help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on February 25, 2003, doi:10.1164/rccm.200209-1119OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200209-1119OCv1
167/10/1374    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ferkol, T.
Right arrow Articles by Davis, P. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ferkol, T.
Right arrow Articles by Davis, P. B.
American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1374-1379, (2003)
© 2003 American Thoracic Society

Targeted Delivery of Antiprotease to the Epithelial Surface of Human Tracheal Xenografts

Thomas Ferkol, Leah A. Cohn, Thomas E. Phillips, Arnold Smith and Pamela B. Davis

Department of Pediatrics, Washington University School of Medicine, St. Louis; Department of Veterinary Medicine and Surgery, College of Veterinary Medicine; Division of Biological Sciences; Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri; and Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio

Correspondence and requests for reprints should be addressed to Dr. Thomas Ferkol, M.D., Division of Pediatric Allergy and Pulmonary Medicine, Department of Pediatrics, 660 South Euclid Avenue, Mailbox 8202, St. Louis, MO 63011. E-mail: ferkol_t{at}kids.wustl.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cystic fibrosis (CF) lung is uniquely susceptible to Pseudomonas aeruginosa, and infection with this organism incites an intense, compartmentalized inflammatory response that leads to chronic airway obstruction and bronchiectasis. Neutrophils migrate into the airway, and released neutrophil elastase contributes to the progression of the lung disease characteristic of CF. We have developed a strategy that permits the delivery of antiproteases to the inaccessible CF airways by targeting the respiratory epithelium via the human polymeric immunoglobulin receptor (hpIgR). A fusion protein consisting of a single-chain Fv directed against secretory component, the extracellular portion of the pIgR, linked to human {alpha}1-antitrypsin is effectively ferried across human tracheal xenografts and delivers the antiprotease to the apical surface to a much greater extent than occurs by passive diffusion of human {alpha}1-antitrypsin alone. Targeted antiprotease delivery paralleled hpIgR expression in the respiratory epithelium in vivo and was not increased by escalating dose, so airway penetration was receptor-dependent, not dose-dependent. Thus, this approach provides us with the ability to deliver therapeutics, like antiproteases, specifically to the lumenal surface of the respiratory epithelium, within the airway surface fluid, where it will be in highest concentration at this site.

Key Words: polymeric immunoglobulin receptor • antiprotease • human tracheal xenograft • airway • epithelium

The respiratory epithelium is the first line of defense of the airways, and many of the nonspecific defenses arise from the epithelial cells. Covering the apical surface, the periciliary fluid layer is a prominent component of the airway surface fluid (ASF) and is crucial to epithelial defenses in the airway, permitting normal cilia function (1, 2). The periciliary fluid layer contains an array of antimicrobial agents, antibacterial enzymes, and antiproteases to protect the host from pathogens (3–5). Secretory immunoglobulins are also transported into this compartment via the polymeric immunoglobulin receptor (pIgR), which is expressed on epithelial cells (6).

These epithelial defenses are breached early in the life of patients with cystic fibrosis (CF), and when bacteria or their exoproducts reach their surface, the epithelial cells direct the initial inflammatory response by releasing proinflammatory mediators. The chemoattractants enhance inflammatory cell migration into the airways, and once there, in an attempt to clear the bacteria, neutrophils release proteases that can damage the epithelium (7–9). If neutrophils remain adherent to respiratory cells, their lytic enzymes are released at the epithelial surface. Neutrophil-derived serine proteases, like neutrophil elastase, stimulate the release of proinflammatory mediators from the respiratory epithelium, and the inflammatory response is further enhanced (10). These enzymes cripple many of the innate defenses and interfere with bacterial clearance, creating the suppurative pulmonary disease characteristic of CF (11, 12).

Several strategies to interrupt this cycle have been proposed. Prevention of the escalation of the inflammatory responses engendered by the neutrophils migrating into the airway could be accomplished by preventing the action of serine proteases at the airway cell surface. However, the use of serine protease inhibitors (serpins) as treatment for CF lung disease has been difficult. Aerosolization of antiproteases, like human {alpha}1-antitrypsin (hA1AT), should permit direct delivery to the airways, but delivery to a diseased lung by this route is uneven and favors less diseased sites. Moreover, the drug is deposited atop the mucus blanket rather than the critical site at the surface of the cell (13). Systemic administration of hA1AT (14) for CF is inefficient because the airway epithelium is relatively impermeable to macromolecules, thereby preventing their diffusion across by the junctional complexes that connect adjacent epithelial cells (15).

