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(Journal of Leukocyte Biology. 2000;68:201-208.)
© 2000 by Society for Leukocyte Biology

Trafficking of neutrophils across airway epithelium is dependent upon both thioredoxin- and pertussis toxin-sensitive signaling mechanisms

Lisa A. Miller*, Jodie Usachenko*,{dagger}, Ruth J. McDonald{dagger} and Dallas M. Hyde*

* The Center for Comparative Respiratory Biology and Medicine and Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, and
{dagger} Department of Pediatrics, School of Medicine, University of California, Davis

Correspondence: Lisa A. Miller, Ph.D., Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616. E-mail: lmiller{at}ucdavis.edu


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ABSTRACT
 
Leukocyte recruitment from the circulation into the airways is a multi-step process, involving both chemotactic and adhesive mechanisms. Using an in vitro model of leukocyte transepithelial trafficking, we show that movement of human peripheral blood neutrophils (PMN) across airway epithelium in the optimal basolateral-to-apical surface direction is partially blocked by pertussis toxin, an inhibitor of G{alpha}i-protein-linked receptors. A neutralizing monoclonal antibody against interleukin-8 (IL-8; constitutively expressed by airway epithelium) did not inhibit PMN transepithelial migration, suggesting that alternative pertussis toxin-sensitive signaling mechanisms are involved in this process. However, a neutralizing antibody against thioredoxin, a redox enzyme with pertussis toxin-insensitive chemoattractant activity, did reduce PMN migration across airway epithelium. We conclude that trafficking of PMN across airway epithelium is mediated by both thioredoxin- and pertussis toxin-sensitive signaling mechanisms that are independent of IL-8.

Key Words: lung • chemokine • transepithelial


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INTRODUCTION
 
Neutrophils (PMN) are the predominant inflammatory cell in a number of pulmonary disease states, including cystic fibrosis and adult respiratory distress syndrome [1 ]. Elevated counts of PMN are typically observed within the respiratory tract lumen at the onset of an inflammatory response, implicating an important role for this leukocyte population as a host defense mechanism in the lung. However, excessive or inappropriate PMN accumulation within the airways and gas-exchanging surfaces of the lung may lead to injury of epithelia and compromise lung function [2 3 4 ]. As such, an understanding of the mechanisms that mediate PMN trafficking within the lung is of important therapeutic value.

To localize within the lumen of the conducting airways, circulating PMN must first diapedese through endothelium and migrate across a barrier of connective tissue and epithelium. Shear forces of the systemic circulation restrict PMN interactions with the endothelial cell walls of the vasculature under normal conditions. At sites of inflammation, extravasation of PMN is mediated by both rapid, transient (selectin/carbohydrate-dependent), and sustained (integrin-dependent) adhesive interactions with activated endothelium [5 6 7 ]. The shear forces that limit peripheral blood PMN rolling interactions with unactivated endothelia are not a factor in PMN migration across epithelia. This would suggest that adhesive mechanisms that regulate transepithelial migration are limited to integrin-mediated events; however, it has been demonstrated that certain carbohydrates are important for polarity-independent movement of PMN across intestinal epithelium [8 ]. Integrin-mediated PMN interactions with endothelia do appear to be distinct from those with epithelia; transendothelial emigration is CD11a/CD18 (LFA-1) dependent [9 ], whereas transmigration of neutrophils across epithelium is CD11b/CD18 (Mac-1) dependent [10 , 11 ].

It is likely that PMN accumulation at sites of inflammation is also dependent upon locally produced chemoattractants and factors that promote random motility. In vitro studies have implicated a role for the chemokine family of small-molecular-weight inflammatory cytokines in the directed recruitment and activation of leukocytes [12 , 13 ]. Airway epithelial cells can express chemokines (particularly in response to proinflammatory mediators) for mononuclear cells and granulocytes, indicating that the epithelial lining of the lung may play an active role in the orchestration of leukocyte trafficking to sites of inflammation. Human airway epithelial cell-derived chemokines for PMN, including interleukin-8 (IL-8) and growth-related oncogene {alpha} (GRO{alpha}), have been detected at the mRNA level [14 , 15 ]. Elevated levels of IL-8 and GRO{alpha} protein within bronchoalveolar lavage fluids have been correlated with airway disease and may be attributed to enhanced chemokine expression by lung epithelium [16 17 18 ]. Airway epithelial cells also secrete chemoattractants that are not categorized as chemokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and platelet-activating factor (PAF) [19 ]. It has been recently demonstrated that thioredoxin, an intracellular redox enzyme expressed by many cell types, including airway epithelia, can have potent leukocyte chemoattractant activity [20 , 21 ]. As yet, the functional role of thioredoxin within airway epithelium, particularly with respect to inflammation, has not been established.

