Introduction

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The epithelium isolates the host from the external environment. In the lung, the epithelium may be only one-cell thick in gas-exchange units. Neutrophils play a vital role in host-defense mechanisms. However, the mechanisms by which mediators and cytokines stimulate neutrophils to leave the peripheral circulation and migrate across the lung epithelium are only now being elucidated (1).

Previously, lung epithelial cells were regarded as barriers that were at best, passive and at worst, hindrances to the stimulated influx of inflammatory cells. However, it has recently been demonstrated that in response to certain stimuli, respiratory epithelial cells play an active and essential role in neutrophil migration responses by producing chemokines (2) and expressing key adhesion molecules (7).

Studies of the mechanisms involved in stimulated neutrophil transepithelial migration have generally been conducted with lung epithelial cells cultured on top of permeable filters (3, 4, 8). However, neutrophil transepithelial migration in the respiratory tract probably occurs in a predominantly basal-to-apical rather than an apical-to-basal direction. We therefore questioned whether there might be a difference in the migration patterns of neutrophils if these cells moved through the lung epithelium in the apical-to-basal as opposed to the basal-to-apical direction. To investigate this, we grew monolayers of the A549 human Type II alveolar epithelial cell line exclusively on the upper or lower surface of collagen-coated 3-µm-pore-size polycarbonate Transwell^® filters (Costar, Cambridge, MA). We then studied whether epithelial orientation had a significant impact on stimulated neutrophil transepithelial migration. Furthermore, we examined the effects on this process of the type of stimulus eliciting migration, comprising the classic chemoattractants formyl-methionylleucylphenylalanine (FMLP), leukotriene B₄ (LTB₄), or interleukin (IL)-8 as opposed to the inflammatory cytokines IL-1 or tumor necrosis factor- (TNF-). We found that the orientation of the epithelium, and thereby the directional movement of the neutrophil, is much more important when inflammatory cytokines are the stimuli.

	Materials and Methods

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Reagents

Bovine serum albumin (BSA), FMLP, and collagen (bovine placental Type IV) were purchased from Sigma Chemical Co., St. Louis, MO. LTB₄ was purchased from Calbiochem, San Diego, CA. Human recombinant IL-1, TNF-, and IL-8 (72-amino-acid form) were purchased from R&D Systems, Minneapolis, MN. Hypaque M-90% was a gift from Winthrop Pharmaceuticals, New York, NY. Ham's F12K medium was purchased from Irvine Biological, Irvine, CA. Fetal calf serum (FCS), trypsin, ethylenediamine tetraacetic acid (EDTA), antibiotics, Hanks' balanced salt solution (HBSS) and phosphate-buffered saline (PBS) were purchased from the University of Iowa Cancer Center, Iowa City, IA. Transwell^® tissue-culture plates were purchased from Costar. (⁵¹Cr)-Na₂CrO₄ was purchased from New England Nuclear Co., Boston, MA.

Cell Culture

A549 human Type II-like epithelial lung cells (A549) (11) (passage 76) were purchased from the American Type Culture Collection, Rockville, MD. A549 cells were grown as monolayers in tissue-culture flasks incubated in 100% humidity and 5% CO₂ at 37°C in Ham's F12K medium supplemented with 10% FCS, penicillin (20 U/ml), and streptomycin (20 µg/ml). Culture flasks and filters were treated with collagen Type IV (50 µg/ml) for at least 30 min at 37°C, and rinsed with HBSS. Cell monolayers in tissue-culture flasks were harvested with trypsin (0.25%) and EDTA (0.1%) in PBS, centrifuged at low speed (250 × g, for 5 min), and resuspended in fresh medium prior to culture on permeable filters in Transwell^® tissue-culture plates. Cells (1.5 × 10⁶/ml) in 100-µl volumes were grown to confluence on the upper or lower surface of these filters in Transwell^® plates over periods of 3 to 5 days, as previously described (3, 4, 8), using the same conditions described for the flasks (Figure 1). Cells grown on the lower surface were first placed on inverted collagen-coated filters and incubated for 2 h at 37°C. The filters were then overturned (righted) and placed in the lower well of the Transwell^® plate. Monolayer integrity was assessed by microscopy, albumin permeability, and transmonolayer electrical resistance, and with negative (buffer) controls. Transmonolayer electrical resistance was the same for monolayers grown on the upper and lower surfaces of the filters. The 24-well Transwell^® plates are separated into upper and lower chambers by a 6.5-mm-diameter polycarbonate filter with a 3.0-µm pore size.

View larger version (26K): [in this window] [in a new window]   Figure 1.   Diagram of the chemotaxis chamber. This figure illustrates how the experiments were done.

