Research Service, Department of Veterans Affairs Medical Center, Omaha 68105; and Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198
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ABSTRACT |
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Bronchial epithelial cell migration,
attachment, and proliferation are important processes in response to
airway injury. We have shown that tumor necrosis factor (TNF)-
stimulates the migration of bovine bronchial epithelial cells (BBEC) in
vitro. We hypothesized that protein kinase C (PKC) may be one of the
intracellular signaling mediators of TNF-
in BBEC. In this study, we
have identified multiple PKC isoforms in BBEC and measured total
cellular PKC activity. Polyclonal antibodies to the PKC-
,
-
2, -
, and -
isoforms
recognized protein bands around 80-90 kDa. BBEC primary cultures
treated with either 500 U/ml TNF-
for 2-4 h or 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate
for 15 min resulted in three- to fivefold increases in PKC activity in
the particulate fractions of crude cell lysates. This activity was
inhibited by 1 µM calphostin C or 10 µM H-7. Similarly,
TNF-
-stimulated BBEC migration was reduced at least twofold in the
presence of H-7 or calphostin C. These studies suggest that the
activation of PKC is necessary for TNF-
-stimulated BBEC migration.
protein kinase C isoenzymes; epithelial cell migration; enzyme activation
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INTRODUCTION |
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BRONCHIAL EPITHELIAL CELL migration, attachment, and proliferation are important processes in the physiological response to wound healing as well as in the pathogenic response to such lung diseases as chronic bronchitis and asthma. Various growth factors and cytokines have been identified that modulate epithelial cell growth rates, extracellular matrix production, and cellular morphology. Dissecting the molecular physiology of those enzymes that regulate signal transduction is essential to understanding the relationship between pharmacological stimuli and the morphological responses observed in these cells.
Protein kinase (PK) C is stimulated by numerous calcium-elevating agents and tumor promoters and is a major activator of the serine-threonine phosphorylation pathway (19). PKC is represented by a family of isoenzymes that have been identified to coexist in many cell types (3, 8). Some of these isoforms have been demonstrated to be calcium and/or phospholipid independent (11). Therefore, each isoenzyme, although similar in structure and sequence, may play a unique role in a compartmentalized state within the cell.
Immune reactions are effected by soluble cytokine mediators such as
tumor necrosis factor (TNF; see Ref. 1). These cytokines are produced
primarily, but not exclusively, by monocytes and act upon their target
cells to induce further proinflammatory mediators. Two classes of
TNF- receptors (high and low affinity) are found to exist
ubiquitously throughout most tissues, although the relationship between
these receptors and the molecular mechanism of TNF-
signal
transduction is unknown.
It has been suggested that PKC is one of the intracellular signaling
mediators of TNF- effects (22). However, it has also been reported
that cell treatment with TNF-
results in the activation of adenylate
cyclase with an accompanying increase in adenosine 3',5'-cyclic monophosphate production (27). We have
previously shown that TNF-
stimulates the migration of bovine
bronchial epithelial cells (BBEC) in cultured monolayers (12), but the signal transduction mechanisms for cytokine-mediated bronchial epithelial cell migration are not understood, and the role of PKC in
these cells has not been investigated extensively. It is our hypothesis
that PKC activation plays a role in the cytokine modulation of
bronchial epithelial cell migration.
We investigated the relationship between in vitro bronchial epithelial
cell migration and PKC activity using kinase inhibitors, immunocytochemical methods, and direct assay of kinase activity. We
have identified, for the first time, several PKC isoforms in BBEC and
correlated BBEC PKC activity with the activation of cell migration in
response to the bioactive cytokine TNF-.