We have recently described an in vitro model for leukocyte transepithelial trafficking into the airways and characterized human peripheral blood mononuclear cell (PBMC) migration across monolayers of human airway epithelium [22 ]. The objective of this study was to investigate PMN transmigration across airway epithelium in a comparative fashion, in order to distinguish differences in epithelial trafficking mechanisms between PMN versus PBMC. Cumulatively, our findings suggest that the epithelium of the lung plays an important regulatory role in the selective recruitment of leukocyte populations into the airways.


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MATERIALS AND METHODS
 
Reagents
Pertussis toxin was obtained from List Biological Laboratories (Campbell, CA). A noncatalytic mutant pertussis toxin (PT9K 129G) was a kind gift from Dr. Rino Rappuoli (Chiron Vaccines, Siena, Italy) [23 ]. A function blocking monoclonal antibody (mAb) against human IL-8 (mouse IgG1) was purchased from R & D Systems (Minneapolis, MN). Purified mouse immunoglobulin controls were obtained from PharMingen (San Diego, CA). Purified recombinant human IL-8 was purchased from Genzyme Diagnostics (Cambridge, MA). Goat anti-human thioredoxin neutralizing antibody was obtained from American Diagnostica (Greenwich, CT). Purified goat immunoglobulin controls were obtained from Chemicon (Temecula, CA).

Cell culture
Monolayers of airway epithelium were cultured from the BEAS-2B S.6 cell line, a gift from Dr. Curtis Harris (Bethesda, MD). The BEAS-2B S.6 cell line was derived from normal human bronchial epithelial cells immortalized using an SV40-adenovirus 12 hybrid virus [24 ]. Cultures of BEAS-2B S.6 cells can develop significant transepithelial resistance and exhibit tight junction formation [25 ]. BEAS-2B S.6 cells were cultured in a serum-free bronchial epithelial growth medium (BEGM; Clonetics, San Diego, CA). For migration assays, 3 to 4 x 103 BEAS-2B S.6 cells were plated onto polycarbonate membranes of Transwell cell culture inserts (3-µm pore size, 6.5-mm diameter; Costar, Cambridge, MA). Depending on the orientation required for an experiment, cells were plated on either the top of membranes (apical surface up) or the bottom of membranes by inversion of Transwell inserts (apical surface down). Cells on inverted Transwell inserts were allowed to settle for 12 h; inserts were then flipped and placed into 24-well plates holding BEGM. Airway epithelial cell monolayers were cultured until confluent (~7 days) before migration experiments. Complete confluency of monolayers was determined by phase-contrast microscopy and confirmed by measurement of transepithelial resistance using a voltmeter equipped with hand-held Ag/AgCl electrodes as previously described [26 ]. At maximal confluence, monolayers achieved a maximal resistance of approximately 100 ohm/cm2, a value consistent for intermediate passage BEAS-2B S.6 cells [25 ]. The total number of epithelial cells per Transwell unit at confluency was determined by incubation of inserts in Hanks’ balanced salt solution (HBSS) containing 0.25% trypsin (Clonetics) at 37°C in a 5% CO2 incubator until all cells were detached (15–20 min). Detached epithelial cells were counted by a standard hemocytometer, using a standard protocol as described previously [27 ].