Isolation of Neutrophils and Chromium Labeling

Neutrophils were isolated from 0.2% EDTA-anticoagulated whole blood collected by venipuncture. Neutrophils were obtained through a modification of the density-gradient technique (1.095 Hypaque-Ficoll) described by Ferrante and Thong (12). The isolated neutrophils were washed once with HBSS without Ca²⁺ or Mg²⁺. Neutrophils were then resuspended in 4 ml of HBSS without Ca²⁺ or Mg²⁺, and residual red cells were lysed with 1% ammonium oxalate (wt/vol) in H₂O. The neutrophils were then washed again in HBSS without Ca²⁺ or Mg²⁺ before cell counts were made. Viability was determined by trypan blue exclusion. The isolated neutrophils were at least 98% pure and 99% viable.

Prior to placing them in the chemotactic chambers, isolated neutrophils were labeled with ⁵¹Cr through a modification of the procedure described by Gallin and colleagues (13). Neutrophils at concentrations of 15 to 45 × 10⁶ cells/ ml were incubated in Ca²⁺/Mg²⁺-free HBSS with one-half mCi of ⁵¹Cr (5 mCi/ml) at 37°C for 1 h, with vigorous mixing. The labeling was terminated at the end of the incubation period by diluting the cells to 50 ml with HBSS without Ca²⁺ or Mg²⁺. The neutrophils were subsequently washed three times in Ca²⁺- and Mg²⁺-free HBSS before being resuspended in HBSS with 0.2% BSA and Ca²⁺ and Mg²⁺. The cells were resuspended at 1.5 × 10⁷/ml prior to use. Aliquots of the cell suspension were removed and assayed for total gamma radiation counts and unbound ⁵¹Cr to determine the cell-associated counts per minute (cpm) as described by Gallin and colleagues (13).

Chemotaxis and Transmigration

Neutrophil chemotaxis through cell monolayers cultured on filters in either the basal or apical orientation was investigated with the tissue-cultures grown in 24-well Transwell^® plates as previously described (3, 4, 8). From 1 to 1.5 million ⁵¹Cr-labeled neutrophils in 100 µl of buffer were placed in the upper chambers of each plate above the filter/monolayer or monolayer/filter complex. The chemoattractant, in 500 µl HBSS with 0.2% BSA, was placed in the lower chambers. Buffer alone was placed in the lower chambers as a negative control in each experiment. Each variable was tested in triplicate. The plates were incubated at 37°C in 5% CO₂ and 100% humidity for 3 h in the dose-response experiments. Both barriers (filter/monolayer and monolayer/filter) were tested simultaneously with the same donor for each of the experiments. The degree of neutrophil migration was determined by mixing the lower chamber contents (500 µl) with 300 µl of 2% (vol/vol) Triton X-100 in H₂O, collecting the fluid, and then counting emissions in a gamma counter to determine cpm. The data were expressed as the percentage net stimulated migration (NSM), using the formula:

The cpm measured in the negative control (HBSS- BSA) samples was generally less than 5% of the total cpm added to the top chamber. There was no significant difference in the negative-control migration values when neutrophils migrated in an apical-to-basal as opposed to a basal-to-apical direction. Figure 1 shows a diagram of the chemotaxis chamber, illustrating how the experiments were done.

	Results

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Effect of Monolayer Orientation on Neutrophil Migration in Response to Chemoattractants

We first measured the NSM of neutrophils through A549 epithelial monolayers grown on the upper surfaces or the lower surfaces of Transwell^® filters in response to varying doses of FMLP, LTB₄, and IL-8 at 3 h (Figure 2). All three chemoattractants induced dose-responsive neutrophil migration through the A549 monolayers grown on the upper and the lower surfaces of the filters. Furthermore, there were no statistically significant differences between values for apical-to-basal and basal-to-apical neutrophil NSM.

View larger version (14K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 2.   Dose response of (a) FMLP-, (b) LTB4-, and (c) IL-8-induced neutrophil migration through A549 monolayers grown on upper versus lower surfaces of Transwell® filters. Results represent means ± SEM of NSM of neutrophils in four experiments, with each variable measured in triplicate.

Effect of Monolayer Orientation on Neutrophil Migration in Response to Cytokines

Both IL-1 and TNF- induced dose-dependent neutrophil migration through the A549 monolayers in an apical-to-basal and basal-to-apical direction. As can be seen in Figure 3a, however, IL-1-induced neutrophil NSM through A549 monolayers was much greater in the basal-to-apical direction than in the apical-to-basal direction. Neutrophil NSM in response to TNF- was also greater in the basal-to-apical direction (Figure 3b).

View larger version (14K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Figure 3.   Dose response of (a) IL-1- and (b) TNF--induced neutrophil migration through A549 monolayers grown on upper versus lower surfaces of Transwell® filters. Results represent means ± SEM of NSM of neutrophils in four experiments, with each variable measured in triplicate. Neutrophil migration through monolayers in a basal-to-apical direction is significantly greater than that in an apical-to-basal direction. *P < 0.05; **P < 0.01 by paired-sample t test.

