(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*,
,
Ruth J. McDonald
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
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
 |
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
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
 |
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
(GRO
), have
been detected at the mRNA level [14
, 15
].
Elevated levels of IL-8 and GRO
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.
 |
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 (1520 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 Students
t test.
 |
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.
View this table:
[in this window]
[in a new window]
|
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.
View this table:
[in this window]
[in a new window]
|
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).

View larger version (20K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
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).

View larger version (31K):
[in this window]
[in a new window]
|
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 910 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 2130%, values comparable to
the pertussis toxin-insensitive migration observed in Figure 1
.

View larger version (64K):
[in this window]
[in a new window]
|
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 (15 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).
|
|
 |
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 35% 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 2550:1
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
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
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
,
suggesting that IL-8 is the more predominant PMN chemokine expressed
[15
]. As such, the function of GRO
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.
 |
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 Tablins laboratory
for contributing a number of blood samples for this study.
Received August 30, 1999;
revised March 15, 2000;
accepted March 16, 2000.
 |
REFERENCES
|
|---|
-
Toews, G. B. (1993) Pulmonary defense mechanisms Semin. Respir. Infect. 8,160-167[Medline]
-
Dorinsky, P. M., Davis, W. B., Lucas, J. G., Weiland, J. E., Gadek, J. E. (1985) Adult bronchiolitis. Evaluation by bronchoalveolar lavage and response to prednisone therapy Chest 88,58-63[Abstract/Free Full Text]
-
Thompson, A. B., Daughton, D., Robbins, R. A., Ghafouri, M. A., Oehlerking, M., Rennard, S. I. (1989) Intraluminal airway inflammation in chronic bronchitis: characterization and correlation with clinical parameters Am. Rev. Respir. Dis. 140,1527-1537[Medline]
-
Weiland, J. E., Davis, W. B., Holter, J. F., Mohammed, J. R., Dorinsky, P. M., Gadek, J. E. (1986) Lung neutrophils in the adult respiratory distress syndrome: clinical and pathophysiologic significance Am. Rev. Respir. Dis. 133,218-225[Medline]
-
Andrian, U. H. V., Chambers, J. D., McEvoy, L. M., Bargatze, R. F., Arfors, K.-E., Butcher, E. C. (1991) Two-step model of leukocyte-endothelial cell interaction in inflammation: Distinct roles for LECAM-1 and the leukocyte ß2 integrins in vivo Proc. Natl. Acad. Sci. USA 88,7538-7542[Abstract/Free Full Text]
-
Ley, K., Gaehtgens, P., Fennie, C., Singer, M. S., Lasky, L. A., Rosen, S. D. (1991) Lectin-like adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo Blood 77,2553-2555[Abstract/Free Full Text]
-
Arfors, K.-E., Lundberg, C., Lindbom, L., Lundberg, K., Beatty, P. G., Harlan, J. M. (1987) A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leakage in vivo Blood 69,338-340[Abstract/Free Full Text]
-
Colgan, S. P., Parkos, C. A., McGuirk, D., Brady, H. R., Papayianni, A. A., Frendl, G., Madara, J. L. (1995) Receptors involved in carbohydrate binding modulate intestinal epithelial-neutrophil interactions J. Biol. Chem. 270,10531-10539[Abstract/Free Full Text]
-
Lu, H., Smith, C. W., Perrard, J., Bullard, D., Tang, L., Shappell, S. B., Entman, M. L. (1997) LFA-1 is sufficient in mediating neutrophil emigration in Mac-1 deficient mice J. Clin. Invest. 99,1340-1350[Medline]
-
Parkos, C. A., Delp, C., Arnaout, M. A., Madara, J. L. (1991) Neutrophil migration across a cultured intestinal epithelium: Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction J. Clin. Invest. 88,1605-1612
-
Liu, L., Mul, F. P. J., Lutter, R., Roos, D., Knol, E. F. (1996) Transmigration of human neutrophils across airway epithelial cell monolayers is preferentially in the physiologic basolateral-to-apical directions Am. J. Respir. Cell Mol. Biol. 15,771-780[Abstract]