We developed a novel strategy to circumvent these difficulties and protect the respiratory epithelial cell surface directly by exploiting properties of the pIgR. The human pIgR (hpIgR) is expressed at high levels in the airway epithelium and serous cells of the submucosal glands and is specifically adapted for the uptake and nondegradative transfer of polymeric antibodies to the lumenal surface of epithelia. We have shown previously that fusion proteins containing a single-chain Fv (scFv) antibody directed at the extracellular domain of hpIgR or human secretory component (hSC), linked to hA1AT, can be transported vectorially from the basolateral surface of cultured epithelial cells to the immediate surface where it can potentially have the greatest impact on endobronchial inflammation by blocking free proteases (16, 17). Moreover, the Fv-based fusion protein secreted in the ASF was fully active as an antiprotease. In this report, we show that such targeted agents are transported across respiratory epithelia lining human tracheal xenografts, and its transcytosis paralleled expression of hpIgR.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Tracheal Xenograft Model
As reported previously (18), tracheal xenografts were established in nude mice by subcutaneously inserting denuded rat tracheal matrix repopulated with human respiratory epithelium isolated from cadaveric airways. Xenografts were studied at least 3 weeks after seeding, and all used to collect these data were judged to be successfully repopulated with human respiratory epithelial cells on the basis of the presence of mucin in the effluent and histologic confirmation of a pseudostratified columnar epithelium at the time of removal. All animal research conformed to National Institutes of Health guidelines, and the protocol was reviewed and approved by the University of Missouri and Case Western Reserve University Institutional Animal Care Committees.

Free and Targeted A1AT
We have developed and characterized bifunctional proteins that consist of single-chain anti-hSC Fv fragments linked to a human antiprotease, thus producing a targeted hA1AT (16, 17). Free and targeted hA1AT were labeled with biotin as described by the manufacturer (Boehringer-Mannheim, Indianapolis, IN) in some experiments to permit their distinction from the endogenous human antiprotease.

Transepithelial Delivery of Targeted hA1AT
We examined the delivery of anti-SC–based fusion proteins to the epithelial surface using the human tracheal xenograft model. In initial experiments, 8 µg (103 pmol) scFv–hA1AT was injected into the tail veins of 6 mice, and 24 hours later the xenografts were washed with phosphate-buffered saline (PBS), pH 7.4. The pre- and post-treatment effluents were collected and assayed for both hSC and hA1AT using ELISA and immunoblot analysis (16, 17).

In subsequent experiments, biotinylated free and scFv–hA1AT were used to permit distinction from endogenous hA1AT secreted by human airway epithelial cells. Again, equivalent amounts of biotinylated free and targeted hA1AT were systemically administered. A low dose of biotinylated hA1AT (64 pmol) or scFv–hA1AT was injected into the tail veins of 6 mice in the first experiments, and fluid samples (6 bronchoalveolar lavage [BAL] and 12 xenograft washings) were then collected one day after treatment. An additional 6 mice underwent intravenous injections with high dose (770 pmol) labeled hA1AT and scFv–hA1AT. The next day, the mice were killed and xenograft washings and BAL fluid were obtained. Three xenografts were unable to be washed due to mucus accumulation and obliteration of the airway lumen, so effluents from only nine xenografts were collected. All of the mice survived the treatment.

Xenograft Washings, BAL, and Histologic Examination
Before and 24 hours after treatment inoculation, xenografts were flushed with 200 µl PBS, pH 7.4, and samples of the effluent were collected and stored at -70°C. BAL was performed 24 hours after treatment by cannulating the trachea, instilling 1.0 ml of sterile PBS, and collecting the fluid by gentle aspiration. The xenografts were also fixed, embedded in an epoxy resin, sectioned, counterstained with uranyl acetate and lead citrate, and examined with a JEOL 1200 EX transmission electron microscope.

Statistical Measures
All values are expressed as means and SEM. Statistical comparisons between control and the various treatment groups were made using unpaired Student's t tests. The relationships between measurable hA1AT and hSC in the xenograft effluents were examined by determining the Pearson product moment correlation coefficient.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modeling a Human Respiratory Epithelium in Xenografts
The putative binding site of SC is highly conserved among mammalian species, and the degree of sequence identity of receptor between humans, rats, and mice is greater than 80%. The monoclonal antibody that we generated against hSC, however, poorly cross-reacted with rodent receptors. We have shown previously that, compared with hSC, the addition of excess rat SC to the basolateral compartment did not inhibit transport of the fusion protein across hpIgR-bearing cells (17). We confirmed this species specificity using immunoblot analyses, which showed that the parental monoclonal anti-SC antibody effectively bound to hSC and human secretory IgA but not to rat or nonhuman primate, rhesus monkey SC (Figure 1A) . This specificity was demonstrated further by the inability of SC from other species to prevent transcytosis of the parental antibody across 16HBEo cells that overexpress hpIgR (Figure 1B). So, despite the apparent receptor homology between species, the scFv component of the fusion protein is highly specific for hSC.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 1. (A) Species specificity of the parental antisecretory component (SC) monoclonal antibody. SC was extracted and purified from human colostrum, primate (rhesus monkey) colostrum, and rat bile, then separated by denaturing sodium dodecyl sulfate–polyacrylamide gel. Western blot analyses revealed specific binding of the parental anti-SC monoclonal antibody to purified human (h) SC, but not to the rat (r) or monkey (m) receptors. Molecular weight standards (kD) are shown in the left margin. (B) Inhibition of anti-hSC transcytosis across respiratory epithelial cells that express human polymeric immunoglobulin receptor (hpIgR). The parental anti-SC antibody and increasing molar excess of SC from different species (human, primate, and rat) were added to media basolateral compartments of hpIgR-bearing 16HBEo cell monolayers grown on permeable filters. After 24 hours, the media from the opposite compartment was collected and assayed for the anti-hSC antibody by sandwich ELISA. Increasing amounts of hSC prevented the penetration of the antibody across the monolayer, whereas primate and rat SC had little effect on trancytosis.

