolayer studies. This study, for the first time, shows transport characteristics of a series of proteins and peptides across monolayers of polarised primary human alveolar epithelial cells. The obtained data differ significantly from previously published reports utilising monolayers from different species. It can be concluded that the use of homologous tissue should be preferred to avoid species differences.
Acknowledgments
Fig. 2 – The relation between apparent permeability coefficients (Papp ) measured across monolayers of human alveolar epithelial cells in absorptive (a-to-b, 䊉) or secretive direction (b-to-a, ) and the molecular weight (MW ) of macromolecules studied. The inferior correlation in absorptive direction may be an indication that absorption of these molecules predominantly may take place via transcellular (e.g., transcytotic), as opposed to paracellular diffusional, processes.
This work was supported by research grants (one from Novo Nordisk A/S and the others from the National Institutes of Health (HL38658 and HL64365, K.J.K.)). The authors thank for the excellent technical assistance by Ms. Susanne Kossek and Katja Klein.
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journal homepage: www.elsevier.com/locate/ejps
Assessment of transport rates of proteins and peptides across primary human alveolar epithelial cell monolayers Michael Bur a , Hanno Huwer b , Claus-Michael Lehr a , Nina Hagen c , Mette Guldbrandt c , Kwang-Jin Kim d , Carsten Ehrhardt a,e,∗ a
¨ Saarland University, Biopharmaceutics and Pharmaceutical Technology, 66123 Saarbrucken, Germany ¨ ¨ Department of Cardiothoracic Surgery, Volklingen Heart Centre, 66333 Volklingen, Germany c Novo Nordisk A/S, Department of Drug Metabolism, 2760 Maløv, ˚ Denmark d Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, Departments of Medicine, Physiology and Biophysics, Biomedical Engineering, and Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA 90033, USA e School of Pharmacy and Pharmaceutical Sciences, University of Dublin, Trinity College, Dublin 2, Ireland b
a r t i c l e
i n f o
a b s t r a c t
Article history:
In this study, we investigated bi-directional fluxes (i.e., in absorptive and secretive direc-
Received 4 October 2005
tions) of human serum proteins [albumin (HSA), transferrin (TF), and immunoglobulin
Received in revised form 30 January
G (IgG)] and peptides/proteins of potential therapeutic relevance [insulin (INS), glucagon-
2006
like peptide-1 (GLP-1), growth hormone (GH), and parathyroid hormone (PTH)] across tight
Accepted 4 February 2006
monolayers of human alveolar epithelial cells (hAEpC) in primary culture. Apparent perme-
Published on line 14 March 2006
ability coefficients (Papp ; ×10−7 cm/s, mean ± S.D.) for GLP-1 (6.13 ± 0.87 (absorptive) versus 1.91 ± 0.51 (secretive)), HSA (2.45 ± 1.02 versus 0.21 ± 0.31), TF (0.88 ± 0.15 versus 0.30 ± 0.03),
Keywords:
and IgG (0.36 ± 0.22 versus 0.15 ± 0.16) were all strongly direction-dependent, i.e., net absorp-
Pulmonary drug absorption
tive, while PTH (2.20 ± 0.30 versus 1.80 ± 0.77), GH (8.33 ± 1.24 versus 9.02 ± 3.43), and INS
Protein absorption
(0.77 ± 0.15 versus 0.72 ± 0.36) showed no directionality. Trichloroacetic acid precipitation
Serum proteins
analysis of tested molecules collected from donor and receiver fluids exhibited very little
Human pneumocytes
