Constitutive nitric oxide production by rat alveolar
macrophages
P. R.
Miles1,2,
L.
Bowman1,
A.
Rengasamy1, and
L.
Huffman1,2
1 Health Effects Laboratory
Division, National Institute for Occupational Safety and Health,
Morgantown 26505; and 2 Department
of Physiology, West Virginia University School of Medicine,
Morgantown, West Virginia 26506
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ABSTRACT |
Results from previous studies suggest that
alveolar macrophages must be exposed to inflammatory stimuli to produce
nitric oxide (· NO). In this study, we report that naive
unstimulated rat alveolar macrophages do produce · NO and
attempt to characterize this process. Western blot analysis
demonstrates that the enzyme responsible is an endothelial nitric oxide
synthase (eNOS). No brain or inducible NOS can be detected. The rate of
· NO production is ~0.07
nmol · 106
cells
1 · h
1,
an amount that is less than that produced by the eNOS found in alveolar
type II or endothelial cells. Alveolar macrophage · NO
formation is increased in the presence of extracellular
L-arginine, incubation medium containing magnesium and no
calcium, a calcium ionophore (A-23187), or methacholine. · NO
production is inhibited by
NG-nitro-L-arginine methyl ester
(L-NAME) but not by
NG-nitro-L-arginine,
L-N5-(1-iminomethyl)ornithine
hydrochloride, or aminoguanidine. Incubation with ATP, ADP, or
histamine also inhibits · NO formation. Some of these
properties are similar to and some are different from properties of
eNOS in other cell types. Cellular · NO levels do not appear
to be related to ATP or lactate content. Alveolar macrophage production
of · NO can be increased approximately threefold in the
presence of lung surfactant or its major component, dipalmitoyl phosphatidylcholine (DPPC). The DPPC-induced increase in · NO formation is time and concentration dependent, can be completely inhibited by L-NAME, and does not appear to be related to
the degradation of DPPC by alveolar macrophages. These results
demonstrate that unstimulated alveolar macrophages produce
· NO via an eNOS and that lung surfactant increases
· NO formation. This latter effect may be important in
maintaining an anti-inflammatory state in vivo.
lung surfactant; dipalmitoyl phosphatidylcholine; nitric oxide
synthase
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INTRODUCTION |
NITRIC OXIDE (· NO) is a free radical that is
produced by a variety of cell types in the lungs. Its synthesis from
L-arginine is catalyzed by nitric oxide synthase (NOS). It
is generally accepted that isoforms of the NOS enzyme fall into one of
two categories. One category is a constitutive form (cNOS), which is
regulated by calcium, and the other is an inducible form (iNOS), which
is inducible by cytokines and/or endotoxin and is
transcriptionally regulated (10). · NO generated by cNOS in
the lungs is found in endothelial cells (7) and airway neurons (4) and
seems to be important in the regulation of vascular and bronchial
smooth muscle activity. Recently, we have shown that alveolar type II cells also contain cNOS (24). On the other hand, other lung cell types
can generate · NO by means of an iNOS. For example, iNOS
activity has been reported in lung fibroblasts (14), pulmonary artery
smooth muscle cells (28), and neutrophils (33) after appropriate
stimulation.
Alveolar macrophages are mobile phagocytic cells located within the
alveoli and small airways of the lungs. These cells represent a primary
line of defense against the adverse effects of inhalation of bacteria
and foreign particles. It is known that alveolar macrophages do produce
· NO and that its synthesis is probably due to an iNOS. Several different investigators have reported that rat alveolar macrophages produce large quantities of · NO in response to
inflammatory stimuli such as interferon-
(IFN-
) and
lipopolysaccharide (LPS; see Refs. 14, 19, 29). These inflammatory
stimuli act by causing expression of iNOS mRNA (20, 21). On the other
hand, it does not appear that naive alveolar macrophages produce
· NO. For example, Persoons et al. (30) reported that
· NO production by these cells could only be detected after
exposure of rats to LPS. Also, Kobzik et al. (17) used
immunocytochemical techniques and found no NOS in normal rat alveolar
macrophages. However, after in vivo or in vitro treatment with LPS,
they found iNOS activity in these cells.
The results from all of these previous studies suggest that naive
alveolar macrophages do not produce measurable amounts of · NO and do not contain measurable amounts of NOS.