 
We also established that human airway epithelial cells continued to express the hpIgR after treatment with antibody ligands (19). Respiratory epithelial cells grown in primary culture at an air–liquid interface spontaneously express human receptors on the basis of immunostaining for the receptor and asymmetric release of hSC into apical secretions. Increasing amounts of ligands, anti-hSC IgG or dimeric IgA, added to the basolateral medium of monolayers of bronchial epithelial cells did not inhibit epithelial release of hSC to the apical surface (Figure 2) .



View larger version (39K):
[in this window]
[in a new window]
  		Figure 2. Polymeric immunoglobulin receptor (pIgR) expression in polarized human respiratory epithelial cells in primary culture in response to ligands. Increasing, equimolar amounts of ligands anti-hSC IgG or dIgA (0.2, 2, and 20 pmol/well) were added to the basolateral medium of bronchial epithelial cell monolayers. The apical surface was washed 24 hours later, and respiratory epithelial lining fluid analyzed for the amount of hSC (open columns) by ELISA (each group, n = 4). The amount of hSC released was expressed as a percentage of nontreated, control levels. There was no difference in apical hSC release at any dose of anti-hSC IgG or dIgA applied.

 
Because the scFv only recognizes the hpIgR, it would be problematic to test the fusion proteins in vivo in a rodent model. However, we were able to examine the transport of the anti-hSC Fv–based fusion proteins from the circulation to the lumenal surface of human epithelia in tracheal xenografts. Human respiratory epithelial cells were seeded onto denuded rat tracheas and implanted subcutaneously into the flanks of recipient nu/nu mice. Morphologic evaluation of tracheal xenografts at different times after implantation in nude mice showed that the denuded rat tracheae were repopulated with human airway epithelial cells, and after 3 weeks, the surface of the rat trachea was covered by a pseudostratified columnar epithelium (Figure 3A) . The morphology of this epithelium was virtually indistinguishable from that of a normal human fully differentiated airway epithelium. Ciliated and goblet cells were present, and the appearance of the epithelium was not significantly different from native human bronchi. Using electron microscopy, ultrastructural examination of the respiratory epithelium at this time revealed the presence of junctional complexes (Figure 3B), thus indicating integrity of the barrier. Even in the absence of infection, some xenograft epithelia produced large quantities of mucus, which accumulated and occasionally obstructed in the airway lumen. The xenografts become vascularized, although the flow is a small fraction of the mouse's . Numerous capillaries, arising from murine arteries, were distributed in the lamina propria, below the respiratory epithelium and basement membrane, in the submucosa (Figure 3B).



View larger version (158K):
[in this window]
[in a new window]
  		Figure 3. (A) Electron microscopy of the human xenograft respiratory epithelium. Four weeks after seeding with human airway epithelial cells and subcutaneous implantation, xenografts were harvested from mice, fixed, sectioned, and underwent histologic examination. The rat tracheal scaffold with cartilaginous rings (r) and basement membrane (bm) has been repopulated with well differentiated, pseudostratified columnar human epithelium (e) containing mucin-filled goblet cells distributed among ciliated cells. Murine capillaries (c) are also present in the subepithelial stroma. Bar indicates 10 µ. (B) Ultrastructure of the apical surface of the xenograft respiratory epithelium, demonstrating the classic terminal bar. The zonula adherens (za) at the terminal web insertion site lies just below the closely opposed membranes of the tight junction (zonula occludens, or zo). Below the zonula adherens is a macula adherens (ma). Bar indicates 100 nm.

 
The pIgR is expressed by the human respiratory epithelium and released as hSC into the lumens of xenografts (Figure 4A) , but the apical release of hSC was highly variable (Figure 4B), with an average concentration in initial xenograft washings 0.44 ± 0.46 µg/ml, or an ASF level of 3.6 ± 3.3 µM (range, 0.5–10.3 µM). For comparison, the concentrations of hSC and sIgA in the ASF from patients with CF are 3.3 ± 2.3 nM and 14.4 ± 1.9 nM, respectively (M. Konstan and T. Ferkol, unpublished data). These values underestimate the actual concentrations in the conducting airways because a high proportion of fluid collected from BAL is from the alveoli. Conversely, the conversion of hSC concentrations in washings to concentrations in ASF measurement may be imprecise because the accumulation of large quantities of mucus in the airway lumen could interfere with our assays by further diluting the epithelial lining fluid. Nevertheless, we were able to ensure that the target receptor exists in the human tracheal xenograft, though different xenograft preparations had marked differences in hSC expression levels.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 4. (A) Immunoblot analysis for hSC in washings from five randomly selected human tracheal xenografts, demonstrating the variability of hSC secretion into airway lumen. Proteins secreted onto the apical surface of human tracheal xenografts were separated by electrophoresis in sodium dodecyl sulfate–polyacrylamide gel, transferred, and hSC was identified by Western blot hybridization. Purified hSC was used as a positive control (hSC). Molecular weight standard (kD) is shown in the left margin. (B) Concentrations of hSC in respiratory epithelial lining fluid from all human tracheal xenograft washings. The lumens of human tracheal xenografts were thoroughly washed with PBS, and 24 hours later, apical secretions were collected and the amount of hSC released was measured using ELISA. Note the variability in hSC secretion into xenograft lumen.