degradation. This is the first study on permeability data for a range of peptides and proteins across an in vitro model of the human alveolar epithelial barrier. These data indicate that there is no apparent size-dependent transport conforming to passive restricted diffusion for the tested substances across human alveolar barrier, in part confirming net absorptive transcytosis. The obtained data differ significantly from previously published reports utilising monolayers from different species. It can be concluded that the use of homologous tissue should be preferred to avoid species differences. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Although immunocytochemical and biochemical approaches have been used to demonstrate the presence of serum proteins (e.g., albumin, transferrin, cerulo-plasmin, and immunoglob-
∗
Corresponding author. Tel.: +353 1 608 2441; fax: +353 1 608 2783. E-mail address: [email protected] (C. Ehrhardt).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.02.002
ulin G) in bronchoalveolar lavage fluid and respiratory tract of lungs of various animal species, the rates and modes for traversing of proteins across the alveolar epithelium remain an issue of debate (Kim and Malik, 2003; Hastings et al., 2004). In a related subject matter, the lung is considered as a potential
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Table 1 – Molecular weight, isoelectric point (IEP), and apparent permeability coefficient (Papp ) of tested proteins and peptides across hAEpC monolayers
GLP-1(7–37) PTH(1–38) Insulin Growth hormone Albumin Transferrin IgG
Molecular weight (Da)
IEP
Papp , a-to-b
Papp , b-to-a
3355 4458 5800 22125 65000 76500 150000
4.9 8.6 5.4 5.0 4.9 5.9 5.8–7.3
6.13 ± 0.87 (n = 12) 2.20 ± 0.30 (n = 10) 0.77 ± 0.15 (n = 8) 8.33 ± 1.24 (n = 8) 2.45 ± 1.02 (n = 10) 0.88 ± 0.15 (n = 8) 0.36 ± 0.22 (n = 8)
1.91 ± 0.51 (n = 12) 1.80 ± 0.77 (n = 11) 0.72 ± 0.36 (n = 8) 9.02 ± 3.43 (n = 12) 0.21 ± 0.31 (n = 8) 0.30 ± 0.03 (n = 9) 0.15 ± 0.16 (n = 8)
Papp , a-to-b, rat AEpC na na 0.21 ± 0.02 (n = 3) 0.041 ± 0.002 (n = 3–6) 0.77 ± 0.32 (n = 3) 1.10 ± 0.35 (n = 3) 0.91 ± 0.06 (n = 3)
Papp , b-to-a, rat AEpC na na 0.12 ± 0.02 (n = 3) 0.074 ± 0.013 (n = 3–6) 0.39 ± 0.01 (n = 3) 0.47 ± 0.02 (n = 3) 0.17 ± 0.09 (n = 3)
Data appearing in the last two columns for rat AEpC monolayers are taken from Matsukawa et al. (2000), Yamahara et al. (1994), and Bosquillon et al. (2004b). All Papp values are expressed as mean ± S.D. (cm/s) × 10−7 . The abbreviations of a-to-b and b-to-a denote the apical-to-basolateral and basolateral-to-apical directions, respectively. na: not available.
alternative route for the systemic delivery of biopharmaceuticals (i.e., proteins and peptides) (Byron and Patton, 1994; Paul et al., 2005). In this study, we investigated proteins and peptides with molecular weights (MW ) ranging from 3300 to 150,000 Da (Table 1) in bi-directional transport studies across an in vitro model of the human alveolar barrier. The molecules tested in this study are briefly described hereafter. Glucagon and related peptides constitute a family included in the proglucagon molecule, which has the identical sequence in the pancreas, intestine and brain. In gut Lcells, the C-terminal portion of proglucagon is predominantly processed to glucagon-like peptide-1 (GLP-1). Further processing produces the truncated and amidated forms of the peptide: GLP-1(1–36) amide (MW 4111 Da), GLP-1(7–36) amide (MW 3297 Da), and GLP-1(7–37) (MW 3355 Da), which all retain biological activity. Upon binding to its receptor, GLP-1 stimulates insulin secretion in a glucose-dependent manner (Richter et al., 1990). Parathyroid hormone (PTH) is secreted by the parathyroid glands and is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. It appears to be a protein containing 84 amino-acid residues, a sequence of which about 33–35 are necessary for biological activity. PTH(1–34) was reported with an