Furthermore, the available evidence suggests that · NO
formation by these cells occurs only after the induction of iNOS, which
can be produced by treatment with inflammatory stimuli, e.g., LPS
and/or IFN-
. Almost all of these previous measurements were
made over relatively long periods of time with the cells in culture. As
part of another investigation, Ischiropoulos et al. (13) did report
that freshly isolated, unstimulated alveolar macrophages do produce
· NO. However, to the best of our knowledge, the production
of · NO by naive alveolar macrophages during relatively short
incubation periods has not been studied in detail. In this paper, we
report that naive rat alveolar macrophages do produce · NO
and that · NO formation can be measured during incubation
periods as brief as 30 min. Therefore, the objectives of this
investigation were to 1) report this
finding, 2) identify the NOS protein
involved, 3) characterize the NOS
involved in terms of factors known to influence NOS isoforms in other
cell types, and 4) study possible
roles of NOS in alveolar macrophage function.
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METHODS |
Isolation of alveolar macrophages.
Specific pathogen-free male Sprague-Dawley rats (225-350 g;
Hilltop Laboratories, Scottdale, PA) were used for all experiments. The
animals were anesthetized with pentobarbital sodium (150 mg/kg body wt)
and exsanguinated by cutting the abdominal aorta. The trachea, heart,
and lungs were then removed from the animals intact. Alveolar
macrophages were obtained via bronchoalveolar lavage according to the
method of Myrvik et al. (27). The lungs from each rat were washed eight times with 5 ml of phosphate-buffered medium (in mM: 145 NaCl, 5 KCl,
9.4 Na2HPO4,
1.9 NaH2PO4,
and 5 glucose; pH 7.4) per gram of lung weight. The cells were
separated from the lavage fluid by centrifugation at 300 g for 10 min and then washed three
times by alternate centrifugation and resuspension in
phosphate-buffered medium. After the washing procedure, the cells were
resuspended in phosphate-buffered medium containing 1.8 mM
CaCl2, 1.0 mM
MgCl2, and 0.5% bovine serum
albumin (BSA) for use in all experiments, except for those done in
calcium- and/or magnesium-free medium in which the
CaCl2 and/or
MgCl2 was omitted from the medium.
The number of cells in the suspensions was determined by using an electronic cell counter (model ZB;
Coulter Electronics, Hialeah, FL). The cells used in these experiments
were >98% alveolar macrophages, with leukocytes as the contaminating
cells. The viability of the alveolar macrophages, as measured by oxygen
consumption and trypan blue exclusion, did not change during the
incubation periods used in these experiments.
Measurement of · NO
production. The amounts of · NO produced by
alveolar macrophages during various time periods were measured as the
stable oxidation products of · NO, nitrate, and nitrite. The
cells (4 × 106/ml) were
incubated at 37°C in phosphate-buffered medium containing 1.8 mM
CaCl2, 1.0 mM
MgCl2, and 0.5% BSA. The
inclusion of BSA in the medium had no effect on the measured
· NO levels. Some experiments were done in the absence of
extracellular calcium and/or magnesium. After the incubation,
cells were removed from the medium by centrifugation, and the
supernatants were saved for analysis. Each sample (0.3 ml) was first
incubated at 37°C for 1 h with Escherichia
coli nitrate reductase (in a 0.3-ml volume) to convert
the nitrate to nitrite. The total nitrite in the samples was then
measured using the Greiss reaction (11). Briefly, an aliquot (0.5 ml)
of each of the samples that had been incubated with the
E. coli enzyme was incubated at room
temperature for 10 min with the Greiss reagent (in a 1.0-ml volume),
and the absorbance at 550 nm was read with a spectrophotometer (model
35; Perkin-Elmer, Norwalk, CT). The amount of nitrate and nitrite in
the samples was calculated from a standard curve that was constructed
from sodium nitrite standards. Conversion of nitrate to nitrite was checked in each assay by using sodium nitrate standards.