 
Delivery of Anti-SC Fv–based Fusion Protein In Vivo
We examined the ability of anti-hSC Fv-based fusion proteins to bind hpIgR and permit selective transport of proteins across human respiratory epithelium in tracheal xenografts. In our initial experiments, five mice underwent tail vein injections with 8 µg (103 pmol) of scFv–hA1AT, and xenograft effluents were obtained immediately before and 24 hours after infusion. The scFv–hA1AT was detectable in the postinfusion xenograft effluents. Targeted hA1AT (20.6 ± 7.8 ng/ml ASF) was measured in xenograft lining fluid using ELISA (Figure 5) , yet endogenous hA1AT was also secreted from the human respiratory epithelium into the xenograft lumen (5.9 ± 3.3 ng/ml ASF). Treatment with the scFv–hA1AT did not affect the release of hSC from the xenografts (data not shown).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 5. Delivery of the targeted, single-chain Fv (scFv)–human {alpha}1-antitrypsin (hA1AT) to the lumen of human tracheal xenografts. Six weeks after tracheal xenografts were implanted subcutaneously, scFv–hA1AT was injected into tail veins of mice, and lumenal contents of the xenografts (n = 5) were collected immediately before and 24 hours after injection. The concentration of the fusion protein in washings pre- and post-treatment was measured using ELISA. The concentration of hA1AT in xenograft washings after treatment was higher, but not significantly different, than endogenous antiprotease secreted by the human respiratory epithelium (unpaired, two-tailed Student's t test, p = 0.12)

 
In subsequent experiments, purified free and targeted hA1AT were labeled with biotin to permit their distinction from the endogenous antiprotease produced by the human epithelium. Immunoassays demonstrated that the scFv portion of the fusion protein recognizes hSC, even after biotinylation, whereas labeled hA1AT or irrelevant proteins did not (data not shown). The investigators were blinded and mice received systemic infusions with either low (64 pmol) or high (770 pmol) doses of biotinylated hA1AT or scFv–hA1AT, with equivalent molar amounts of injected into the tail veins. The xenografts were washed the next day with PBS, pH 7.4, and their lumenal contents collected. In the low-dose experiments, scFv–hA1AT was found in five of the six xenografts (408.2 ± 138.3 ng/ml washing), and the amount of scFv–hA1AT correlated with hSC, with the exception of a single outlier. In contrast, free hA1AT was below the level of detection in the effluents (Figure 6A) , and neither hA1AT or scFv–hA1AT was detected in the BAL fluid from any of the animals. Free hA1AT was measured in the xenograft washings of mice injected with the higher dose of antiprotease, but consistently more scFv–hA1AT was found in the effluents. Approximately sevenfold more scFv–hA1AT (49.7 ± 19.1 ng/ml) was detected in the xenograft washings as compared with free hA1AT (7.7 ± 3.6 ng/ml), which was relatively excluded from passage through the respiratory epithelium (Figure 6B). Curiously, the amount of targeted hA1AT measured in the xenografts of mice that received the high dose of targeted hA1AT was far less than that measured in the low dose group, but the xenografts also released much less hSC into the lumen, suggesting that there was markedly less hpIgR expression in these xenografts on the basis of hSC concentrations of the effluents. Indeed, the measured hSC concentration in xenograft washings from the low-dose experiment was 17,174 ± 27,272 ng/ml (n = 12 xenografts), whereas the level measured in the high-dose experiment was 661.2 ± 609 ng/ml (n = 10 xenografts). When detected, the concentration of the scFv–hA1AT measured in both low- and high-dose experiments correlated (r2 = 0.87) with hSC measured in the washings (Figure 7) , and the targeted hA1AT in the washings consistently amounted to 10% of measured hSC (Figure 8) . There was no such relationship between free hA1AT and hSC. Thus, lumenal delivery of the targeted antiprotease depended more on the level of hpIgR expression in the airway than on the injected dose. In contrast, diffusion of free hA1AT was dose-dependent. Finally, the concentrations of hA1AT or scFv–hA1AT in the BAL fluid of mice that received the high dose of human antiprotease were similar (7.3 and 7.7 ng/ml, respectively). Thus, the addition of a targeting scFv neither facilitated nor excluded diffusion of hA1AT into the mouse lung.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. Delivery of targeted scFv–hA1AT to the lumens of human tracheal xenografts. (A) The xenografts were washed, then low-dose biotinylated free hA1AT or scFv–hA1AT (64 pmol) was injected into the tail veins of six mice (12 xenografts total). Washings were collected the next day and analyzed for hA1AT using ELISA. Compared with free hA1AT (bhA1AT, open circles, n = 6), significantly more scFv–hA1AT (bthA1AT, filled circles, n = 6) was measured in the surface washings 24 hours after treatment (unpaired Student's t test, p = 0.02). (B) In a second experiment, six mice (12 xenografts total) underwent systemic injections with high-dose (770 pmol) biotinylated free and targeted hA1AT, and lumenal contents of the xenografts were collected 24 hours after injection. The concentration of free A1AT (bhA1AT, open circles, n = 6) and scFv–hA1AT (bthA1AT, filled circles, n = 6) in washings was measured by ELISA. Again, considerably more targeted hA1AT was delivered to the airway surface (unpaired Student's t test, p = 0.03)