absolute bioavailability of ∼34% in in vivo rat lung studies, making it a promising candidate for pulmonary drug delivery (Codrons et al., 2003, 2004). Insulin (INS) is a twochain polypeptide hormone of 5800 Da produced by the -cells of pancreatic islets. It regulates the cellular uptake, utilisation, and storage of glucose, amino acids, and fatty acids and inhibits the breakdown of glycogen, protein, and fat (Morishige et al., 1977). The human growth hormone (GH) is a member of the somatotropin/prolactin family of hormones which play an important role in growth control. The isoform I has 191 amino acid residues and a molecular weight of 22,125 Da. Action of GH is regulated upon binding to the growth hormone receptor (GHR) expressed at cytoplasmatic membranes of various cells and/or the soluble growth hormone binding protein (GHBP) (Allen et al., 2000). Albumin (HSA) is a soluble, monomeric protein which comprises about one-half of the blood serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones and plays a role in stabilising extracellular fluid volume (Kim et al., 2003). It is a globular unglycosylated serum protein of a molecular weight 65,000 Da. Albumin is synthesised in the
liver as preproalbumin which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted form of albumin. Transferrin (TF) is a glycoprotein of an approximate molecular weight of 76,500 Da. It transports iron from the intestine, reticuloendothelial system, and liver parenchymal cells to all proliferating cells in the body. In addition to its function in iron transport, this protein may also have a physiologic role as granulocyte/pollen-binding protein (GPBP) involved in the removal of certain organic matter/allergens from serum (Widera et al., 2003a). Immunoglobulin G (IgG) antibody molecules have biological properties conferred by transport across the maternal–foetal membranes, interaction with the classical complement system, and fixation to heterologous tissues via the Fc fragment of IgG. IgG molecules have a molecular weight of ∼150,000 Da (Spiekermann et al., 2002). Although we know peptides and proteins get transported across the rodent alveolar epithelium via various mechanisms, information specific to human alveolar epithelium is lacking to date (Kim and Malik, 2003). Thus, in this study, we measured in vitro permeability characteristics of a series of compounds across monolayers of primary cultured human alveolar epithelial cells (hAEpC) grown on tissue culturetreated filter inserts. These monolayers comprise alveolar epithelial type I-like cells and develop a high barrier resistance (>1000 cm2 ) (Elbert et al., 1999; Ehrhardt et al., 2005). We found that there is no apparent size-dependent transport conforming to passive restricted diffusion for serum proteins (HSA, TF, and IgG) across human alveolar barrier, in part confirming the net absorptive transcytosis of these macromolecules. Some of the investigated therapeutic proteins and peptides (PTH, GH, and INS) appear to be passively transported, while GLP-1 exhibited a significant net absorption.
2.
Materials and methods
2.1.
Peptides and proteins
Unlabelled human recombinant insulin, fluorescein isothiocyanate (FITC)-labelled human serum albumin (HSA) and FITC-labelled human immunoglobulin G were all obtained
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from Sigma (Deisenhofen, Germany). 125 I-labelled human parathyroid hormone (1–38) (PTH) and glucagon-like peptide-1 (7–37) (GLP-1) were purchased from Phoenix Pharmaceuticals (Karlsruhe, Germany) and 125 I-labelled human growth hormone (GH) and transferrin (TF) from Perkin-Elmer (Rodgau, Germany).
2.2.