Effect of Monolayer and TNF- Orientation on Neutrophil Migration

We next designed a series of experiments to examine whether the enhanced cytokine-induced neutrophil NSM in a basal-to-apical direction was dependent upon the direction of the neutrophil migration or the polarity of the stimulus. We grew A549 monolayers on either the upper or lower surface of Transwell^® filters and compared neutrophil migration in response to TNF- placed in the upper versus the lower chamber. Figures 4a and 4b show that neutrophil migration in the basal-to-apical direction was greater, regardless of whether the TNF- was above or below the filter/monolayer complex. In Figures 4c and 4d, the data are transformed to compare neutrophil NSM when the stimulus was in an apical versus a basal location. As can be seen, the degree of neutrophil migration was relatively equivalent when the TNF- was above or below a monolayer grown either on the upper (Figure 4c) or on the lower (Figure 4d) surface of the filter. Irrespective of the polarity of the stimulus, the amount of neutrophil migration was always greater when the A549 monolayers were grown on the lower surface of the Transwell^® filters.

View larger version (12K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Figure 4.   Effect of monolayer and TNF- orientation on neutrophil migration. Neutrophil migration through A549 monolayers grown on upper versus lower surfaces of Transwell® filters with TNF- located either above or below the filter/monolayer complex. (a and b) Effects of monolayer orientation. (c and d) Transformation of data from a and b in order to examine the effects of stimulus polarity. Results represent means ± SEM of NSM of neutrophils in four experiments, with each variable measured in triplicate. *P  0.05; **P  0.01 by paired-sample t test.

Inhibitory Effect of Actinomycin D

We next examined the effects of pretreating the A549 monolayers with the metabolic inhibitor actinomycin D. A549 monolayers grown either on the upper or lower surface of Transwell^® filters were pretreated for 30 min with 2 µg/ml of actinomycin D or control buffer, and were then washed before a 3-h migration assay with FMLP, TNF-, or IL-1 (Figure 5). Actinomycin D had no effect on FMLP-induced neutrophil migration, but did significantly inhibit both TNF-- and IL-1-induced neutrophil migration. The inhibitory effects of actinomycin D were equivalent for monolayers grown on the upper or on the lower surfaces of the filters.

View larger version (13K): [in this window] [in a new window]   Figure 5.   Inhibitory effect of actinomycin D on FMLP-, TNF--, and IL-1-induced neutrophil migration through A549 monolayers grown on upper or lower surfaces of Transwell® filters. Data are expressed as inhibition of NSM of neutrophils ± SEM in two experiments, with each variable measured in triplicate.

	Discussion

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To our knowledge, there have been no published reports of studies of neutrophil migration through respiratory epithelial cells in a manner analogous to the influx of neutrophils into the airway lumen (i.e., in a basal-to-apical direction). We examined the migration of neutrophils through A549 cells grown on the upper or on the lower surfaces of filters, in response to low-molecular-weight chemoattractants, the chemokine IL-8, and two proinflammatory cytokines. We found marked differences in the effects of the orientation of the epithelium on the capacity of these agents to induce neutrophil transepithelial migration. However, these differences were largely dependent upon the agent used.

In the studies reported herein, the potent chemoattractants FMLP, LTB₄, and IL-8 induced the same degree of neutrophil transepithelial migration regardless of the orientation of the cultured cell monolayers (Figure 2). In contrast to TNF- and IL-1, these agents are truly chemotactic, do not mediate the production of secondary chemoattractants by the epithelium, and do not appear to be potent inducers of key adhesion molecules (8). Thus, in contrast to the case with TNF- and IL-1, the presence of an epithelial monolayer does not lead to migration greater than that measured through naked filters when the migration is induced by FMLP, LTB₄, or IL-8 (3, 4, 6, 8). It is likely that these potent chemotactic agents exert greater effects on the neutrophils themselves. Indeed, the epithelial monolayer may play a more passive role in neutrophil transepithelial migration mediated by FMLP, LTB₄ or IL-8. This concept is supported by our findings that: (1) neutrophil transepithelial migration in an apical-to-basal direction was equivalent to that in a basal-to-apical direction with these chemoattractants; and (2) actinomycin D treatment of the epithelium had no inhibitory effect on FMLP-induced neutrophil migration (Figure 5).

Neutrophil migration through the A549 monolayers was significantly greater in the basal-to-apical than in the apical-to-basal direction in response to IL-1 and TNF- (Figure 3). This enhanced neutrophil transepithelial migration occurred throughout the dose range tested for these agents.