PMN transepithelial migration assay
PMN were purified from peripheral blood from healthy human volunteers by dextran sedimentation and density gradient centrifugation; preparations were used within 1 h of isolation [28 ]. Viability of PMN was determined for each preparation by measurement of phorbol myristate acetate-stimulated hydrogen peroxide production. Approximately 1 h before initiation of migration assays, both upper and lower chambers of Transwell inserts containing epithelial cell monolayers were changed to fresh, preequilibrated/prewarmed (37°C) BEGM. Just before initiation of migration assays, PMN were resuspended in preequilibrated/prewarmed BEGM at a concentration of 3 to 5 x 106 cells/mL. For some experiments, HBSS with 1% bovine serum albumin (BSA) was substituted for BEGM in all of the aforementioned steps. Unless otherwise indicated, PMN were added directly to upper chambers of Transwell inserts at a concentration of 3 to 5 x 105 cells/0.1 mL/insert. Transwell inserts were incubated at 37°C in 5% CO2 for 2 h. Transmigration was quantified by collection of PMN from lower chambers and counting with a standard hemocytometer. Calculation of PMN numbers was based on a standard protocol described previously [27 ].

Pertussis toxin/neutralizing antibody experiments
For experiments utilizing pertussis toxin, PMN were isolated as described above and suspended in RPMI 1640 supplemented with 10% fetal calf serum at a concentration of 5 x 106 cells/mL. PMN were incubated with either pertussis toxin or mutant pertussis toxin (100 ng/mL) for 2 h at 37°C in 10% CO2 as previously described [29 ]. After treatment with either pertussis toxin or mutant pertussis toxin, PMN were washed twice with HBSS and resuspended in preequilibrated/prewarmed BEGM as described above.

For neutralization experiments utilizing function-blocking antibodies against IL-8 or thioredoxin, epithelial cell monolayers were preincubated with either 1 or 5 µg/mL IgG for 1 h at 37°C in 5% CO2 before addition of PMN to Transwell inserts. Appropriate nonspecific mouse or goat immunoglobulins were utilized as controls. In addition to testing by the manufacturer, the IL-8 neutralizing antibody was independently tested at 1 µg/mL and confirmed for biological activity (inhibition of migration) in 2-h PMN migration assays across naked Transwell inserts to 0.1 µg/mL recombinant human IL-8 in BEGM (data not shown).

PMN migration to conditioned medium
Twenty-four-hour conditioned media were collected from apical surfaces of confluent epithelial cell Transwell cultures (cultured on inverted surfaces) immediately before PMN migration assays. Conditioned media were filtered, diluted 1:1 with fresh BEGM, and added to the lower chamber of 3-µm-pore 6.5-mm Transwell units. Conditioned and control media were maintained in a 37°C, 5% CO2 incubator before addition of PMN (resuspended in fresh BEGM) to the upper chamber of Transwells.

Statistics
Unless indicated, all data are presented as the mean ± SEM. Statistical significance was determined by Student’s t test.


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RESULTS
 
Optimization of PMN transmigration across airway epithelium in vitro
The effects of airway epithelial cell monolayer polarity and culture conditions on efficiency of PMN transmigration were examined using a previously established model for PBMC transepithelial migration in vitro [22 ]. PMN migration across confluent airway epithelial cell monolayers was measured in the apical-to-basolateral direction (apical) or the basolateral-to-apical direction (basal). To determine the optimal experimental conditions for efficient migration, PMN transepithelial movement was compared using either HBSS (a bicarbonate-based buffer) supplemented with 1% BSA or BEGM (a serum-free culture medium) to incubate epithelial cell monolayers. Table 1 shows that substantial numbers of PMN migrated across airway epithelial cell monolayers within a 2-h assay, regardless of the type of experimental incubation medium utilized. For both HBSS 1% BSA and BEGM, PMN transepithelial migration was significantly more efficient in the basolateral-to-apical direction, compared with apical-to-basolateral movement (P < 0.01 and P < 0.05, respectively). The efficiency of PMN transmigration in the basolateral-to-apical direction was slightly higher for HBSS 1% BSA compared with BEGM (mean ± SEM percentage of transmigration: 36 ± 6.5 versus 28 ± 8.4, respectively). However, the overall ratio of basolateral-to-apical percent transmigration versus apical-to-basolateral percent transmigration using BEGM was enhanced, compared with HBSS 1% BSA [basal/apical ratio: 5 (BEGM) versus 3.3 (HBSS 1% BSA)]. Because these results suggested that serum-free culture medium can enhance polarized mechanisms of PMN movement across airway epithelium, subsequent experiments utilized BEGM for transmigration assays.