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 7. The relationship between targeted scFv–hA1AT delivery and hSC release in all experiments was examined. The amount of transported free hA1AT (open symbols) and scFv–hA1AT (filled symbols) 24 hours after low-dose (circles) and high-dose (squares) injections was compared with hSC measured in apical washings. With the exception of one exceptional value, scFv–hA1AT transported correlated with hSC released.

 


View larger version (33K):
[in this window]
[in a new window]
  		Figure 8. Regardless of the dose, the targeted scFv–hA1AT (bthA1AT) in the washings consistently amounted to 10% of measured hSC, but no relationship existed between free hA1AT (bhA1AT) and hSC. The delivery of scFv–hA1AT measured in human tracheal xenograft washings was not limited by the hpIgR in the airway, as determined by the concentration of hSC washings.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown previously that recombinant fusion proteins consisting of a scFv directed against the hpIgR, linked to hA1AT, can be specifically transported across receptor-bearing cells in vitro (16, 17). In this report, using human tracheal xenografts, we extend this same observation to respiratory epithelial cells lining human tracheal xenografts. The level of hpIgR expression in the epithelium determined delivery of the targeted antiprotease, which confirms that airway penetration was receptor-dependent and not dose-dependent. Thus, this strategy provides us with the ability to deliver therapeutics, including antiproteases, specifically to the lumenal surface of the respiratory epithelium, beneath the mucus layer and within the ASF, where it will be in highest concentration at this critical site.

Airway epithelium is a barrier that prevents access of pathogens, toxins, and other noxious agents to the host. It also prevents egress of macromolecules from the host to the lumenal surface, potentially hindering delivery of systemically administered drugs to the airway. We have shown previously that there is little transcellular or paracellular passage of proteins, like hA1AT, across a respiratory epithelium in the basolateral-to-apical direction in vitro (16, 17). This barrier function is maintained by a variety of junctional complexes between epithelial cells. Desmosomes and intermediate junctions (zonula adherens) effectively maintain cell-to-cell adhesion, whereas tight junctions regulate the opening and occlusion of intercellular spaces and promote intercellular communication (20–22). We have shown that the pIgR, specifically adapted for the transcytosis of polymeric antibodies across epithelia, permits drugs to penetrate the airway epithelial barrier.

Epithelia in human tracheal xenografts also act as a barrier. Using low molecular weight tracers, the integrity of epithelia lining the implanted xenograft has been examined at the ultrastructural level. Respiratory epithelium was remarkably impermeable 4 weeks after seeding (20). The xenograft respiratory epithelia secrete copious amounts of mucus into their lumen, which did not interfere with targeted hA1AT transport. The bioelectric properties of these human tracheal xenografts are stable for months, and the well-differentiated lining epithelium act as a selective barrier to ion transport (23). Indeed, human tracheal xenografts have been used to study bioelectrical properties (24), secretions (23, 25), epithelial regeneration (26), and defenses of respiratory epithelium (18, 27, 28). This model has also been used to test molecular therapies, especially gene therapy (29–31) directed at the respiratory epithelium because one of the advantages of the tracheal xenograft model is human airway epithelium is exposed to the air environment similar to native airways. Whereas other investigators have focused on apical delivery of drugs, the studies reported here show that this model epithelium can be used as well to examine access of systemically administered therapeutics to the airway surface.

The epithelial cell is an important regulator of the inflammatory response in the conducting airway, especially in CF, and signals at its surface can profoundly alter the inflammatory milieu in the airway. Neutrophil elastase, an abundant serine protease contained within primary granules, is a potent stimulus for epithelial production of interleukin-8 (10) and induces the release of macromolecules into the airway lumen, increasing the protein content in respiratory secretions (32). For this reason, local defenses have evolved to protect the airway. Low molecular weight, antimicrobial peptides secreted by epithelial cells protect the airway from bacteria (3, 4), and secretory leukocyte protease inhibitor protects the airway surface from lytic enzymes and other noxious agents (33, 34). Our studies indicate that hA1AT is also secreted by airway epithelial cells, thus contributing to the antiprotease capacity of the ASF at the lumenal surface. Nevertheless, these defenses are overwhelmed in CF as the lung disease progresses and the proteases go uninhibited, further destroying structure and defenses of the airway. Under these circumstances, it is important to neutralize inflammatory stimuli at the epithelial surface.