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METHODS |
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Reagents. Laboratory of Human
Carcinogenesis (LHC) basal medium was purchased from Biofluids
(Rockville, MD). RPMI 1640, Dulbecco's modified Eagle's medium
(DMEM), minimum essential media (MEM), streptomycin-penicillin, and
fungizone were purchased from GIBCO (Chagrin Falls, OH). Extraction of
frozen bovine pituitaries from Pel Freez (Rogers, AR) was performed as
previously described and yielded an extract containing 10 mg/ml protein
(15). Recombinant human TNF- was purchased from Genzyme (Cambridge,
MA). Purified PKC, PKC isoform marker peptides, and polyclonal rabbit
antisera to the PKC isoenzymes were obtained from Calbiochem (San
Diego, CA). Peroxidase-conjugated goat anti-rabbit immunoglobulin G was purchased from Cappel (Durham, NC). All other reagents not
specified were purchased from Sigma Chemical (St. Louis, MO).
Cell preparation. As previously described (25), the cells were prepared from bovine lung obtained fresh from a local abattoir. Bronchi were necropsied from the lung, cleaned of adjoining lung tissue, and incubated overnight at 4°C in 0.1% bacterial protease (type IV) in MEM. After the overnight incubation, the bronchi were rinsed in DMEM with 10% fetal calf serum repeatedly to collect the cells lining the lumen. These cells were then filtered through a 250-µm nylon mesh and were washed again in DMEM. This technique typically produces a high-viability cell preparation of >95% epithelial cells (24). The cells were then washed in DMEM, counted with a hemacytometer, and plated in 1% collagen-coated 100-mm polystyrene culture dishes at a density of 1 × 104 cells/cm2 in a 1:1 medium mixture of LHC-9 and RPMI (15). Cell incubations were performed at 37°C in humidified 95% air-5% CO2. Confluent monolayers of cells were obtained every 3 days. At this time, each 100-mm dish contained ~2 mg of total cellular protein. Primary cultures of BBEC were used for these studies because it has been suggested that tissue culture artifacts may induce the downregulation of enzyme activity in the late-passaged cell (7).
DEAE chromatography of BBEC fractions.
Because the relative amounts of some PKC isoforms may exist in
concentrations beyond detectability by Western blot of crude whole cell
fractions, we attempted large-scale fractionation and concentration of
PKC by ion-exchange chromatography. Chromatography of PKC isoforms was performed as follows. Confluent monolayers of BBEC (>1 × 108 cells) were extracted in 10 ml
of buffer containing 10 mM
KH2PO4, 1 mM EDTA, and 25 mM 2-mercaptoethanol (KPEM). The cells were sonicated
and centrifuged at 10,000 g for 30 min
at 4°C. The pellet fraction was sonicated further in 12 mM ethylene
glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA) and 0.1% Triton X-100. Both supernatants were applied to a
DEAE-Sephacel column (0.9 × 10 cm) equilibrated in KPEM buffer.
The column was washed with 100 ml of KPEM and was developed with a
30-ml NaCl linear gradient (0-500 mM), and 1-ml fractions were
collected. Individual fractions collected were analyzed for protein
content [by the method of Bradford (5)], NaCl
concentration, and immunoreactivity for PKC isoforms.
Western blotting. PKC distribution was
determined by Western blot of BBEC subcellular fractions. After
stimulation of the cells, the medium was removed, and the cells were
sonicated (20 s, 1 pulse) at 4°C in 0.5 ml of lysis buffer
consisting of 35 mM tris(hydroxymethyl)aminomethane
(Tris) · HCl (pH 7.4), 0.2 mM phenylmethylsulfonyl
fluoride, 10 mM MgCl2, 10 mM
2-mercaptoethanol, and 5 µg/ml leupeptin. The lysis buffer also
contained 3 mM CaCl2 or 9 mM EGTA
in the presence or absence of 1% Triton X-100, depending upon which
fraction was being homogenized. Sonicates were then centrifuged for 30 min at 10,000 g at 4°C. Cell
fractions were solubilized in sodium dodecyl sulfate
(SDS)-2-mercaptoethanol reducing buffer, and equal amounts of protein
per sample (20-200 µg) were resolved on 10% SDS-polyacrylamide
gels and transferred to polyvinylidene difluoride membranes.