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  		Table 1. Effect of Incubation Medium on Efficiency of Polarized PMN Transmigration Across Airway Epithelial Cell Monolayersa

To further characterize the limitations of PMN transepithelial movement across airway epithelium, the optimal seeding ratio of PMN to epithelial cells per Transwell insert was determined in this study. As shown in Table 2 , the efficiency of PMN transmigration in the basolateral-to-apical direction across airway epithelial cell monolayers was dependent upon the initial starting concentration of PMN per Transwell unit. Migration in the basolateral-to-apical direction was optimal at 5 x 105 PMN per Transwell unit. In contrast, PMN migration across monolayers in the apical-to-basolateral direction showed minimal differences in response to seeding density. Although the increased number of PMN migrating at 5 x 105 versus 5 x 104 was not statistically significant (P > 0.05), comparisons of the basolateral-to-apical percent transmigration versus apical-to-basolateral percent transmigration ratio (basal/apical ratio) for 5 x 105 versus 5 x 104 or 5 x 106 were statistically significant (Table 2 ; P < 0.05). Based on the total airway epithelial cell number per Transwell unit at confluency (n = 6 monolayers, mean cell number ± SEM per Transwell unit: 134,458 ± 20,513) the optimal seeding density of PMN to epithelial cells was determined to be approximately 4:1. Because these findings showed that a PMN/epithelial cell seeding density of 4:1 can further enhance polarized responses of PMN movement across an airway epithelial cell monolayer in BEGM, subsequent experiments utilized this optimized arrangement.


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  		Table 2. Effect of PMN Seeding Density on Efficiency of Polarized Transmigration Across Airway Epithelial Cell Monolayersa

PMN transmigration across airway epithelium is partially inhibited by pertussis toxin
Signaling through chemokine receptors can trigger a biphasic pattern of cytoplasmic Ca2+ mobilization in leukocytes [30 ]. Pertussis toxin specifically blocks an initial, transient flux of Ca2+ associated with chemotaxis, but does not affect a secondary sustained Ca2+ mobilization associated with cellular activation. The inhibitory effects of pertussis toxin on chemotaxis are mediated via ADP ribosylation of G-proteins, leading to uncoupling from receptors [31 ]. To determine whether such a signaling pathway could mediate PMN transepithelial migration, we examined the effect of pertussis toxin on PMN migration across airway epithelium. As a control for cellular toxicity, a mutant pertussis toxin that lacks ADP-ribosyltransferase activity was utilized in these experiments. Because movement of PMN across airway epithelium in vivo is from basolateral-to-apical surfaces, experimental assessment of epithelial cell-associated chemotactic gradients utilized a physiological monolayer configuration. As shown in Figure 1 , treatment of PMN with pertussis toxin inhibited transepithelial migration across airway epithelial cell monolayers approximately threefold compared with PMN treated with mutant pertussis toxin (P < 0.01). Although inhibition of migration was significant, it was not complete; approximately 10% of the starting population of PMN were capable of crossing the epithelial barrier. To determine whether pertussis toxin-sensitive and/or pertussis toxin-insensitive PMN migration were mediated by a secreted factor produced by airway epithelium, 24-h conditioned media (CM) were collected from Transwell cultures and tested for capacity to promote PMN migration across naked Transwell membranes. A 1:1 mixture of CM and BEGM significantly enhanced mutant pertussis toxin-treated PMN migration across naked Transwell membranes by approximately 2.5-fold, compared with control BEGM (Fig. 2 , P < 0.01 compared with mutant pertussis toxin/control BEGM). Treatment of PMN with pertussis toxin reduced migration to both BEGM controls and CM; however, residual pertussis toxin-insensitive PMN attractant activity remained in the CM (P < 0.05 compared with pertussis toxin/control BEGM).



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  		Figure 1. Effect of pertussis toxin on PMN transmigration across airway epithelial cell monolayers. Before transmigration across epithelial cell monolayers, PMN were treated with either PTX or mPTX as described in Materials and Methods. Untreated PMN (control) were incubated under identical conditions as mutant and wild-type pertussis toxin before transmigration. Transmigration across epithelial monolayers in the basolateral-to-apical direction was measured after a 2-h incubation using 5 x 105 PMN per Transwell insert. The calculated percentage of transmigrated PMN was based on the starting number of PMN added to individual Transwell inserts. Columns represent the mean percentage ± SEM transmigration from three independent experiments using three different blood donors. Each experiment utilized three Transwell inserts per treatment group. *P < 0.01 compared with mutant pertussis toxin treatment.