By exploiting the pIgR, a receptor specifically adapted for the internalization and nondegradative transfer of polymeric antibodies, we have been able to deliver a targeted antiprotease to the epithelial surface. Once internalized, the receptor–ligand complex is transported across the cell to the apical surface, where the receptor is cleaved, releasing dIgA bound to the ectoplasmic domain of the receptor, or SC, into the lumen (35). The receptor does not require the natural ligand, which most likely interacts with domain I (36), for endocytosis, and antibodies (or fragments) directed against hSC also undergo efficient transcytosis (37). The natural ligand is not an obstacle to airway delivery because we have shown that dIgA does not interfere with binding to or transport via the hpIgR (17). In humans, the pIgR is found in airway epithelial cells that reach the lumenal surface (38). The human receptor is also expressed in cells of the submucosal glands, especially serous cells, a potentially important yet inaccessible target for CF (39). Thus, the pIgR in humans is well suited for the delivery of fusion proteins to the bronchi and bronchioles and could permit the delivery drugs to the apical surface of the respiratory epithelium after systemic administration.

As we demonstrate, accessing the hpIgR with its ligand does not downregulate transport via the receptor. Indeed, the natural ligand, dIgA, itself has been found to stimulate transcytosis though the pIgR (19). Targeting therapeutic proteins through the pIgR may have additional advantages for delivery to the CF airway because various cytokines, including TNF-{alpha}, IF-{gamma}, and interleukin-4, can increase receptor expression in the airway (40–42). The secretory component itself in bronchial secretions can be protective. The natural ligand in secretions is exposed to a hostile environment, but the binding of SC to dIgA shelters the antibody from protease degradation, enhancing its biological effectiveness (43, 44).

Although human tracheal xenografts provide a relevant and accessible target for the anti-hSC–based fusions, the model has potential limitations for modeling systemic delivery of reagents. The xenograft structure may not precisely model the spatial relationship of blood vessels, basement membrane, and epithelium that exists in the native airway. An increase in distance between the feeding capillary and epithelium will reduce delivery to the surface. In future studies, some measure of antiprotease accumulation in the interstitial space could better define this obstacle to airway delivery. Even so, in this model 10% of the hpIgR undergo transcytosis carrying an anti-hSC Fv-based fusion protein molecule.

In summary, we have developed a strategy to deliver active proteins to human tracheal xenografts by targeting the respiratory epithelial surface via the hpIgR. Even though the amount of antiprotease delivered to the apical surface might be insufficient to block the overwhelming protease burden of the late-stage CF lung, it would be protective in earlier stages of the disease. Our data indicate that there are advantages to unidirectional trafficking of proteins and delivery into the remarkably small volume of the ASF. Such an approach may yield therapeutic levels of active agent at the cell surface because our results suggest that less (about one-fourth the dose) of the targeted hA1AT would need to be administered to achieve the same level as the unmodified antiprotease at the immediate airway surface.

Therapeutic payloads of this approach are not limited to antiproteases. Indeed, the approach is generic, and this system could preferentially deliver other functional proteins, such as antiinflammatory or antibacterial agents, to the respiratory epithelial surface in the CF lung. The fusion protein reported here is a prototype for a much broader class of agents, for which the therapeutic index is improved by targeting them preferentially to the epithelial surface.


   Acknowledgments
 
The authors thank Shadi Swaidani, Frank Mularo, Sheri Miller, Jerry Chipuk, Catherine Silski, and David Fletcher for their expert technical support. They also acknowledge Michael Welsh, M.D., Philip Karp, and the University of Iowa CF Cell Culture Core (supported by NIH grant HL51670 and the Cystic Fibrosis Foundation) for their invaluable assistance to this project.


   FOOTNOTES
 
Supported by the Cystic Fibrosis Foundation and National Institutes of Health grants DK48996, HL64044, P50HL60293, and P30DK27651.

No part of this research was funded by tobacco industry sources.