Cell culture
Fresh human type II alveolar epithelial cells (hAEpC) were isolated from non-tumour lung tissue which was obtained from patients undergoing lung resection. The use of human material for isolation of primary cells was reviewed and approved by the local ethical committees (Saarland State Medical Board, Germany). Isolation of primary human type II pneumocytes was performed according to a protocol modified from those of Elbert et al. (1999) and Ehrhardt et al. (2005). Briefly, finely minced lung tissues were digested for 40 min at 37 ◦ C using a combination of 150 mg trypsin type I (Sigma, Seelze, Germany) and 0.641 mg elastase (CellSystems, St. Katharinen, Germany) in 30 ml HEPES-buffered balanced salt solution (BSS; 137 mM NaCl, 5.0 mM KCl, 0.7 mM Na2 HPO4 ·7H2 O, 10 mM HEPES (N-[2hydroxy-ethyl]piperazine-N -[2-ethanesulfonic acid]), 5.5 mM glucose, and preservatives (penicillin (100 units/ml) and streptomycin (100 g/ml)), pH 7.4). The human alveolar epithelial type II cell (ATII) population was purified from the crude cell mixture, using a combination of differential cell attachment, centrifugation with a percoll density gradient, and cell sorting with magnetic beads (anti-HEA (EpCAM) MicroBeads, Miltenyi Biotec, Bergisch Gladbach, Germany). The average yield of ATII cells was 0.8 × 106 cells/g tissue (n = 19) with a purity of >90% determined by staining cells for alkaline phosphatase. Purified ATII cells were then seeded at a density of 600,000 cells/cm2 on collagen/fibronectin coated polyester filter inserts (Transwell Clear, 6.5 mm in diameter, 0.4 m pore size, Corning, Wiesbaden, Germany) using small airways growth medium (Cambrex Bio Science, Verviers, Belgium) supplemented with penicillin (100 units/ml), streptomycin (100 g/ml), and 1% foetal calf serum. Under the chosen culture conditions, the type II pneumocytes transdifferentiate into monolayers of type I-like phenotype (Fuchs et al., 2003; Demling et al., 2006). Integrity of cell monolayers was routinely determined by measuring transepithelial electrical resistance (TEER) using an epithelial voltohmmeter (EVOM, WPI, Berlin, Germany). We used hAEpC monolayers of >1000 cm2 on days 7–9 post plating. Formation of tight junctions was also monitored by immunolabelling for a tight junctional protein, occludin.
2.3.
Transport studies
Transport experiments were conducted using hAEpC monolayers from days 7 to 9 obtained from two different isolations, when TEER values peaked. Both sides of monolayers were washed twice with pre-equilibrated bicarbonated Krebs–Ringer’s solution (KRB; 15 mM HEPES, 116.4 mM NaCl, 5.4 mM KCl, 0.78 mM NaH2 PO4 , 25 mM NaHCO3 , 1.8 mM CaCl2 , 0.81 mM MgSO4 , and 5.55 mM glucose, pH 7.4). Monolayers were then placed in new 24-well cluster plates containing 800 l per well of KRB, pre-warmed to 37 ◦ C. After 60 min of equilibration, transport experiments were initiated (i.e., t = 0)
by replacing the donor fluid with 220 l (apical) or 820 l (basolateral) of KRB containing the respective proteins or peptides to be tested for transport. The initial concentrations of test molecules in the donor fluid were assayed by drawing 20 l samples immediately after t = 0. Receiver samples (100 l) were drawn serially from the respective downstream fluids at t = 30, 60, 120, 180, and 240 min. After each sampling, fresh transport buffer of an equal volume was returned to the receiver side to maintain a constant volume. At the end of the transport experiments, 20 l samples were drawn from the donor fluids for assay. Each transport experiment was performed using four to six monolayers (obtained from two cell preparations) for flux measurements in either the apical-to-basolateral (i.e., absorptive, a-to-b) or basolateral-to-apical (i.e., secretive, bto-a) direction. In order to assess the integrity of monolayers during the flux studies, TEER was measured before and after each transport experiment. Unidirectional fluxes (J) were determined from steady-state appearance rates of each compound accumulating in the receiver fluid. The apparent permeability coefficient, Papp , is calculated according to the equation Papp =
J A × Ci
where Ci is the initial concentration of the substance under investigation in the donor fluid and A is the nominal surface area of cell monolayers (0.33 cm2 ) utilised in this study.
2.4.