The effects of different NOS inhibitors, L-arginine, a
calcium ionophore, dipalmitoyl phosphatidylcholine (DPPC) vesicles, lung surfactant, and other substances on · NO production by
alveolar macrophages were determined. Four different NOS
inhibitors, NG-nitro-L-arginine
methyl ester (L-NAME; Sigma Chemical, St. Louis, MO),
NG-monomethyl-L-arginine
[L-NMMA; Research Biochemicals International (RBI),
Natick, MA],
L-N5-(1-iminomethyl)ornithine
hydrochloride (L-NIO; RBI), or aminoguanidine (RBI), were
included in some incubations with alveolar macrophages. The final
concentration of each inhibitor was 1 mM. In some experiments, various
amounts of L-arginine were included in the incubation medium. Five different compounds known to affect NOS in other cell
types, acetyl-
-methylcholine chloride (methacholine), bradykinin (acetate salt), histamine (diphosphate salt), ATP, or ADP (all from
Sigma), were included in some incubations with alveolar macrophages. The final concentration of all compounds was 0.1 mM. In other experiments, the effects of the calcium ionophore 4-bromo-A-23187 (Calbiochem, La Jolla, CA) on cellular · NO production were
determined. A-23187 was dissolved in dimethyl sulfoxide (DMSO) and
added to the incubation mixture so that the final concentration was 1 µM. The amount of DMSO in the final incubation mixture was only 1 µl/ml, a level that has no adverse effects.
In some experiments, alveolar macrophages were incubated with DPPC
vesicles or purified lung surfactant. The DPPC vesicles were formed as
described previously (16). Briefly, DPPC (Sigma) was dissolved in
ethanol. Liposomes were formed by injecting the dissolved lipids into
phosphate-buffered medium warmed to 48°C. Then the dispersion was
sonicated to form vesicles, and the vesicles were added to the alveolar
macrophage incubation mixtures. The amount of ethanol used has no
effect on alveolar macrophage function. Pulmonary surfactant was
obtained according to a modification (24) of the method of King and
Clements (15). Briefly, a concentrated form of cell-free alveolar
lavage materials, which was obtained as we described previously (24),
was spun at 100,000 g for 2 h. The
resultant pellet was resuspended in phosphate-buffered medium and
applied to a linear sodium bromide density gradient. The gradient was
then spun at 81,500 g for 15 h in a
swinging bucket rotor. The band containing the surfactant was removed
and spun at 66,000 g for 1 h. This
pellet was washed and resuspended in phosphate-buffered medium for use
as the surfactant preparation in these experiments. The surfactant
disaturated phosphatidylcholines (DSPC) were isolated according to the
method of Mason et al. (22) and were measured as DSPC phosphorus (3).
There was no nitrate and nitrite in the DPPC vesicle or surfactant
preparations. Finally, experiments were performed to determine whether
or not the substances that were used in an attempt to alter
· NO production interfered with the assay. None of the
substances that we used in this study had any effect on any aspect of
the assay for nitrate and nitrite.
Detection of NOS protein. To determine
if NOS protein could be detected in alveolar macrophages, Western blot
analysis was used. The cells were first sonicated to disrupt cell
membranes. Sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis was performed on 100-µg aliquots of cell sonicate
protein with 7.5% (wt/vol) polyacrylamide gels. Proteins were
transferred to nitrocellulose paper by using an electrophoretic
transfer unit (Hoefer Scientific Instruments, San Francisco, CA). The
blots were then blocked for 1 h at room temperature in a medium
(blocking buffer) containing 50 mM tris(hydroxymethyl)aminomethane
(Tris) · HCl, 150 mM NaCl, 2% (vol/vol) BSA, and
0.1% (vol/vol) Tween 20 (pH 7.4). These blots were then incubated for
an additional hour at room temperature in blocking buffer containing
anti-NOS antibody. Two primary antibodies against two forms of cNOS,
i.e., mouse [immunoglobulin (Ig)
G1] monoclonal
anti-endothelial NOS (eNOS) and mouse
(IgG2a) monoclonal anti-brain
NOS (bNOS), and one primary antibody against iNOS, i.e., mouse
macrophage (IgG2a) monoclonal
anti-iNOS, were used. All antibodies were obtained from Transduction
Laboratories in Lexington, KY. These antibodies were diluted 1:500 in
blocking buffer. After incubation with the primary antibodies, the
blots were washed six times (5 min/wash) at room temperature with
medium containing 50 mM Tris · HCl, 150 mM NaCl, and
0.1% Tween 20 (TBS-T; pH 7.4). Then the blots were incubated for 1 h
at room temperature in blocking buffer containing the secondary
antibody, anti-mouse IgG coupled to horseradish peroxidase (Amersham
Life Sciences; Cleveland, OH). After incubation with the secondary
antibody, the blots were washed six times (5 min/wash) at room
temperature with TBS-T. Protein bands detected by the antibodies were
visualized by enhanced chemiluminescence (Amersham). The standards that
were carried through the entire procedure were human endothelial cell
lysate (for eNOS), rat pituitary cell lysate (for bNOS), and macrophage
lysate prepared from RAW 264.7 cells that had been stimulated with
IFN-
and LPS (for iNOS). All standards were obtained from
Transduction Laboratories.
