(Received for publication, December 4, 1996)
From the Department of Microbiology and Immunology,
Institute of Molecular Biology, Jagiellonian University, 31-120 Kraków, Poland and § Department of Biochemistry and
Molecular Biology, University of Georgia, Athens, Georgia 30602
Destruction of components of the extracellular
matrix of the lung by neutrophil elastase is believed to be a critical
event in the development of obstructive lung disease. The local
synthesis of 1-proteinase inhibitor, the
controlling inhibitor of this enzyme, may provide a partial mechanism
for neutrophil elastase regulation, especially during inflammation,
when proteolytic enzymes are released from phagocytes. In this study,
we show that lung-derived epithelial cells not only have the capacity
to synthesize functional
1-PI but also to increase the
rate of its production when stimulated by specific inflammatory
mediators, including oncostatin M, interleukin-1, and the
glucocorticoid analogue, dexamethasone.
1-Proteinase inhibitor
(
1-PI)1 is a major serine
proteinase inhibitor in human plasma and the archetype of the serpin
(Serine proteinase inhibitors) superfamily, which in humans
includes among others;
1-antichymotrypsin (Achy),
antithrombin III,
2-antiplasmin, C1-inhibitor, plasminogen activator inhibitor,
heparin cofactor II, protein C inhibitor, cortisol-binding globulin,
thyroxine-binding globulin, and angiotensinogen (1). As a principal
inhibitor of neutrophil elastase (HNE), an enzyme which degrades
components of the extracellular matrix,
1-PI is involved
in the control of turnover of connective tissue. The respiratory tract
is particularly vulnerable to damage by proteolytic enzymes, and
deficiency has been correlated with disturbances in lung function (2).
Although the lung also contains other antiproteases capable of
inhibiting HNE,
1-PI contributes >90% of the
functional anti-elastase protection of the alveolar walls (3). During
tissue injury or inflammation when elevated proteinase activity is
present, the role of
1-PI seems to be particularly
important and is reflected by an increase in the plasma concentration
of this inhibitor under such conditions, as part of the acute phase
reaction. Although the liver appears to be the primary source of
1-PI, this inhibitor has also been shown to be
synthesized by extrahepatic cells, including cells of epithelial origin
(4).
Some data have already been accumulated that indicate that the extrahepatic production of specific serpins is likely to be more significant than had previously been believed. For example, Achy, which increases dramatically in concentration in plasma during the acute phase reaction can be produced by epithelial cells originating from jejunum (4), lung, breast, or skin (5). Indeed, model lung-derived epithelial HTB55 cells synthesize comparable amounts of Achy to those from hepatic-derived HepG2 cells. Furthermore, such synthesis can be up-regulated by a number of factors, including oncostatin M (OSM), interleukin-1 (IL-1), and glucocorticoid analog, dexamethasone (5).
The human airway epithelium, with a surface of 1-2 m2, has
the potential of exposure to a variety of factors that may trigger an
inflammatory response (6). However, the contribution of lung epithelial
cells to the local production of 1-PI has never been
clarified. In the current study, a variety of inflammatory mediators
were tested as potential stimulators of
1-PI in a
lung-derived HTB55 cell line, as well as in normal bronchial epithelial
cells.
Human recombinant interleukin-1 (IL-1)
(specific activity, 1 × 107 units/mg) was donated by
Dr. D. Schenk, Athena Neurosciences, Inc. (San Francisco, CA). Human
recombinant interleukin-6 (IL-6) (specific activity, 1 × 107 units/mg), and OSM (specific activity, 4.7 × 107 units/mg) were kindly provided by Immunex (Seattle,
WA). Human leukemia inhibitory factor (LIF) from conditioned media of
Chinese hamster ovary cells, expressing recombinant LIF at
105 units/ml, was a generous gift of Dr. H. Baumann
(Buffalo, NY). Dexamethasone, concanavalin A (ConA; type IV),
-methyl-D-mannoside, and
methyl-
-D-glucopyranoside were purchased from
Sigma. Serum and lung lavage fluid (LLF) (a generous gift of
Dr. R. Senior, Washington University, St. Louis, MO) were obtained from
a single, 29-year-old Caucasian male smoker in good health.