-
Rot, A. (1992) Endothelial cell binding of NAP-1/IL-8: role in neutrophil emigration Immunol. Today 13,291-294[Medline]
-
Tanaka, Y., Adams, D. H., Hubscher, S., Hirano, H., Siebenlist, U., Shaw, S. (1993) T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1ß Nature 361,79[Medline]
-
Nakamura, H., Yoshimura, K., Jaffe, H. A., Crystal, R. G. (1991) Interleukin-8 gene expression in human bronchial epithelial cells J. Biol. Chem. 266,19611-19617[Abstract/Free Full Text]
-
Becker, S., Quay, J., Koren, H. S., Haskill, J. S. (1994) Constitutive and stimulated MCP-1, GRO
, ß, and
expression in human airway epithelium and bronchoalveolar macrophages Am. J. Physiol. 266,L278-L286[Abstract/Free Full Text]
-
Villard, J., Dayer-Pastore, F., Hamacher, J., Aubert, J. D., Schlegel-Haueter, S., Nicod, L. P. (1995) GRO alpha and interleukin-8 in Pneumocystis carinii or bacterial pneumonia and adult respiratory distress syndrome Am. J. Respir. Crit. Care Med. 152,1549-1554[Abstract]
-
Nocker, R. E. T., Schoonbrood, D. F. M., Graaf, E. A. v. d., Hack, C. E., Lutter, R., Jansen, H. M., Out, T. A. (1996) Interleukin-8 in airway inflammation in patients with asthma and chronic obstructive pulmonary disease Int. Arch. Allergy Immunol. 109,183-191[Medline]
-
Goodman, R. B., Strieter, R. M., Martin, D. P., Steinberg, K. P., Milberg, J. A., Maunder, R. J., Kunkel, S. L., Walz, A., Hudson, L. D., Martin, T. R. (1996) Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome Am. J. Respir. Crit. Care Med. 154,602-611[Abstract]
-
Cromwell, O., Hamid, Q., Corrigan, C. J., Barkans, J., Meng, Q., Collins, P. D., Kay, A. B. (1992) Expression and generation of IL-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1ß and tumour necrosis factor-
Immunology 77,330-337[Medline]
-
An, G., Wu, R. (1992) Thioredoxin gene expression is transcriptionally up-regulated by retinol in monkey conducting airway epithelial cells Biochem. Biophys. Res. Commun. 183,170-175[Medline]
-
Bertini, R., Howard, O. M. Z., Dong, H.-F., Oppenheim, J. J., Bizzarri, C., Sergi, R., Caselli, G., Pagliei, S., Romines, B., Wilshire, J. A., Mengozzi, M., Nakamura, H., Yodoi, J., Pekkari, K., Gurunath, R., Holmgren, A., Herzenberg, L. A., Herzenberg, L. A., Ghezzi, P. (1999) Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells J. Exp. Med. 189,1783-1789[Abstract/Free Full Text]
-
Miller, L. A., Butcher, E. C. (1998) Human airway epithelial monolayers promote selective transmigration of memory T cells: a transepithelial model of lymphocyte migration into the airways Am. J. Respir. Cell Mol. Biol. 19,892-900[Abstract/Free Full Text]
-
Pizza, M., Covacci, A., Bartoloni, A., Perugini, M., Nencioni, L., Magistris, M., Villa, L., Nucci, D., Manetti, R., Bugnoli, M., Giovannoni, F., Olivieri, R., Barbieri, J. T., Sato, H., Rappuoli, R. (1989) Mutants of pertussis toxin suitable for vaccine development Science 246,497-500[Abstract/Free Full Text]
-
Ke, Y., Reddel, R. R., Gerwin, B. I., Miyashita, M., McMenamin, M., Lechner, J. F., Harris, C. C. (1988) Human bronchial epithelial cells with integrated SV40 virus T antigen genes retain the ability to undergo squamous differentiation Differentiation 38,60-66[Medline]
-
Noah, T. L., Yankaskas, J. R., Carson, J. L., Gambling, T. M., Cazares, L. H., McKinnon, K. P., Devlin, R. B. () Tight junctions and mucin mRNA in BEAS-2B cells. In Vitro Cell. Dev. Biol. 31,738-740
-
Cheek, J. M., McDonald, R. J., Rapalyea, L., Tarkington, B. K., Hyde, D. M. (1995) Neutrophils enhance removal of ozone-injured alveolar epithelial cells in vitro Am. J. Physiol. 269,L527-L535[Abstract/Free Full Text]
-
Freshney, R. I. (1987) Culture of Animal Cells: A Manual of Basic Technique Liss New York.
-
Nielson, C. P., Vestal, R. E., Sturm, R. J., Heaslip, R. (1990) Effects of selective phosphodiesterase inhibitors on the polymorphonuclear leukocyte respiratory burst J. Allergy Clin. Immunol. 86,801-808[Medline]
-
Bargatze, R. F., Butcher, E. C. (1993) Rapid G protein-regulated activation event involved in lymphocyte binding to high endothelial venules J. Exp. Med. 178,367-372[Abstract/Free Full Text]
-
Bacon, K. B., Premack, B. A., Gardner, P., Schall, T. J. (1995) Activation of dual T cell signaling pathways by the chemokine RANTES Science 269,1727-1730[Abstract/Free Full Text]
-
Bokoch, G. M., Gilman, A. G. (1984) Inhibition of receptor-mediated release of arachidonic acid by pertussis toxin Cell 39,301-308[Medline]
-