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  		Figure 2. Effect of pertussis toxin on PMN migration to conditioned medium from airway epithelial cell cultures. For migration assays, 24-h-conditioned medium was collected from apical surfaces of epithelial cell cultures and diluted 1:1 with fresh BEGM. Before migration, PMN were treated with either PTX or mPTX as described in Materials and Methods. Migration across naked Transwell membranes in response to either diluted conditioned medium (hatched bars) or control fresh BEGM (open bars) was measured after a 2-h incubation with 5 x 105 PMN/Transwell insert. The calculated percentage of transmigrated PMN was based on the starting number of PMN added to individual Transwell inserts. Columns represent the mean percentage ± SEM transmigration from six separate experiments using four different blood donors. Each experiment utilized three Transwell inserts per treatment group. *P < 0.01 compared with mutant pertussis toxin/control BEGM; **P < 0.05 compared with petussis toxin/control BEGM; (*)P < 0.01 compared with mutant pertussis toxin/conditioned medium.

PMN transmigration across airway epithelium does not require IL-8
To study the pertussis toxin-sensitive mechanisms of PMN movement across airway epithelium, we initially focused on the evaluation of known chemokines as potential mediators of transepithelial migration. IL-8 is a potent PMN chemoattractant and constitutively expressed by human bronchial epithelium, therefore an IL-8 monoclonal antibody was utilized to evaluate its role in PMN transepithelial migration. To confirm the neutralizing efficacy of the antibody used in our experiments, PMN transmigration across airway epithelium was evaluated in the presence of exogenous IL-8. Addition of IL-8 (0.01 µg/mL) to the lower chamber of Transwell inserts resulted in a threefold increase in percentage of PMN that migrate across airway epithelium in the basolateral-to-apical direction (Fig. 3A , P < 0.001 compared with controls). Incubation of exogenous IL-8 with anti-IL-8 antibody significantly inhibited IL-8-induced PMN transmigration across airway epithelium (P < 0.001 compared with IL-8-induced transmigration). It is important that, in four independent experiments, treatment of airway epithelial cell monolayers with IL-8 antibody alone had no significant effect on subsequent PMN transepithelial migration (Fig. 3B) . In additional experiments, increasing the concentration of neutralizing antibodies utilized had no effect on PMN transepithelial migration (data not shown). Addition of anti-IL-8 antibody to CM did not reduce PMN transmigration across Transwell membranes in two experiments (data not shown).



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  		Figure 3. Effect of IL-8 neutralizing antibody on PMN transmigration across airway epithelial cell monolayers. (A) Before initiation of migration assays, IL-8 (0.01 µg/mL) or IL-8 + IL-8 mAb (1 µg/mL) were incubated in BEGM for 2 h. Preincubated IL-8 or IL-8 + mAb in BEGM were placed in lower wells of Transwell inserts containing epithelial monolayers. Preincubated BEGM without IL-8/mAb was used as a control. PMN transmigration was measured in the basolateral-to-apical surface direction after a 2-h incubation with 5 x 105 PMN/Transwell insert. Three independent experiments using two different blood donors are represented. The percentage of transmigrated PMN was calculated based on the starting number of PMN added to individual Transwell inserts. Each column represents the mean percentage ± SEM transmigration from 9–10 Transwell inserts. *P < 0.001 compared with control transmigration; **P < 0.001 compared with IL-8-induced transmigration. (B) Before initiation of migration assays, IL-8 mAb (1 µg/mL) in BEGM was added to lower wells of Transwell inserts containing epithelial cell monolayers and incubated for 1 h. Alternatively, monolayers were preincubated with nonspecific mouse IgG at 1 µg/mL as a control. Transmigration across epithelial monolayers in the basolateral-to-apical surface direction was measured after a 2-h incubation with 5 x 105 PMN/Transwell insert; immunoglobulins remained present throughout the assay. Four independent experiments using four different blood donors are represented. The percentage of transmigrated PMN was calculated based on the starting number of PMN added to individual Transwell inserts. Each column represents the mean percentage ± SEM transmigration from 12 Transwell inserts.