Transferred gels were stained with Coomassie R-250 to determine
completeness of transfer. Blots were blocked overnight with a blocking
buffer of 3% bovine serum albumin in 50 mM Tris (pH 7.5), 150 mM NaCl,
and 0.1% NaAz at 4°C with constant rocking. Membranes were
incubated with rabbit anti-PKC-,
-
1,
-
2, -
, -
, -
, -
,
-
, or -
(1:2,000 dilution in blocking buffer) for 1 h at room
temperature. As a control, membranes were incubated in either rabbit
preimmune serum or in the absence of primary antibody. Membranes were
washed three times in blocking buffer with 0.02% Nonidet P-40 for 20 min during each wash. Blots were incubated with horseradish
peroxidase-labeled goat anti-rabbit secondary antibody diluted 1:10,000
in blocking buffer for 30 min at room temperature. Membranes were
washed again as described, and immunoreactivity was visualized by
chemiluminescence as detected on autoradiographic film. Any
immunoreactive bands were identified as occurring in the cytosolic or
particulate (membrane) fraction, and their relative molecular masses
(kDa) were calculated for each isoform blotted.
PKC activity assay. PKC activity was
determined in both DEAE fractions as well as in crude whole cell
fractions of bronchial epithelial cells. The assay employed was a
modification of procedures previously described (9, 10) using 50 µg/ml PKC substrate peptide, 12 mM
Ca(C2H3O2)2,
8 µM phosphatidyl-L-serine, 24 µg/ml 12-O-tetradecanoyl-phorbol-13-acetate
(PMA), 30 mM dithiothreitol, 150 µM ATP, 45 mM
Mg(C2H3O2)2,
and 10 µCi/ml
[-32P]ATP in a
Tris · HCl buffer (pH 7.5). Samples (20 µl) were
added to 40 µl of the above reaction mixture and were incubated for 15 min at 30°C. Incubations were halted by spotting 50 µl of each sample onto P-81 phosphocellulose papers (Whatman). Papers were then
washed five times for 5 min each in phosphoric acid (75 mM) and one
time in ethanol and then were dried and counted in nonaqueous scintillant as previously described (21). Negative controls consisted
of similar assay samples with 12 mM EGTA or without substrate peptide.
A positive control of 0.35 mg/ml purified rat brain PKC (Calbiochem)
was included as a sample. Kinase activity was expressed in relation to
total cellular proteins assayed and was calculated in picomoles per
minute per milligram. All samples were assayed in triplicate, and no
less than three separate experiments were performed per unique
parameter. Data were analyzed for statistical significance using
Student's t-test.
Bronchial epithelial cell migration. Bronchial epithelial cell migration assay was performed with the Boyden chamber technique (4, 6, 20, 23) using a 48-well multiwell chamber (Neuroprobe, Bethesda, MD). Polycarbonate membranes with 8-µm pores (Neuroprobe) were used. Membranes were coated with 0.1% gelatin (Bio-Rad, Richmond, CA) as previously described (6, 20, 23). Varying concentrations of fibronectin were used in the bottom wells as attractants. Bronchial epithelial cells were placed into each of the top wells above the filter. The chambers were then incubated at 37°C with 5% CO2 for 6 h. After incubation, cells on the top of the filter were removed by scraping. The filter was then stained with a modified Wright stain (Leukostat; Fisher Scientific, Fairlawn, NJ). Epithelial cell migration activity was quantified as the number of migrated cells on the lower surface of the filter in 10 high-power fields (HPF) using a light microscope at ×400 magnification. The results represent means ± SE for triplicate wells.
As an additional control, the above assays incorporated the treatment
of epithelial cells with various concentrations of TNF- or PMA in
the presence of PKC inhibitors (H-7 and calphostin C) for various time
points. Controls included unstimulated cells and incubation of cells
with PKC inhibitors without TNF-
or PMA stimulation. Triplicates
were performed for each assay. Data are expressed as mean ± SE
cells migrated per HPF from triplicates assayed. Statistical
significance was determined by Student's t-test and was considered significant
at P < 0.01.