The technology for this research has been licensed from Case Western Reserve University by Copernicus Therapeutics, Inc; P.D. and T.F. currently hold or have had equity in the company.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form September 30, 2002; accepted in final form February 16, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002;109:571–577.[CrossRef][Medline]
  2. Matsui H, Randell SH, Peretti SW, Davis CW, Boucher RC. Coordinated clearance of periciliary liquid and mucus from airway surfaces. J Clin Invest 1998;102:1125–1131.[Medline]
  3. McCray PB, Bentley L. Human airway epithelia express a ß-defensin. Am J Respir Cell Mol Biol 1997;16:343–349.[Abstract]
  4. Travis SM, Conway BA, Zabner J, Smith JJ, Anderson NN, Singh PK, Greenberg EP, Welsh MJ. Activity of abundant antimicrobials of the human airway. Am J Respir Cell Mol Biol 1999;20:872–879.[Abstract/Free Full Text]
  5. Rice WG, Weiss SJ. Regulation of proteolysis at the neutrophil-substrate interface by secretory leukoprotease inhibitor. Science 1990;249:178–181.[Abstract/Free Full Text]
  6. Lamm ME. Current concepts of mucosal immunity. IV: how epithelial transport of IgA antibodies relates to host defense. Am J Physiol 1998;271:G566–G571.
  7. Sommerhoff CP, Nadel JA, Basbaum CB, Caughey GH. Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells. J Clin Invest 1990;85:682–689.
  8. Breuer R, Christensen TG, Lucey EC, Bolbochan G, Stone PJ, Snider GL. Elastase causes secretory discharge in bronchi of hamsters with elastase-induced secretory cell metaplasia. Exp Lung Res 1993;19:273–282.[Medline]
  9. O'Connor CM, Gaffney K, Keane J, Southey A, Byrne N, O'Mahoney S, Fitzgerald MX. {alpha}1-Proteinase inhibitor, elastase activity, and lung disease severity in cystic fibrosis. Am Rev Respir Dis 1993;148:1665–1670.[Medline]
  10. Nakamura H, Yoshimura K, McElvaney NG, Crystal RG. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J Clin Invest 1992;89:1478–1484.
  11. Tosi MF, Zakem H, Berger M. Neutrophil elastase cleaves C3bi on opsonized Pseudomonas as well as CR1 on neutrophils to create a functionally important opsonin receptor mismatch. J Clin Invest 1990;86:300–308.
  12. Konstan MW, Berger M. Infection and inflammation of the lung in cystic fibrosis. In: Davis PB, editors. Cystic fibrosis. New York: Marcel Decker, Inc.; 1993. p. 219–276.
  13. McElvaney NG, Hubbard RC, Birrer P, Chernick MS, Caplan DB, Frank MM, Crystal RG. Aerosol {alpha}1-antitrypsin treatment of cystic fibrosis. Lancet 1991;337:392–394.[CrossRef][Medline]
  14. Wewers MD, Casolaro MA, Sellers SE, Swayse SC, McPhaul KM, Wittes JT, Crystal RG. Replacement therapy for alpha1-antitrypsin deficiency associated with emphysema. N Engl J Med 1987;316:1055–1062.[Abstract]
  15. Cereijido M, Ponce A, Gonzalez-Marical J. Tight junctions and apical-basolateral polarity. J Membr Biol 1989;110:1–9.[CrossRef]
  16. Eckman E, Mallender W, Szegletes T, Silski C, Schreiber J, Davis P, Ferkol T. In vitro transport of active alpha1-antitrypsin to the apical surface of epithelia by targeting the polymeric immunoglobulin receptor. Am J Respir Cell Mol Biol 1999;21:246–252.[Abstract/Free Full Text]
  17. Ferkol T, Eckman E, Swadani S, Silski C, Davis P. The transport of bifunctional proteins across respiratory epithelial cells via the polymeric immunoglobulin receptor. Am J Respir Crit Care Med 2000;161:944–951.[Abstract/Free Full Text]
  18. Cohn LA, Weber A, Phillips T, Lory S, Kaplan M, Smith A. Pseudomonas aeruginosa infection of respiratory epithelium in a cystic fibrosis xenograft model. J Infect Dis 2001;183:919–927.[CrossRef][Medline]
  19. Cardone MH, Smith BL, Memmitt PA, Mochely-Rosen D, Silver SB, Mostov KE. Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis. J Cell Biol 1996;133:997–1005.[Abstract/Free Full Text]
  20. Dupuit F, Gaillard D, Hinnrasky J, Mongodin E, de Bentzmann S, Copreni E, Puchelle E. Differentiated and functional human airway epithelium regeneration in tracheal xenografts. Am J Physiol Lung Cell Mol Physiol 2000;278:L165–L176.[Abstract/Free Full Text]
  21. Montefort S, Herbert CA, Robinson C, Holgate ST. The bronchial epithelium as a target for inflammatory attack in asthma. Clin Exp Allergy 1992;22:511–520.[CrossRef][Medline]
  22. Rennard SI, Romberger DJ, Sisson JH, Von Essen SG, Rubinstein I, Robbins RA, Spurzem JR. Airway epithelial cells: functional roles in airway disease. Am J Respir Crit Care Med 1994;150:S27–S30.
  23. Baconnais S, Tirouvanziam R, Zahm JM, de Bentzmann S, Peault B, Balossier G, Puchelle E. Ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts. Am J Respir Cell Mol Biol 1999;20:605–611.[Abstract/Free Full Text]
  24. Tirouvanziam R, Desternes M, Saari A, Puchelle E, Peault B, Chinet T. Bioelectric properties of human cystic fibrosis and non-cystic fibrosis fetal tracheal xenografts in SCID mice. Am J Physiol 1998;274:C875–C882.