The mechanisms involved in the enhanced IL-1- and TNF--induced neutrophil transepithelial migration in a basal-to-apical direction were not definitively elucidated by the present study. The data suggest several possible mechanisms, including the production of secondary peptide chemoattractants and/or enhanced polarized expression of key adhesion molecules.

The marked inhibitory effects of actinomycin D on IL-1- and TNF-- but not on FMLP-induced transepithelial migration support the importance of the epithelium in cytokine-induced transepithelial migration (Figure 5). Regardless of the monolayer orientation, both IL-1- and TNF--induced neutrophil transepithelial migration depend upon the intact metabolic capacity of the A549 monolayer. Actinomycin D has its greatest effect on ribosomal RNA synthesis, leading to the cessation of most protein synthesis. We have shown in other experiments that actinomycin D is capable of inhibiting the production of soluble chemotactic factors by TNF--stimulated A549 epithelial cells (4). We have also demonstrated by fluorescence-activated cell sorting (FACS) analysis (data not shown) that TNF--induced expression of intercellular adhesion molecule-1 (ICAM-1) is inhibited by actinomycin D. In the data shown in Figure 5, actinomycin D inhibited TNF-- and IL-1-induced neutrophil transepithelial migration by about 75%, regardless of whether the monolayers were grown on the upper or lower surface of the filters. Although the percent inhibition of neutrophil transepithelial migration was equivalent, the actual amount of migration inhibited was greater for cells grown on the lower surface of the filters, since the amount of neutrophil transepithelial migration is about 2-fold greater in a basal-to-apical than in an apical-to-basal direction. Nonetheless, since actinomycin D inhibits both chemoattractant release (e.g., IL-8) (3, 4) and adhesion molecule expression (e.g., ICAM-1) (14), these data do not specifically define the mechanisms involved in enhanced basal-to-apical transepithelial neutrophil migration.

Several recent studies have examined the polarized production and/or secretion of chemoattractants. Stimuli including TNF- and IL-1 have been shown to induce greater apical than basal secretion of IL-8 and monocyte chemotactic peptide-1 from cultured human mesothelial cells and rat Type II alveolar epithelial cells, respectively (20, 21). However, different stimuli of rat Type II alveolar cells resulted in a preferential basolateral secretion of granulocyte-macrophage-colony stimulating factor (22). Furthermore, both TNF- and IL-1 have been shown to induce IL-8 secretion by HT 29/19A enterocytes in a polarized fashion according to the direction of stimulation (23). Since TNF- and IL-1 can induce IL-8 production by A549 cells (3, 4, 8), the polarized release of this or other chemokines could either enhance or inhibit transepithelial migration (24). We attempted to examine this possibility, but our results were inconclusive, owing to the intrinsic difficulty in compartmentalizing secreted product with the system used in our study (Figure 1).

In the experiments whose results are shown in Figure 4, we addressed the importance of epithelial orientation versus stimulus polarity with regard to the polarity of neutrophil migration. These experiments suggested that epithelial orientation was more important than the polarity of TNF- to the polarity of neutrophil migration. Indeed, the degree of neutrophil transepithelial migration was always greater in a basal-to-apical direction, regardless of whether the stimulus was applied from an apical or basal orientation. These experiments suggest that the polarized secretion of a chemoattractant from cytokine-stimulated epithelial cells may not be the sole explanation for the preferential basal-to-apical transepithelial migration of neutrophils. However, because of the limitations of our model system, we were unable to definitively prove or disprove the importance of polarized secretion of chemoattractants to our findings.

It has previously been shown that TNF- and IL-1 can cause lung epithelial cells to express adhesion molecules, including ICAM-1 (7, 15). Because of the interposed filter in our experiments, we were unable to study whether these cytokines induced adhesion-molecule expression in a polarized fashion. Moreover, we are unaware of any studies using analogous systems to examine this possibility. Nonetheless, protein trafficking pathways leading to an apical or basolateral expression of proteins have been demonstrated (19). Thus, the polarized expression of key adhesion molecules remains in part a possible explanation for our results with IL-1 and TNF-.

In summary, we found greater IL-1- and TNF--induced neutrophil transepithelial migration in a basal-to-apical than in an apical-to-basal direction. Although the exact mechanisms involved in this preferentiality are not fully clear, the critical role of a metabolically active epithelium is clear. Migration in the basal-to-apical direction is probably more physiologic, and may account for the accumulation of neutrophils in the airway lumen following inhalation of a noxious stimulus. Indeed, the stimulated release of IL-1 and TNF- by alveolar macrophages probably contributes to the directed migration of neutrophils across the lung epithelium in a basal-to-apical direction. Future studies, aimed at further defining the role of the epithelium in this process, will probably aid in understanding the events underlying neutrophil-rich inflammatory responses in the lung.