Measurement of alveolar macrophage ATP and lactate
levels. The effects of L-NAME,
L-arginine, and DPPC vesicles on alveolar macrophage ATP
levels were determined. ATP content was measured with the firefly
luciferase assay adapted from the method of Wulff and Doppen (32).
Cells (1 × 106/ml) were
incubated (37°C) for either 2 or 4 h in the absence (control) or
presence of L-NAME (1 mM), L-arginine (100 µM), or DPPC vesicles (150 µg/ml). After incubation, the cells were
spun at 1,000 g for 10 min, and the
medium was removed by aspiration. The cells were washed one time and
then resuspended so that there were 1 × 106 cells in 0.125 ml of 0.5 M
Tris-acetate (pH 7.4). Triton X-100 (0.125 ml; 1:200 in Tris-acetate)
was added to disrupt cell membranes. The samples were mixed by
vortexing for 10 s and then were analyzed immediately for ATP content.
The ATP concentration was determined by measuring the emission of light
when 0.05 ml of the sample was mixed with 0.05 ml of firefly lantern
extract (Sigma) in 0.4 ml of Tris-acetate. Light emission was recorded
with a Lumiaggregometer (model 400; Chrono-Log, Havertown, PA).
Cellular ATP content was calculated from a curve of ATP standards
(Sigma) and is expressed as nanomoles per
106 cells.
The metabolism of glucose via glycolysis was estimated by measuring
alveolar macrophage levels of lactate, the major end product of
glycolysis. Alveolar macrophages (4 × 106/ml) were incubated (37°C)
for 4 h in the absence (control) or presence of L-NAME (1 mM) or L-arginine (100 µM). After incubation, an aliquot
(0.3 ml) of the cell suspension was added to perchloric acid (final
concentration = 3%) to precipitate cellular protein. The samples were
thoroughly mixed and centrifuged in an Eppendorf microcentrifuge at
12,800 g for 2 min. Lactate
concentrations in the perchloric acid extracts were measured
spectrophotometrically at 340 nm with enzymatic methods (12). Results
are expressed as nanomoles per 106
cells.
Measurement of degradation of DPPC vesicles by
alveolar macrophages. The degradation of DPPC vesicles
by alveolar macrophages was measured in the absence or presence of
L-NAME as we described previously (25). Briefly, alveolar
macrophages (2 × 106/ml)
were incubated (37°C) in phosphate-buffered medium containing 1.8 mM CaCl2, 1.0 mM
MgCl2, and 0.5% BSA. DPPC
vesicles were formed as described elsewhere in this manuscript and were
added to the incubation mixture so that the final concentration was 50 µg/ml. L-3-Phosphatidyl-[N-methyl-3H]choline,1,2-dipalmitoyl
(specific activity = 80 Ci/mmol; Amersham Life Science, Arlington
Heights, IL) was also included with DPPC at a final concentration of
0.2 µCi/ml. Some samples were incubated in the absence (control) and
other samples were incubated in the presence of 1 mM
L-NAME. Immediately before and after a 2-h incubation period, DPPC was extracted from the samples (22), and labeled DPPC was
determined by counting in a liquid scintillation counter. The amount of
labeled DPPC that was degraded during the incubation period is
expressed as a percentage of that present before the incubation. The
level of · NO in the samples was also measured as described
elsewhere in this manuscript.
Statistical analyses. All comparisons
of statistical significance were made by comparing each treatment with
the control value (100%) using a one-sample Student's
t-test.
P < 0.05 was taken as the limit to
indicate significance.
 |
RESULTS |
Time course for · NO
production. There is some endogenous · NO in
unincubated, freshly isolated alveolar macrophages. The nitrate and
nitrite in supernatant from these cells, which had just been washed
three times by centrifugation and resuspension, is 0.013 ± 0.006 nmol/106 cells (Fig.
1). The time course for production of
additional · NO by the alveolar macrophages is shown in Fig.