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RESULTS |
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Identification of PKC isoforms by Western
blot. We have utilized Western blotting to identify the
various PKC isoforms expressed in DEAE-Sephacel fractions of primary
BBEC. Monolayers of BBEC were lysed and Triton extracted, and
supernatants were loaded onto a DEAE-Sephacel column. The column was
then developed in a linear NaCl gradient of 0-0.5 M. The
subsequent chromatograph revealed three distinct peaks of PKC activity
eluting at ~0.1, 0.3, and 0.4 M NaCl. Utilizing polyclonal antibodies
to the specific unique peptide regions of nine different PKC
isoenzymes, we have identified the presence of -,
2-, and
-isoforms as major
immunoreactive bands ranging in molecular mass from 78 to 82 kDa in the
BBEC (Fig. 1). Additionally, we have
identified a 90-kDa immunoreactive band for the
-isoform (Fig. 1).
The presence of multiple bands within the same cell type was
not surprising and has been previously reported in other cell types (3,
14, 17).
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PKC activation assays. In an attempt
to identify PKC activity in crude cell fractions, we have assayed PKC
activity in cytosolic and particulate fractions of BBEC. Primary
cultures of BBEC were treated with various concentrations of TNF-
(0-500 U/ml) for various time points (2-48 h) in multiple
100-mm tissue culture dishes. The cells were then separated into a
cytosolic fraction and a detergent-extracted fraction from the
particulate. Both fractions were assayed for PKC kinase activity. PKC
activity changes in TNF-
-stimulated epithelial cells were compared
with unstimulated control cells. We determined the concentration curve
for this TNF-
-mediated response to maximally stimulate PKC activity
at a concentration of 500 U/ml (Fig. 2). At
this concentration of TNF-
, no significant cell death was observed
because BBEC viability was determined to be >96% by trypan blue
exclusion assay.
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Additionally, we observed that a time-dependent translocation (2-4
h) of peak PKC activity occurs in primary cultures of BBEC treated with
500 U/ml TNF- from the cytosol to the particulate fraction (Fig.
3). TNF-
-stimulated PKC activity reached
its maximal levels from 2 to 4 h and subsided to near-unstimulated
levels by 24 h. We observed that PKC inhibitors such as calphostin C and H-7 abrogate this TNF-
-mediated PKC activity (Fig.
4). As expected, calphostin C (1 µM)
completely inhibited the activity of PKC in both the cytosolic and
particulate fractions, whereas H-7 (10 µM) reduced PKC activity well
below unstimulated resting cells. At these concentrations of calphostin
C and H-7, no significant cell death was observed by the cellular
uptake of trypan blue dye (viability >92%). Compared with early
primary cells (>1 wk old), older cultures of BBEC (<2 wk old) as
well as passaged cells demonstrate a distinct loss of particulate
fraction PKC activity in response to TNF-
stimulation (data not
shown).
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As a positive control, cells were treated with PMA (100 ng/ml for 15 min) to activate PKC. We observed a significant increase in PKC activity in the particulate fraction of BBEC treated with PMA compared with the cytosolic fraction, suggesting that a translocation of PKC from the cytosol to a component of the particulate fraction was occurring in those BBEC under conditions of activated PKC (data not shown).
TNF--stimulated BBEC migration. To
examine the role of PKC in TNF-
-stimulated bronchial epithelial cell
migration to fibronectin, bronchial epithelial cells were treated with
TNF-
for various times and were evaluated for migration in Boyden
blind-well chamber assays. Bronchial epithelial cells treated with
TNF-
for 4 h before assay showed significantly increased migration
to fibronectin compared with control cells (Fig.
5). Exposure of the cells for up to 24 h
with TNF-
resulted in the stimulation of increased BBEC migration to
fibronectin. However, long-term stimulation of the cells with TNF-
(24-50 h) led to a significant decrease in cell migration compared
with 4 h of TNF-
treatment. Similar to the inhibition of PKC
activity, BBEC pretreated with the PKC inhibitors H-7 (10 µM) or
calphostin C (1 µM) for 30 min before TNF-
stimulation resulted in
a 50% reduction in cell migration (Fig.