Moser, R., Schleiffenbaum, B., Groscurth, P., Fehr, J. (1989) Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage J. Clin. Invest. 83,444-455
-
Smart, S. J., Casale, T. B. (1994) Pulmonary epithelial cells facilitate TNF-alpha-induced neutrophil chemotaxis J. Immunol. 152,4087-4094[Abstract]
-
Huang, A. J., Furie, M. B., Nicholson, S. C., Fischbarg, J., Liebovitch, L. S., Silverstein, S. C. (1988) Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules J. Cell Physiol. 135,355-366[Medline]
-
Bazzoni, F., Cassatella, M. A., Rossi, F., Ceska, M., Dewald, B., Baggiolini, M. (1991) Phagocytosing neutrophils produce and release high amounts of the neutrophil-activating peptide-1/interleukin 8 J. Exp. Med. 173,771-774[Abstract/Free Full Text]
-
Spangrude, G. J., Sacchi, F., Hill, H. R., Van Epps, D. E., Daynes, R. A. (1985) Inhibition of lymphocyte and neutrophil chemotaxis by pertussis toxin J. Immunol. 135,4135-4143[Abstract]
-
Nourshargh, S., Williams, T. J. (1990) Evidence of a receptor-operated event on the neutrophil mediates neutrophil accumulation in vivo. Pretreatment of 111In-neutrophils with pertussis toxin in vitro inhibits their accumulation in vivo J. Immunol. 145,2633-2638[Abstract]
-
Kelly, C. P., Becker, S., Linevsky, J. K., Joshi, M. A., OKeane, J. C., Dickey, B. F., LaMont, J. T., Pothoulakis, C. (1994) Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit J. Clin. Invest. 93,1257-1265
-
Brito, G. A., Souza, M. H., Melo-Filho, A. A., Hewlet, E. L., Lima, A. A., Flores, C. A., Ribeiro, R. A. (1997) Role of pertussis toxin A subunit in neutrophil migration and vascular permeability Infect. Immun. 65,1114-1118[Abstract]
-
Chang, M., Wu, R., Plopper, C. G., Hyde, D. M. (1998) IL-8 is one of the major chemokines produced by monkey airway epithelium after ozone-induced injury Am. J. Physiol. 275,L524-L532[Abstract/Free Full Text]
-
McCormick, B. A., Colgan, S. P., Delp-Archer, C., Miller, S. I., Madara, J. L. (1993) Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils J. Cell Biol. 123,895-907[Abstract/Free Full Text]
-
Parkos, C. A. (1997) Molecular events in neutrophil transepithelial migration BioEssays 19,865-873[Medline]
-
Holtkamp, G. M., Van Rossem, M., de Vos, A. F., Willekens, B., Peek, R., Kijlstra, A. (1998) Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells Clin. Exp. Immunol. 112,34-43[Medline]
-
Crippen, T. L., Klasing, K. C., Hyde, D. M. (1995) Cytokine-induced neutrophil chemoattractant production by primary rat alveolar type II cells Inflammation 19,575-586[Medline]
-
Rossi, D. L., Hurst, S. D., Xu, Y., Wang, W., Menon, S., Coffman, R. L., Zlotnik, A. (1999) Lungkine, a novel CXC chemokine, specifically expressed by lung bronchoepithelial cells J. Immunol. 162,5490-5497[Abstract/Free Full Text]
-
Abdelaziz, M. M., Devalia, J. L., Khair, O. A., Calderon, M., Sapsford, R. J., Davies, R. J. (1995) The effect of conditioned medium from cultured human bronchial epithelial cells on eosinophil and neutrophil chemotaxis and adherence in vitro Am. J. Respir. Cell Mol. Biol. 13,728-737[Abstract]
-
Harakawa, N., Sasada, M., Maeda, A., Asagoe, K., Nohgawa, M., Takano, K., Matsuda, Y., Yamamoto, K., Okuma, M. (1997) Random migration of polymorphonuclear leukocytes induced by GM-CSF involving a signal transduction pathway different from that of fMLP J. Leukoc. Biol. 61,500-506[Abstract]
-
Nakamura, H., Nakamura, K., Yodoi, J. (1997) Redox regulation of cellular activation Annu. Rev. Immunol. 15,351-369[Medline]
-
Das, K. C., Guo, X. L., White, C. W. (1999) Induction of thioredoxin and thioredoxin reductase gene expression in lungs of newborn primates by oxygen Am. J. Physiol. 276,L530-L539[Abstract/Free Full Text]
This article has been cited by other articles:

|
 |

|
 |
 
K. L. Oslund, L. A. Miller, J. L. Usachenko, N. K. Tyler, R. Wu, and D. M. Hyde
Oxidant-Injured Airway Epithelial Cells Upregulate Thioredoxin but Do Not Produce Interleukin-8
Am. J. Respir. Cell Mol. Biol.,
May 1, 2004;
30(5):
597 - 604.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
V. B. Serikov, H. Choi, K. J. Chmiel, R. Wu, and J. H. Widdicombe
Activation of Extracellular Regulated Kinases Is Required for the Increase in Airway Epithelial Permeability during Leukocyte Transmigration
Am. J. Respir. Cell Mol. Biol.,
March 1, 2004;
30(3):
261 - 270.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
T. Papayannopoulou, G. V. Priestley, H. Bonig, and B. Nakamoto
The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization
Blood,
June 15, 2003;
101(12):
4739 - 4747.
[Abstract]
[Full Text]
[PDF]
|
 |
|