Thioredoxin plays a functional role in PMN transmigration across airway epithelium
It has been recently demonstrated that human thioredoxin can function as a chemoattractant for neutrophils, monocytes, and T lymphocytes [21 ]. To investigate whether this intracellular redox enzyme may represent the putative pertussis toxin-insensitive component secreted by airway epithelium in this study, we treated epithelial monolayers with a neutralizing antibody against human thioredoxin before PMN transmigration. In three independent experiments using different blood donors, anti-thioredoxin antibody significantly inhibited PMN migration across airway epithelial cell monolayers, compared with immunoglobulin controls (Fig. 4 , P < 0.05 compared with goat IgG control). Inhibition of PMN migration ranged from 21–30%, values comparable to the pertussis toxin-insensitive migration observed in Figure 1 .



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  		Figure 4. Effect of thioredoxin neutralizing antibody on PMN transmigration across airway epithelial cell monolayers. Before initiation of migration assays, epithelial cell monolayers within Transwell inserts were preincubated with thioredoxin neutralizing antibody (1–5 µg/mL) for 1 h. Alternatively, control monolayers were preincubated with nonspecific goat IgG. Transmigration across epithelial monolayers in the basolateral-to-apical surface direction was measured after a 2-h incubation with 3 x 105 PMN/Transwell insert; immunoglobulins remained present throughout the assay. The calculated percentage of transmigrated PMN was based on the starting number of PMN added to individual Transwell inserts. Columns represent the mean percentage ± SEM transmigration from three independent experiments using three different blood donors. Each experiment utilized three to four Transwell inserts per treatment group. Two experiments utilized IgG at 1 µg/mL, one experiment utilized IgG at 5 µg/mL. *P < 0.05 compared with goat IgG (control).


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DISCUSSION
 
The recruitment of circulating PMN into the conducting airways requires successive migration from the vasculature, through connective tissue, and finally across an epithelial barrier. Under normal conditions, it is likely that the endothelial cell component of the vasculature functions as a critical gatekeeper to restrict movement of PMN within the airways. Indeed, it has been reported that PMN movement across unstimulated endothelial cell monolayers is limited; proinflammatory cytokine treatment of endothelium is required for significant transmigration to occur [32 ]. This current investigation focuses on characterization of PMN migration across conducting airway epithelium using an in vitro model for leukocyte transepithelial trafficking. Results from this study suggest that the epithelium of the upper airways may function as a permissive barrier for influx of PMN into the lung. It is important to note the data indicate that PMN transmigration across airway epithelium is dependent upon both a pertussis toxin-sensitive and pertussis toxin-insensitive component secreted by epithelial cells.

A report by Liu et al. [11 ] has suggested that PMN movement across unstimulated airway epithelium is limited; exogenously applied chemoattractants were required for significant migration to occur within a 30-min time period. In agreement with these authors and others, we have demonstrated that polarity of epithelial cell monolayers has a profound effect on efficiency of leukocyte transmigration [10 , 11 ]. For the model of airway epithelia utilized in this study, PMN movement in the basolateral-to-apical direction was enhanced from 3- to 19-fold over movement in the apical-to-basolateral direction; these values are consistent with observations by C. Parkos et al. [10 ] for polarized transmigration of PMN across intestinal epithelia. However, in contrast to Liu and colleagues, we have observed significant numbers of PMN migrating across airway epithelium without an exogenous source of chemoattractants. This discrepancy is likely due to technical differences; the experimental migration assay used within our study is longer (2 h instead of 30 min) and culture conditions were optimized for maximal efficiency of PMN transmigration. Furthermore, we have demonstrated that PBMC trafficking in the basolateral-to-apical surface direction within our in vitro model of leukocyte transepithelial migration is limited to 3–5% of the starting population; in comparison with values obtained with PMN, this confirms that the airway epithelial cell monolayers were not leaky [22 ]. Previously described in vitro models for transepithelial leukocyte migration have utilized a number of different media, including HBSS and RPMI 1640 supplemented with serum albumin [10 , 11 , 33 ]. Our rationale for comparing a bicarbonate-based buffer versus a serum-free culture medium was based on concerns about potential background chemotactic activity that may be found in components of serum-free culture medium. Yet, it was also important to maintain epithelial cell monolayers under conditions in which metabolic processes were functioning at optimal levels for a 2-h period of incubation. A direct comparison of HBSS and BEGM as incubation media showed that PMN migration in the basolateral-to-apical direction was slightly increased in the presence of HBSS. However, comparison of PMN movement in the basolateral-to-apical direction versus apical-to-basolateral direction showed that the preference for polarity was greater when experimental assays were performed with BEGM. These results suggested that culture/growth conditions of epithelial cell monolayers are important for polarized mechanisms, possibly for optimal production of chemokine gradients and/or presence of appropriate localized receptors.