HTB55 (Calu-3) human lung adenocarcinoma and HepG2 human hepatoma cell lines were obtained from the American Type Culture Collection (Rockville, MD). Normal human bronchial epithelial cells were purchased from Clonetics (San Diego, CA). HTB55 cells were cultured in Eagle's MEM supplemented with 0.1 mM nonessential amino acids and 1 mM sodium pyruvate (all from Life Technologies, Inc., whereas HepG2 cells were cultured in Dulbecco's modified Eagle's medium. Both media contained, in addition, 100 units/ml penicillin G, 100 µg/ml streptomycin (Life Technologies, Inc.), and 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA). Bronchial cells were cultured in serum-free bronchial epithelial cell basal medium containing 0.5 ng/ml human epidermal growth factor, 5 µg/ml insulin, 0.5 µg/ml each hydrocortisone and epinephrine, 10 µg/ml transferrin, 0.5 ng/ml triiodothyronine, and 0.4% v/v bovine pituitary extract (all from Clonetics, San Diego, CA). Cells were plated, allowed to grow to confluency before assay, and then treated with various stimulating factors.
Northern Blot AnalysisTotal RNA was isolated as described
previously (7, 8). Northern blot analysis was carried out by
electrophoresis of RNA samples in 1% agarose gels containing 2.2 M formaldehyde, followed by capillary transfer (9) to
Hybond-N membranes (Amersham Corp.). Hybridization with
32P-labeled probes was performed overnight at 65 °C in a
mixture containing 1 M NaCl, 1% SDS, and 10% dextran
sulfate. The following probes were used; 1.4-kilobase
EcoRI-EcoRI restriction fragment of human
1-PI cDNA (10) and 0.5-kilobase
PstI-PstI restriction fragment of human GAPDH
cDNA (8). These probes were labeled using the Megaprime labeling
kit (Amersham). Autoradiographs were scanned by quantitative
densitometry (PDI System, Huntington, NY), and
1-PI
mRNA was normalized to amounts of GAPDH.
Culture medium was
collected either at 24 or 48 h after factor(s) addition. In the
latter case, however, the culture medium was replaced at 24 h by
fresh medium containing stimulating factors. Thus, in all experiments,
the actual, quantitated stimulation of 1-PI production
was measured during a 24-h incubation period. The amount of secreted
1-PI was determined by rocket immunoelectrophoresis using polyclonal rabbit antibodies against human
1-PI
(Dako, Carpinteria, CA). Purified human plasma
1-PI
(kindly provided by Miles, Inc., Berkeley, CA) was used as a
standard.
The microheterogeneity
of both 1-PI and
1-acid glycoprotein
(AGP) was analyzed by crossed affinoimmunoelectrophoresis (CAIE) with
ConA, using 1% agarose in Tris-barbital buffer (11). At first,
proteins were resolved by electrophoresis in agarose gel (3 h at 10 V/cm) incorporated with ConA (1. 5 mg/ml). A second dimension
electrophoresis (overnight, 2 V/cm), vertical to the first separation,
was carried out in a gel containing either specific anti-
1-PI or anti-AGP antibodies and 60 mg/ml
methyl-
-D-glucopyranoside. The concentration of
antibodies and the amounts of sample were adjusted in different
experiments to produce precipitation arcs of comparable intensity and
height. Gels were stained with Coomassie Blue to demonstrate precipitin
lines. The area covered by
1-PI glycoforms was
determined by planimetry and the amount of ConA-nonreactive fraction
expressed as a percentage of total
1-PI.
The
separation of glycoforms of 1-PI was achieved by
affinity chromatography using ConA-4B Sepharose column. After
application of aliquots of serum, LLF, or HTB55 cell medium, the
ConA-nonbinding fraction was eluted with Tris-HCl buffer, pH 7.6, containing 0.15 M NaCl and 1 mM each of
MnCl2, MgCl2, and CaCl2. ConA-bound
material was desorbed with 0.2 M
-methyl-D-mannoside in the same buffer. Fractions were
dialyzed against 25 mM ammonium bicarbonate, lyophilized, and dissolved in 1:10 of the original volume. The total amount of
recovered
1-PI in each fraction was measured by rocket
immunoelectrophoresis using a specific antiserum against
1-PI, whereas the composition of glycoforms was
determined by affinoimmunoelectrophoresis with ConA.
Aliquots of
serum, LLF, the ConA-nonbinding fraction of LLF, and HTB55 cell medium,
all containing defined amounts of 1-PI, were incubated
with equimolar amounts of HNE (a generous gift of Athens Research and
Technology, Athens, GA) for 30 min at room temperature in 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl. Untreated or
HNE-treated samples were then subjected to 9% SDS-PAGE (12).