Sample analysis
Samples of 125 I-labelled proteins (GLP-1, PTH, GH, and TF) were collected in scintillation vials which were mixed with 2 ml each of Ultima Gold scintillation cocktail (Perkin-Elmer). Activity of these samples was assessed with a Tri-Carb liquid scintillation counter (Perkin-Elmer). Fluorescence of FITC-labelled HSA and IgG samples was analysed in 96-well plates using a fluorescence plate reader (Cytofluor II, PerSeptive Biosystems, Wiesbaden, Germany) at excitation and emission wavelengths of 485 and 530 nm, respectively. Samples from insulin flux studies were analysed by an enzyme-linked immunosorbent assay (ELISA, Active Insulin, Diagnostic Systems Laboratories, Sinsheim, Germany) according to the manufacturer’s instructions.
2.5.
Trichloroacetic acid precipitation assay
The fraction of intact proteins labelled with either 125 I or FITC was determined by precipitation assay. Briefly, an equal volume of ice cold 20% trichloroacetic acid (TCA) was added to the respective protein samples and incubated for 30 min on ice. Subsequent to centrifugation at 14,000 × g for 15 min at 4 ◦ C, the supernatant was carefully removed, 300 l cold acetone was added to the pellet, and centrifuged again with the same settings. Supernatants were then assessed for either radioactivity or fluorescence.
2.6.
Data analyses
Data are presented as mean ± standard deviation (n), where n is the number of observations. Differences among group
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 196–203
means were determined by one-way analysis of variance followed by post hoc Newman–Keuls–Student procedures and p < 0.05 taken as the level of significance.
ues were 2976 ± 1257 cm2 (absorptive) and 3336 ± 661 cm2 (secretive).
3.1.7.
3.
Results
3.1.
Transport studies
3.1.1.
Glucagon-like peptide 1
Unidirectional flux studies of GLP-1(7–37) using hAEpC monolayers from two cell preparations are shown in Fig. 1 and Table 1. Mean TEER values during flux studies were 1965 ± 589 and 1966 ± 411 cm2 for monolayers in absorptive (n = 12) and secretive (n = 12) directions, respectively. GLP1(7–37) showed a significant (p < 0.05) net absorption across the monolayers.
3.1.2.
Insulin
As seen in Fig. 1 and Table 1, insulin did not exhibit a significant (p < 0.05) asymmetry in permeability. The observed insulin Papp values were in the same order of magnitude as those reported for FITC-labelled dextran (MW 4000) across hAEpC monolayers by Elbert et al. (1999), indicating that no active transport process is involved. Cells from two different isolations were used. Mean TEER values were 1430 ± 310 cm2 for absorptive and 1220 ± 190 cm2 for secretive direction. A similar magnitude of insulin Papp with apparent degradation was reported for rat alveolar epithelial cell monolayers (Yamahara et al., 1994).
3.1.4.
Growth hormone
In our unidirectional transport studies with growth hormone, 8 monolayers of hAEpC were used for absorptive transport experiments and 12 monolayers for secretive transport experiments (Fig. 1 and Table 1). The cells were cultured from two cell isolations. Respective mean TEER values were 1702 ± 598 cm2 (absorptive) and 2243 ± 460 cm2 (secretive). GH showed no significant directionality.
3.1.5.
Albumin
Unidirectional fluxes of human serum albumin across hAEpC monolayers are shown for absorptive (n = 10) and secretive (n = 8) directions in Fig. 1 and Table 1. The monolayers were cultured from two isolations. Respective mean TEER values were 4278 ± 1779 cm2 (absorptive) and 2056 ± 535 cm2 (secretive). HSA is net absorbed (p < 0.05) at the concentration tested.
3.1.6.
Immunoglobulin G
The transport of human IgG was conducted using eight hAEpC monolayers each for absorptive and secretive directions. The monolayers were cultured from two isolations. Respective mean TEER values were 1523 ± 274 cm2 (absorptive) and 1767 ± 463 cm2 (secretive). In Fig. 1 and Table 1, Papp values for IgG transport are shown. IgG showed net absorption, albeit small, across hAEpC monolayers.