  25. Zhang Y, Engelhardt JF. Airway surface fluid volume and Cl content in cystic fibrosis and normal bronchial xenografts. Am J Physiol 1999;276:C469–C476.
  26. Puchelle E, Peault B. Human airway xenograft models of epithelial cell regeneration. Respir Res 2000;1:125–128.[CrossRef][Medline]
  27. Goldman MJ, Anderson M, Stolzenberg ED, Kari UP, Zazloff M, Wilson JM. Human ß-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997;88:553–560.[CrossRef][Medline]
  28. Tirouvanziam R, de Bentzmann S, Hubeau C, Hinnrasky J, Jacquot J, Peault B, Puchelle E. Inflammation and infection in naive human cystic fibrosis airway grafts. Am J Respir Cell Mol Biol 2000;23:121–127.[Abstract/Free Full Text]
  29. Pilewski JM, Engelhardt JF, Bavaia JE, Kaiser LR, Wilson JM, Albelda SM. Adenovirus-mediated gene transfer to human bronchial submucosal glands using xenografts. Am J Physiol 1995;268:L657–L665.
  30. Goldman MJ, Yang Y, Wilson JM. Gene therapy in a xenograft model of cystic fibrosis lung corrects chloride transport more effectively than the sodium defect. Nat Genet 1995;9:126–131.[CrossRef][Medline]
  31. Engelhardt JF, Yang Y, Stratford-Perriaudet LD, Allen ED, Kozansky K, Perriaudet M, Yankaskas JR, Wilson JM. Direct gene transfer of human CFTR into bronchial epithelium of xenografts with E1-deleted adenoviruses. Nat Genet 1993;4:27–34.[CrossRef][Medline]
  32. Nadel JA. Role of enzymes from inflammatory cells on airway submucosal gland secretion. Respiration 1991;58:3–5.
  33. Abe T, Kobayashi N, Yoshimura K, Trapnell BC, Kim H, Hubbard RC, Brewer MT, Thompson RC, Crystal RG. Expression of the secretory leukoprotease inhibitor gene in epithelial cells. J Clin Invest 1991;87:2207–2215.
  34. Jin FY, Nathan C, Radzioch D, Ding A. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 1997;88:417–426.[CrossRef][Medline]
  35. Childers NK, Bruce MG, McGhee JR. Molecular mechanisms of immunoglobulin A defense. Annu Rev Microbiol 1989;43:503–536.[CrossRef][Medline]
  36. Bakos M, Kurosky A, Woodard CS, Denney RM, Goldblum RM. Probing the topography of free and polymeric Ig-bound human secretory component with monoclonal antibodies. J Immunol 1991;146:162–168.[Abstract]
  37. Mostov KE, Friedlander M, Blobel G. The receptor for transepithelial transport of IgA and IgM contains multiple immunoglobulin-like domains. Nature 1984;308:37–43.[CrossRef][Medline]
  38. Fiedler MA, Kaetzel CS, Davis PB. Sustained production of secretory component by primary tracheal epithelial cells in primary culture. Am J Physiol 1991;261:L255–L261.
  39. Engelhardt JF, Yankaskas JR, Ernst SA, Yang Y, Marino CR, Boucher RC, Cohn JA, Wilson JM. Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat Genet 1992;2:240–248.[CrossRef][Medline]
  40. Loman S, Radl J, Jansen HM, Out TA, Lutter R. Vectorial transcytosis of dimeric IgA by the Calu-3 human lung epithelial cell line: upregulation by IFN-{gamma}. Am J Physiol 1997;272:L951–L958.
  41. Phillips JO, Everson MP, Moldoveanu Z, Lue C, Mestecky J. Synergistic effect of IL-4 and IFN-{gamma} on the expression of polymeric Ig receptor (secretory component) and IgA binding by human epithelial cells. J Immunol 1990;145:1740–1744.[Abstract]
  42. Blanch VJ, Piskurich JF, Kaetzel CS. Coordinate regulation of IFN regulatory factor-1 and the polymeric Ig receptor by proinflammatory cytokines. J Immunol 1999;162:1232–1235.[Abstract/Free Full Text]
  43. Lindh E. Increased resistance of immunoglobulin A dimers to proteolytic degradation after binding to secretory component. J Immunol 1975;112:949–955.
  44. Brandtzaeg P. Transport models for secretory IgA and secretory IgM. Clin Exp Immunol 1981;44:221–232.[Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
R. Braathen, A. Sandvik, G. Berntzen, S. Hammerschmidt, B. Fleckenstein, I. Sandlie, P. Brandtzaeg, F.-E. Johansen, and V. Lauvrak
Identification of a Polymeric Ig Receptor Binding Phage-displayed Peptide That Exploits Epithelial Transcytosis without Dimeric IgA Competition
J. Biol. Chem., March 17, 2006; 281(11): 7075 - 7081.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
S. Gupta, M. Heacock, A. Perez, and P. B. Davis
Antibodies to the Polymeric Immunoglobulin Receptor with Different Binding and Trafficking Patterns
Am. J. Respir. Cell Mol. Biol., October 1, 2005; 33(4): 363 - 370.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. Tobin
Pediatrics, Surfactant, and Cystic Fibrosis in AJRCCM 2003
Am. J. Respir. Crit. Care Med., January 15, 2004; 169(2): 277 - 287.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200209-1119OCv1
167/10/1374    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ferkol, T.
Right arrow Articles by Davis, P. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ferkol, T.
Right arrow Articles by Davis, P. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2003 American Thoracic Society