1-PI was visualized by enhanced chemiluminescence (ECL;
Amersham) after electrotransfer to a nitrocellulose membrane and
incubation with antibodies. 1:2000 dilution of rabbit
anti-
1-PI antibodies (Dako) and 1:2000 dilution of
donkey anti-rabbit IgG antibodies conjugated to horseradish peroxidase
(Amersham) were used for Western blot analysis.
Confluent monolayers of HTB55 or normal bronchial
cells were stimulated for 18 h with 50 ng/ml OSM. The cells were
then rinsed and incubated for 4 h in the presence of
methionine-free medium containing OSM and 200 µCi/ml
[35S]methionine/cysteine (Tran35S-label) (ICN
Biomedicals, Inc., Costa Mesa, CA). Aliquots of medium were pretreated
with preimmune serum and Pansorbin (Calbiochem-Novabiochem, La Jolla,
CA), as described previously (9). The supernatants were then incubated
overnight at 4 °C in 20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1% Triton X-100, with excess
anti-1-PI antibody. Immune complexes were precipitated
with protein A-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), washed, released by boiling in Laemmli sample buffer, and examined
on SDS-PAGE. Bands were detected by fluorography as described elsewhere
(13).
Both inflammatory cytokines and the glucocorticoid analog
dexamethasone were tested for their ability to regulate the synthesis of 1-PI in lung adenocarcinoma HTB55 cells. As
demonstrated in Fig. 1, A and B,
expression of
1-PI was stimulated with OSM and to a
lesser extent by dexamethasone. Although the up-regulation of
1-PI synthesis by OSM was also noted at the protein
level, the effect of dexamethasone on
1-PI protein
synthesis was variable as demonstrated by a high S.D. value (Table
I). As might be expected, these two factors acted
additively for the up-regulation of
1-PI expression,
which was also observed at the protein level (Fig. 1; Table I).
However, no stimulation of
1-PI synthesis was obtained when IL-1 was given alone, although this cytokine appeared to enhance
the effect of either OSM or dexamethasone. In fact, the most
significant up-regulation of
1-PI synthesis was noted
when OSM, dexamethasone, and IL-1 were given in combination. No
significant stimulation of
1-PI expression was observed
following either IL-6 or LIF treatment (Fig. 1), although both
cytokines were capable of stimulating
1-PI production in
HepG2 cells (Table I).
|
Northern blot analysis of RNA extracted from
normal bronchial epithelial cells indicated that 1-PI
mRNA levels were substantially increased by OSM or a combination of
OSM and IL-1, and somewhat by IL-1 alone, whereas IL-6 had no effect
(Fig. 2A). The stimulatory effect of OSM and
OSM in combination with IL-1 on
1-PI message expression
correlated with corresponding changes in secretion (Fig.
2C). In the case of OSM, the up-regulation of
1-PI synthesis was observed as early as 2 h after
addition of this cytokine, with maximum stimulation occurring after
8 h, and no further significant increase in
1-PI
production after prolonged time of cell exposure to OSM (Fig.
3). The increase in synthesis of
1-PI by
OSM-exposed cells was also concentration-dependent and
occurred only at cytokine concentrations above 10 ng/ml, reaching a
maximum at about 50 ng/ml and remaining approximately at the same level
at higher OSM concentrations (Fig. 3A).
Analysis of
CAIE with ConA revealed that three microheterogeneous forms
of 1-PI were secreted by liver-derived HepG2 cells,
whereas only one was secreted by lung-derived HTB55 cells (Fig.
4). ConA binds with bi- but not with tri- or
tetra-antennary carbohydrate structures, and in the case of
multiheteroglycan proteins, the degree of reactivity with this lectin
depends on the number of bi-antennary structures present on the
molecule (14). The three variants of
1-PI present in
HepG2 medium can thus be described as either nonreactive (the electrophoretic migration of this form in a gel containing ConA is not
affected), weakly reactive (retarded electrophoretic mobility in
ConA-agarose gel), and strongly reactive with ConA (immobilized in
ConA-agarose gel electrophoresis), respectively. The
1-PI secreted by HTB55 cells was only of the
ConA-nonbinding type, as confirmed by affinity chromatography using
ConA-4B Sepharose, where all of the applied
1-PI passed
directly through the column (0.6 × 7 cm) without being bound or
even retarded.