6). Incubation of BBEC with these
inhibitors alone resulted in no significant change in migration from
control unstimulated cells.
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DISCUSSION |
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The presence of multiple PKC isoforms in BBEC may represent a
multifaceted role for both calcium-dependent and calcium-independent PKC signaling in highly compartmentalized BBEC. Heterogeneous subpopulations of BBEC may exist in our cultures, each containing unique isoenzymes. However, the presence of various numbers of ciliated
and nonciliated BBEC in these primary cultures has not contributed to
significant differences in the magnitude or response of our BBEC
cultures to TNF--mediated PKC activation. Continued studies designed
to answer this question consist of immunofluorescence microscopy of
both primary cultures of BBEC as well as immunohistochemistry of
Formalin-fixed bronchial thin sections utilizing antibodies to the PKC
isoforms.
Our data suggest the presence of at least two distinct peaks of
calcium-dependent kinase activity in the elution profile of BBEC DEAE
fractions. These peaks may represent the distinct elution of the
calcium-dependent isoform- and -
2. Another smaller
peak of PKC activity corresponding to PKC-
demonstrated
calcium-independent activity. The identification of PKC-
primarily comes from Western blots of crude cell lysates. This isoform
appears to be localized to the particulate fraction, possibly the
cytoskeleton, and therefore may be lost in the pellet fraction before
the DEAE column was loaded. It has been previously reported that
certain PKC isoforms are localized on the microfilaments and
intermediate filaments (18).
The presence of conventional PKC isoforms is also demonstrated by the
calcium-dependent kinase activity observed in BBEC extracts. The
presence of PKC- and PKC-
2
isoforms could be responsible for the translocatable PKC activity
observed in the particulate fractions of BBEC stimulated with TNF-
.
Additionally, such isoforms as PKC-
and PKC-
could be responsive
to phorbol ester stimulation. Maximal or peak PKC activity is detected
in the particulate fractions of cells treated with the phorbol ester
PMA or with TNF-
. This suggests that a translocatable form of PKC is
present in the BBEC, which may represent
- and/or
2-enzyme activity in the BBEC. However, this study does not establish that translocation of one or
more PKC isoforms is entirely responsible for TNF-
-stimulated migration. We feel that the coincident activation of PKC with the
stimulation of migration and the inhibition of that migration by PKC
inhibitors link the requirement of PKC activation with TNF-
-stimulated migration. Whether or not the targeting of specific isoforms to specific regions of the cells occurs must be addressed in a
future study.
The time of maximal BBEC-stimulated migration (~4-6 h)
correlates well with the time of peak PKC particulate fraction
activity. However, as PKC activity begins to subside after 6 h of
continual stimulation with TNF-, migration of the cells to
fibronectin continues at an increased rate up to 24 h of TNF-
treatment. Inhibition of PKC activity by calphostin C or H-7 resulted
in the inhibition of TNF-
-stimulated migration. Together, these data
suggest that PKC activation is required for migration to be initiated
in response to TNF-
. PKC activation is apparently required as an
upstream initiator of BBEC migration but is not necessary for continued
long-term migration in response to TNF-
. Although TNF-
has been
shown to activate other kinases in other cell types, we found no
inhibition of TNF-
-stimulated cell migration in the presence of the
protein tyrosine kinase inhibitor genistein (25-100 µM) nor did
we detect any TNF-
-stimulated activation of PKA or PKG in our
control studies (data not shown).
When BBEC are stimulated with TNF- over long periods of time
(24-48 h), the rate of cell migration to fibronectin begins to
decrease. TNF-
receptor shedding may play a role in this longer time
course TNF-
insensitivity. It has been reported that normal human
airway epithelial cells shed soluble type I TNF-
receptors upon
activation of PKC by PMA (16). Levine et al. (16) demonstrated that
maximal TNF-
receptor shedding occurred at 24 h of stimulation by
PMA and that receptor shedding could be halted by PKC inhibitors. This
delayed loss of TNF-
receptors was observed despite the fact that
PKC was most likely fully activated within minutes of PMA treatment.