3.2.
Trichloroacetic acid precipitation assay
None of the supernatant fractions showed any appreciable activity for 125 I or FITC, indicating that the observed fluxes for these macromolecules represent intact solutes (data not shown).
Parathyroid hormone
Apparent permeability coefficients (Papp ) of PTH(1–38) using 10 hAEpC monolayers in absorptive direction and 11 monolayers in secretive direction obtained from two cell preparations are shown in Fig. 1 and Table 1. Respective mean TEER values were 2982 ± 1310 cm2 (absorptive) and 2938 ± 800 cm2 (secretive). PTH(1–38) showed no significant directionality.
3.1.3.
199
Transferrin
In Fig. 1 and Table 1, net absorption (p < 0.05) of TF across hAEpC monolayers is shown. Eight monolayers for absorptive and nine for secretive transport experiments were used from two different cell preparations. Respective mean TEER val-
4.
Discussion
In this study, transport characteristics of a series of proteins and peptides across monolayers of polarised primary human alveolar epithelial cells (hAEpC) have been assessed. Permeability data of these molecules across hAEpC monolayers have not been reported to date, however, some of the compounds have been used in transport experiments in other in vitro and/or in situ models (Kim and Malik, 2003; Hastings et al., 2004; Matsukawa et al., 2000). For glucagon-like peptide-1 (7–37) and parathyroid hormone (1–38) no permeability studies are available at all, while reports on the permeability of insulin, growth hormone, serum albumin (HSA), transferrin, and immunoglobulin G are available to a certain extent. Of the investigated compounds, GLP-1, HSA, TF, and IgG showed net absorptive transport behaviour, while PTH, GH, and INS exhibited no distinct directionality. None of the compounds revealed net secretion or any significant breakdown during the flux studies. Due to the high potential of inhalational application as opposed to injections required, permeability characteristics of insulin have been widely investigated in several respiratory in vitro models. The results of these investigations, however, are not always very consistent. Pezron et al. (2002) reported net insulin secretion across Calu-3 bronchial epithelial cells, while other groups, using 16HBE14o-cell layers, observed symmetric transport (Ahsan et al., 2003; Ehrhardt, unpublished data), suggesting paracellular diffusion of insulin. As for GH transport, receptors for GH (GHR) have been reported in Caco-2 cells (Bogazzi et al., 2004), where previously an asymmetric, but P-glycoprotein inhibitor-dependent, absorption of GH was shown (Wu and Robinson, 1999). Over the last 15 years, the absorption of GH via the pulmonary route has been reported with relatively good bioavailability (8–45%) (Folkesson et al., 1992). Regional deposition as well as formulation appears to make significant impact on the absolute bioavailability of GH (Colthorpe et al., 1995; Bosquillon et al., 2004a). Non-passive (i.e., transcytotic) transport of albumin across monolayers of human intestinal epithelial Caco-2 cells
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Fig. 1 – Time courses of cumulative amount of 125 I-GLP-1(7–37), 125 I-PTH(1–38), insulin (INS), 125 I-growth hormone (GH), FITC-albumin (HSA), 125 I-transferrin (TF), and FITC-IgG in apical () or basolateral (䊉) receiver fluids of primary human alveolar epithelial cell monolayers. Each data point represents the mean ± S.D. for n = 8–12.