Analysis of
1-PI appears to enter the lung by diffusion
from blood plasma (2). However, CAIE analysis of
1-PI
from serum and LLF of the same healthy donor demonstrated a higher
proportion of the ConA-nonbinding
1-PI glycoform in LLF,
which may reflect its local synthesis (Fig. 5). Using
planimetry, it was found that the amount of the ConA-nonbinding
1-PI fraction, expressed as percentage of total
1-PI, was about 12% higher in LLF (17.2 ± 1.5 in
LLF versus 4.8 ± 0.9 in serum, mean ± S.D. of
three separate determinations), and the difference was independent of
the concentration of ConA and methyl-
-D-glucopyranoside,
parameters known to affect the separation on CAIE (11). ConA CAIE
analysis of serum mixed with HTB55 cell medium resembled that of LLF
with respect to
1-PI (Fig. 5). A decrease in ConA
reactivity appeared to be specific for
1-PI, because
there was no difference in the ConA reactivity of AGP derived from the
same serum and LLF (Fig. 5). It is important to note that, in contrast
to
1-PI, AGP was not synthesized by lung-derived HTB55
cells (data not shown). To exclude a possibility that differences in
ConA reactivity observed between
1-PI from serum and LLF
were due to inactivation of this inhibitor,
1-PI from
LLF was separated on a ConA-Sepharose 4B column, and the nonbinding
fraction of the inhibitor was tested for the ability to form a complex
with HNE. As demonstrated in Fig. 6, this fraction of
1-PI did form such a complex.
Inhibition of HNE by
To examine if lung epithelial cell-derived
1-PI exhibits inhibitory activity comparable to serum
1-PI, the formation of an SDS-stable complex with HNE
was studied in detail. For this experiment, the condition medium from
OSM-stimulated HTB55 cells was subjected to rocket
immunoelectrophoresis to determine the concentration of
1-PI, and the medium was then incubated with various
amounts of HNE. At 0.2 HNE:
1-PI molar ratio, a shifted (
1-PI-HNE complex) band appeared (Fig.
7A). At higher concentrations of HNE, the
intensity of the shifted band increased, and native
1-PI
decreased, with all detectable
1-PI being in complex
with HNE when the concentration of this enzyme was equimolar to
1-PI (Fig. 7A). This result was similar to
that observed when human serum
1-PI was reacted with HNE
(Figs. 6 and 7).
To determine if 1-PI secreted by normal bronchial
epithelial cells was functionally active, we compared its ability to
form a complex with HNE to that exhibited by HTB55 cell-derived
1-PI. As illustrated in Fig. 7B, at a molar
excess of HNE over
1-PI, both HTB55- and normal
bronchial cell-derived
1-PI was detected in complex with
HNE, concomitant with the disappearance of the band corresponding to
native inhibitor. The additional band visible in case of bronchial
cells most probably represents nonspecific binding because it can be
removed by an additional preclearing step using preimmune serum (data
not shown).
At sites of inflammation or tissue injury, proteolytic enzymes,
released from phagocytes, may directly contribute to the host inflammatory response as well as to tissue destruction. The implication of these proteinases, including HNE, in the pathogenesis of lung emphysema as well as the finding that extrahepatic cells produce 1-PI suggest that locally synthesized
1-PI is likely to have an effect on the antielastase
screen of lung tissue.
In this study, we demonstrated that cells originating from the
respiratory tract epithelium are capable of 1-PI
synthesis and that several mediators of inflammation increase the
expression of this inhibitor in these cells. However, regulation of
1-PI in HTB55 and normal bronchial epithelial cells
differs significantly from that in hepatic cells. IL-6-type cytokines
(IL-6, OSM, LIF, IL-11, and ciliary neurotrophic factor) up-regulate
the production of
1-PI in hepatocytes (Ref. 15; Table
I), whereas OSM and to a lesser extent IL-1, but not IL-6 or LIF, exert
a significant increase in
1-PI levels in lung-derived
epithelial cells (Figs. 1 and 2). This strong effect of OSM and the
lack of effect of IL-6 and LIF on production of another inhibitor of
serine proteinases, Achy, was also observed in epithelial cells
originating from the lung (16). However, IL-1 appears to be a much more
potent stimulator of Achy than
1-PI synthesis in these
cells. It is important to note that this difference in the effect of
OSM and IL-1, on Achy expression, compared with that of IL-6, was also
observed in breast-derived epithelial cells (5).