In addition to culture conditions, we also examined how seeding density of PMN to epithelial cell monolayers may affect overall efficiency of transmigration. It was surprising to note that, when PMN seeding densities were increased by approximately 40 PMN to one airway epithelial cell, the efficiency of transepithelial migration (as measured by percent of PMN migrated from the starting population) was markedly reduced. Moreover, at 40:1, numbers of PMN migrating in the basolateral-to-apical direction were similar to numbers migrating in the apical-to-basolateral direction. In contrast, when the ratio of PMN to airway epithelial cells was kept at approximately 4:1, polarized PMN transmigration was optimal. In addition, we found that seeding densities of PMN/epithelial cells at less than 1:1 resulted in slightly less efficient migration in the basolateral-to-apical direction, and basal/apical ratios were significantly less than that obtained by 4:1 densities (PMN/epithelial cells). It is unclear how PMN numbers may quantitatively and qualitatively affect subsequent movement across an epithelial barrier. It has been reported that when seeding densities of PMN to endothelial cells are 5:1 or less, no measurable changes in permeability of monolayers to ions or albumin occurs; at 25–50:1 seeding ratios, transendothelial electrical resistance significantly drops [34 ]. Although we did not measure the electrical resistance of monolayers immediately after PMN transmigration, our results from seeding ratios at 40:1 indicate a loss of polarized movement and overall efficiency of migration across airway epithelium, which may be due to changes in permeability (i.e., loss of chemotactic gradients). The optimal migration observed at 4:1 seeding densities (as opposed to < 1:1) may also reflect a supportive function of PMN in the recruitment of additional leukocytes; indeed, stimulated PMN can produce chemoattractants for granulocytes [35 ].

Pertussis toxin can inhibit PMN chemotaxis in vitro and depress PMN accumulation at sites of inflammation in vivo [36 37 38 39 ]. To characterize epithelial cell-derived components that mediate PMN transepithelial migration, we utilized pertussis toxin as a pharmacological tool to determine whether G{alpha}1 protein-mediated signaling events were a contributing component of this process. We have previously shown that pertussis toxin has a potent inhibitory effect on PBMC migration across airway epithelium [22 ]. In the present study, we found that treatment of PMN with pertussis toxin significantly, but not completely, inhibited migration across airway epithelium. These findings indicate that PMN movement across airway epithelium is mediated by both pertussis toxin-sensitive and pertussis toxin-insensitive mechanisms. In addition, it appeared that both pertussis toxin-sensitive and insensitive chemotactic properties for PMN were retained by conditioned medium derived from airway epithelial cell cultures, indicating that these components are secreted.