Our findings in BBEC support those of Levine et al. (16). Activation of
PKC by TNF-
may result in an eventual decrease in PKC activity over
a period of several hours due to the downregulation of the TNF-
receptor. Thus the very signal produced by TNF-
stimulation (PKC
activation) may lead to the downregulation of TNF-
responsiveness by
stimulating TNF-
receptor shedding. As the maximal amount of TNF-
receptors are shed beginning at 24 h, TNF-
is no longer able to
stimulate BBEC migration, and the decrease in cell migration to
fibronectin is observed at these extended time points.
Alternatively, the loss of TNF--stimulated cell migration over very
long periods of time could be explained through the loss of expressed
PKC activity in BBEC. It has been previously reported that BBEC extrude
filopodia in response to PKC-activating agents such as phorbol esters
and calcium ionophores (2). It was noted that a steady loss in the
filopodia-inductive response occurred over the first several days in
culture. Although no kinases were assayed, the loss of phorbol
ester-stimulated filopodia could be the result of a downregulation of
PKC expression or a loss of PKC activity. The loss of an enzyme
activity over the course of several passages of certain cultured cell
lines has been previously reported in numerous studies because cell
adaptation is often associated with changes in specific proteins (7).
In this report, our studies were limited to primary cultures of BBEC
used at ~3 days ex vivo. However, we have observed diminished PKC
activity in response to TNF-
as well as to PMA in passaged cells and
in cells that are >1 wk old in culture (unpublished observation). This possible artifact of tissue culture leading to decreased PKC
activity over time may be involved in the decreased stimulation of cell
migration over several days of TNF-
treatment.
The widespread application of kinase inhibitors instead of direct
kinase activity assays has often led to a lack of kinase specificity
and cytotoxicity in the cellular model (13, 26). Because of these
limitations, we have chosen to measure the PKC activation state of BBEC
in the presence of both TNF- and PKC inhibitors rather than only
assaying BBEC migration in the presence of PKC inhibitors. As
isoform-specific substrates and inhibitors become available, it will be
important to dissect the role of these PKC isoenzymes in regulating
TNF-
-stimulated migration, receptor shedding, and substrate
phosphorylation. Until activity assays specific for individual PKC
isoforms can be performed, it is difficult to make any quantitative
comparison of isoform activity within the intact cell in response to an
agent such as TNF-
. Although the separation of the individual
isoforms is possible by immunoprecipitation, our preliminary studies
have shown that the binding of specific antibody to PKC isoforms
inhibits PKC activity (data not shown).
This report represents the initial observation of multiple PKC isoforms
in airway epithelial cells and the composite PKC activity in response
to TNF- stimulation. Our data also show that inhibition of PKC
activity results in the inhibition of BBEC migration to fibronectin in
response to TNF-
. This observation, combined with previous studies
of other PKC effects on the airway epithelial cell (i.e., receptor
shedding), suggests a multifunctional role for multiple PKC isoenzymes
found in the BBEC. Elucidating those roles for PKC in the bronchial
epithelial cell may contribute to a better understanding of how
cytokines affect the airway cells in both the physiological and
pathological states. Understanding the mechanisms that regulate
epithelial cell migration are tantamount to elucidating the processes
of airway injury and repair. These mechanisms are thought to reflect
complex interactions between the inflammatory cells, proinflamatory
cytokines, and epithelial cells. Determining which
intracellular kinases are involved in the responses of bronchial
epithelial cells to cytokines and under what conditions those kinases
are activated may lead to the development of pharmacological agents
that can be employed to modulate such responses at the level of
phosphate signaling.
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ACKNOWLEDGEMENTS |
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This work was supported by a Department of Veterans Affairs Merit Review grant (to J. R. Spurzem) and by the American Lung Association (T. A. Wyatt).
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FOOTNOTES |
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Address for reprint requests: T. A. Wyatt, Dept. of Internal Medicine, Pulmonary and Critical Care Medicine Section, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-5300.
Received 9 January 1997; accepted in final form 7 August 1997.
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