(Caillard and Tome, 1995), rat alveolar epithelial cells (Kim et al., 2003), and endothelial cells (John et al., 2003) has been reported. A concentration-dependent absorption of albumin was also shown for the intact rat lung (Hastings et al., 2004). Although the underlying mechanisms are slow to emerge, it appears that albumin transport is most likely dominated by the transcytosis pathway, in that albumin transport saturates
with physiological concentrations and that binding of albumin to a limited number of high-affinity sites at the caveolae on the epithelial and endothelial cell may constitute albumin transport across lung air–blood barrier (John et al., 2001, 2003; Ghinea et al., 1988). It should be noted that most experiments were conducted using serum albumin of bovine origin, and not homologous type as in our studies.
european journal of pharmaceutical sciences
Receptors for TF have been found in Caco-2 cells (Shah and Shen, 1994), human bronchial epithelial cells (Franklin et al., 1996), and rat alveolar type II epithelial cells (Widera et al., 2003b). In all cases, the receptor is mostly expressed on the basal aspect of the cells. In Calu-3 cells, TF was found to be net secreted into apical fluid (Foster et al., 2000), while significant net absorption was observed in rat alveolar epithelial cell monolayers (Matsukawa et al., 2000). Intriguingly, an enhanced absorption of TF-conjugates has been demonstrated, despite the basal localisation of the transferrin receptor in Caco-2 and rat pneumocytes (Foster et al., 2000; Widera et al., 2003c, 2004). Exactly how IgG crosses epithelial barriers to function in host defence and mucosal immunity remains unclear, although MHC class I-related Fc receptors (FcRn) are reported to be expressed at the epithelial barriers of the intestine (Shah et al., 2003), bronchi (Spiekermann et al., 2002), and alveoli (Kim et al., 2004). Transcytosis of IgG mediated by FcRn across rat alveolar epithelial cell monolayers and other barriers has been published recently (Kim et al., 2004; Sakagami et al., 2006). It should be noted that almost all proteins were transported at rates which were faster than values reported earlier from our laboratory for FITC-labelled dextrans of similar molecular weights (Elbert et al., 1999). The only exception to this general observation was insulin, whose permeability values were similarly low as those of comparably sized dextrans. It is entirely possible that enzymatic degradation, as previously reported for rat pneumocyte monolayers (Yamahara et al., 1994), might not have been detected by the ELISA method used in our study. This suggests that biologically active intact insulin is transported across the human alveolar barrier by passive sizedependent diffusion only. When permeability data were contrasted with molecular weight values, no clear cut “inverse” relation can be found
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Fig. 3 – The relation between apparent permeability coefficients (Papp ) measured across human (current study) and rat (taken from Matsukawa et al., 2000; Bosquillon et al., 2004b and Yamahara et al., 1994) alveolar epithelial cell (AEpC) monolayers in absorptive (a-to-b, 䊉) or secretive direction (b-to-a, ). The pronounced difference observed for some compounds, especially in the a-to-b direction may be attributable to species difference.
using data generated in our studies (Fig. 2). This may be related to involvement of non-passive transport processes, at least for some of the macromolecules. In general, the observed permeability values of the peptides and proteins followed the same trend and were in the same order of magnitude as those observed in an in vitro model of rat pneumocyte monolayers (Table 1; Fig. 3). Apparent differences in the absorption profile (i.e., poor correlation between human versus rat monolayer studies) might be attributable to the use of homologous proteins used in the present study, as opposed to non-homologous proteins (e.g., bovine serum albumin and human growth hormone) used in the rat monolayer studies. This study, for the first time, shows transport characteristics of a series of proteins and peptides across monolayers of polarised primary human alveolar epithelial cells. The obtained data differ significantly from previously published reports utilising monolayers from different species. It can be concluded that the use of homologous tissue should be preferred to avoid species differences.
Acknowledgments
Fig. 2 – The relation between apparent permeability coefficients (Papp ) measured across monolayers of human alveolar epithelial cells in absorptive (a-to-b, 䊉) or secretive direction (b-to-a, ) and the molecular weight (MW ) of macromolecules studied. The inferior correlation in absorptive direction may be an indication that absorption of these molecules predominantly may take place via transcellular (e.g., transcytotic), as opposed to paracellular diffusional, processes.
This work was supported by research grants (one from Novo Nordisk A/S and the others from the National Institutes of Health (HL38658 and HL64365, K.J.K.)). The authors thank for the excellent technical assistance by Ms. Susanne Kossek and Katja Klein.
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