To evaluate the pertussis toxin-sensitive components that mediate PMN transepithelial migration, we focused on characterization of chemokines produced by airway epithelium. We previously reported that the BEAS-2B cell line can constitutively express IL-8 protein, at a concentration of approximately 1 ng per 250,000 cells within a 2-h period [40 ]. Here, we have demonstrated that a concentration of neutralizing antibody that effectively inhibited 5 ng of IL-8 in vitro did not inhibit PMN transmigration across BEAS-2B monolayers consisting of >200,000 cells. This observation was somewhat surprising, in lieu of a previous report implicating IL-8 as an important mediator of neutrophil transmigration across airway epithelium in vitro, and several clinical observations that correlate the presence of IL-8 in bronchoalveolar lavage fluid with PMN accumulation in the airways [16 17 18 ]. Again, discrepancies in findings by in vitro model systems may be explained by differences in culture media used (RPMI 1640 versus BEGM), as well as relative length of migration assays. IL-8 may play an important role in the initial migration of PMN into the airways; other epithelial cell-derived factors may promote moderate levels of migration via alternative mechanisms. It should be noted that phagocytic leukocytes (PMN, alveolar macrophages) are an additional source of IL-8; leukocytes that accumulate within the airways may be responsible for elevated levels of chemokines in bronchoalveolar lavage fluid. In addition, in vitro infection of intestinal epithelia with Salmonella typhimurium results in accumulation of IL-8 on basolateral surfaces of epithelial cell monolayers, which does not mediate PMN transepithelial migration [41 ]. Based on the aforementioned data, it has been proposed that IL-8 expression by epithelium may be important for migration of PMN from the vasculature or subepithelial space and may also serve to functionally activate PMN before trafficking across epithelial barriers [42 ]. This notion is supported by evidence of basolateral accumulation of IL-8 by other types of epithelia, such as retinal pigment and distal airway (Type II) [43 , 44 ]. As such, the findings presented here suggest the existence of an alternative mediator(s) that is responsible for the trafficking of PMN across airway epithelium. mRNA for GRO{alpha} has been demonstrated in human bronchial epithelial cells by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, however, secreted protein is barely detectable regardless of stimulation by proinflammatory mediators [15 ]. Using a semi-quantitative RT-PCR approach, Becker and colleagues have further demonstrated that IL-8 mRNA expression levels in human bronchial epithelial cells are at least twofold higher than that of GRO{alpha}, suggesting that IL-8 is the more predominant PMN chemokine expressed [15 ]. As such, the function of GRO{alpha} in the lung is not clear; it may have a supportive chemotactic role in certain inflammatory states. Recently, Rossi and colleagues have characterized a new mouse chemokine, designated as Lungkine, specifically expressed by conducting airway epithelium and chemotactic for neutrophils [45 ]. We speculate that the yet to be identified human homolog of Lungkine will have a similar pattern of expression and function as in the mouse and, as such, be responsible for the pertussis toxin-sensitive PMN transmigration observed across airway epithelium.

Airway epithelial cells express several pertussis toxin-insensitive mediators of PMN motility, including GM-CSF, PAF, and thioredoxin [19 , 20 ]. It has been reported that PMN chemotactic properties of CM derived from bronchial epithelial cultures may be inhibited by antibodies against GM-CSF [46 ], yet recent reports suggest that GM-CSF functions primarily to increase PMN motility but not chemotaxis [47 ]. PMN transmigration across proinflammatory cytokine-stimulated airway epithelium cannot be inhibited with a PAF-receptor antagonist, indicating that the contribution of epithelial cell-derived PAF is limited in this process [11 ]. In this study, we have determined that thioredoxin plays a role in PMN trafficking across airway epithelium. Thioredoxin is a ubiquitous enzyme that functions as an intracellular antioxidant and, when secreted, has cytokine-like activity [48 ]. It has been reported that conditions of hyperoxia can induce thioredoxin gene expression within primate lung, although the cellular source for mRNA has not been identified [49 ]. Recently, Bertini and colleagues have demonstrated that purified thioredoxin protein is chemotactic for PMN, as well as monocytes and T lymphocytes [21 ]. It has been postulated that thioredoxin functions as a chemoattractant via enzymatic activity on cell-surface substrates, presumably on leukocytes [21 ]. Our findings suggest that thioredoxin is an important mediator for PMN trafficking across epithelium, and also demonstrate a function for airway epithelial cell-derived thioredoxin.

In summary, these data indicate that transepithelial trafficking of PMN into the airways is mediated by multiple signaling pathways via direct (interaction with pertussis toxin-sensitive receptors) or indirect (enzymatic modification of substrates) mechanisms. Given the limited contributions of IL-8 to PMN transepithelial migration, subsequent studies will focus on identification of additional chemotactic mediators produced by airway epithelium.


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ACKNOWLEDGEMENTS
 
This work was supported by NRSA HL09177-02 (L. A. M.), University of California Tobacco-Related Disease Research Program Grants 6KT-0411 & 8IT-0054 (L. A. M.), American Lung Association Research Grant RG-070 (L. A. M.), and NIEHS ES-00628 (D. M. H.). We thank Dr. Fern Tablin’s laboratory for contributing a number of blood samples for this study.

Received August 30, 1999; revised March 15, 2000; accepted March 16, 2000.


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