Many of the overlapping biological responses of IL-6-related cytokines,
including stimulation of 1-PI production in hepatoma cells, have been explained by the presence of a common subunit, gp130,
in cytokine receptors, or in the case of OSM and LIF, by the finding
that both cytokines bind with high affinity to the same receptor, the
latter consisting of two subunits, the LIF receptor
chain and gp130
(17). However, the regulation of
1-PI expression in
lung-derived epithelial cells, relative to hepatocytes, illustrates
significant functional differences among IL-6-type cytokines. The
exclusive effect of OSM on induction of several biological responses
has been described previously (18). However, the mechanism of this
process is not yet fully understood, and the stimulation of different
patterns of tyrosine-phosphorylated proteins by OSM and LIF, as well as
the finding that some cells bind OSM but not LIF, confirms the
existence of an OSM-specific receptor (19). It is important to note
that in lung-derived epithelial cells OSM, but not LIF or IL-6, exerted
an effect on synthesis of another inhibitor of serine proteinases, Achy
(16). Analysis of the mechanism(s) by which OSM wields its stimulatory action may help to explain the sensitivity of lung-derived epithelial cells to this cytokine.
Apart from the differential regulation of 1-PI synthesis
in hepatic and epithelial cells, a significant difference in the glycosylation pattern of the inhibitor was also found. The latter effect was clearly demonstrated by ConA reactivity, which indicated three hepatic and a single ConA-nonbinding epithelial glycoform (glycoforms) of
1-PI (Fig. 4). This observation may
suggest a different clearance mechanism for lung-derived epithelial
1-PI, because the half-life of the liver form of this
inhibitor is, at least in part, dictated by the number and type of
carbohydrate side chains (2). Furthermore, hepatocytes respond to
inflammatory stimuli by changing the synthesis and type of
N-glycosylation of many proteins, including
1-PI (20, 21). It has been shown previously that IL-6,
LIF, and interferon-
decreased the ConA reactivity of HepG2-derived
1-PI (21). A similar effect was observed when HepG2
cells were stimulated with OSM (data not shown). However, although this
cytokine was the most potent in up-regulation of
1-PI in
lung-derived epithelial cells, it did not change the ConA reactivity of
the inhibitor (data not shown). A significant difference in the binding
to ConA of
1-PI secreted by HepG2 and HTB55 cells
suggested that it could be useful in determining the contribution of
epithelial cells to the local synthesis of
1-PI in the
lung. Because
1-PI is believed to diffuse from serum
into most organs (2), the proportion of ConA-nonreactive
1-PI glycoform should be higher in lung fluids compared
with serum if lung epithelial cells are a local source of this
inhibitor. Such a difference was clearly observed between LLF and serum
from a single healthy donor (Fig. 5). To mimic the postulated
contribution of lung-derived epithelial cells to the synthesis of
1-PI, the serum was mixed with HTB55 cell medium, and
the mixture was examined using CAIE. The results obtained clearly
confirm the suggested interpretation because the separation of
1-PI in this synthetic mixture resembled that in
LLF.
It is possible that the difference in the proportion of
ConA-nonreactive 1-PI in LLF when compared with serum is
due to selective transportation into, or concentration within, the
lung. This is, however, less likely because the ConA reactivity of AGP
was unchanged in LLF. We also examined whether the faster migration of
LLF-derived
1-PI on ConA CAIE might be due to
modifications of serum-derived
1-PI within the lung.
This was important in view of the fact that differences in the
migration of
1-PI on agarose gel electrophoresis and
crossed immunoelectrophoresis in lung-derived fluids, when compared
with serum, have been described, and in those studies,
1-PI originating from the respiratory tract appeared to
be inactive (22, 23). However, our results of SDS-PAGE analysis
demonstrate that the ConA-nonreactive fraction of
1-PI
was active as an inhibitor of HNE (Fig. 6), which suggests that
although
1-PI is modified within the lung, such a
modification does not seem to be manifested by an increase in the
amount of ConA-nonreactive
1-PI.
The data we have obtained also show that 1-PI is
produced as a fully active inhibitor by lung-derived epithelial cells
(Fig. 7), which strongly supports a role for these cells in the local synthesis of
1-PI. It is, however, a matter of further
analysis to establish other sources of
1-PI in the lung,
as well as to examine the significance of local production of this
inhibitor in protecting the lung tissue from irreversible destruction
during inflammatory episodes.
We thank C. Land for technical assistance and Dr. T. Kordula for review of the manuscript.