1. · NO formation proceeds along an approximately linear time
course during the 4-h incubation period. The amount of · NO
produced in 4 h is 0.29 ± 0.02 nmol/106 cells. These results
demonstrate that unstimulated, naive alveolar macrophages do produce
· NO.

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Fig. 1.
Time course for production of nitric oxide by alveolar macrophages.
Cells (4 × 106/ml) were
incubated (37°C) in phosphate-buffered medium containing calcium
(1.8 mM), magnesium (1.0 mM), and bovine serum albumin (BSA; 0.5%) for
varying times up to 4 h. After the appropriate incubation period, cells
were centrifuged, and the supernatants were saved for analysis. Nitrate
and nitrite were measured as described in METHODS. Points
are mean values for 6 experiments, and bars represent SE.
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Immunochemical detection of NOS
protein. Experiments were performed in an attempt to
identify which type(s) of NOS may be present in naive alveolar
macrophages. Western blot analysis was used. Antibodies against two
known types of cNOS, i.e., eNOS and bNOS, and an antibody against iNOS
were used. The only positive result was obtained with the anti-eNOS
antibody and is shown in Fig. 2. This
result demonstrates the presence of an alveolar macrophage protein that
reacts with a monoclonal anti-eNOS protein (Fig. 2,
lane 3). The molecular mass of this
protein corresponds to that of an eNOS standard (Fig. 2,
lane 2). However, no bNOS or iNOS
could be detected with the monoclonal anti-bNOS or anti-iNOS antibodies, respectively. Furthermore, no bNOS could be detected when
another antibody, a rabbit polyclonal anti-bNOS antibody (Transduction
Laboratories), was used. The results of these experiments show that
there is an endothelial type of cNOS present in unstimulated, naive
alveolar macrophages.

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Fig. 2.
Western blot analysis of alveolar macrophage proteins with
anti-endothelial nitric oxide synthase (eNOS) antibody. Cells were
sonicated to disrupt cell membranes. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was used to fractionate
100-µg aliquots of cell sonicate protein. Proteins were then
transferred to a nitrocellulose membrane and immunodetected with a
monoclonal anti-eNOS antibody (lane
3). A standard preparation of eNOS from human
endothelial cell lysate was used as a positive control and was carried
through the entire procedure (lane
2). Molecular-mass markers are shown in
lane 1. Unlabeled arrow at
right shows location of the eNOS at a
molecular mass of ~140 kDa. Blot is representative of results
obtained from 6 different alveolar macrophage preparations.
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Effects of inhibitors, calcium, magnesium, and other
compounds that affect eNOS. Because eNOS activity has
not been previously reported in alveolar macrophages, experiments were
carried out to characterize cellular · NO production. The
relationship between extracellular L-arginine, the
substrate for NOS, and · NO formation is shown in Fig.
3. As the external concentration of
L-arginine is increased from 0 to 100 µM, there is a
progressive increase in · NO production. In fact, at 50 and
100 µM L-arginine, the formation of · NO is
increased by ~50 and 75%, respectively. On the other hand, addition
of D-arginine has no effect on formation of · NO;
i.e., in the presence of D-arginine (100 µM),
· NO production is 94 ± 7% of control
(n = 5). The effects of four
different NOS inhibitors on alveolar macrophage · NO
production are shown in Table 1. Incubation
of the cells with L-NAME leads to a 32% inhibition of
· NO formation. The L-NAME-induced inhibition of
· NO production can be reversed by including
L-arginine in the incubation medium; i.e., in the presence
of L-NAME and L-arginine (10 mM),
· NO formation is 123 ± 11% of control
(n = 5). However, none of the other
inhibitors, L-NMMA, L-NIO, or aminoguanidine,
has any effect on the formation of · NO. The results of these
experiments show that · NO production by alveolar macrophages
is dependent on extracellular L-arginine and can be
inhibited, at least partially, by L-NAME.

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Fig. 3.
Effects of extracellular L-arginine on nitric oxide
production by alveolar macrophages. Cells (4 × 106/ml) were incubated (37°C)
in phosphate-buffered medium containing calcium (1.8 mM), magnesium
(1.0 mM), and BSA (0.5%) for 4 h in the absence (control) and presence
of various concentrations of L-arginine. After the
incubation period, cells were centrifuged, and the supernatants were
saved for analysis. Nitrate and nitrite were measured as described in
METHODS. Nitrate and nitrite production by control cells
was 0.25 ± 0.05 nmol/106
cells. Values are means ± SE for 6 experiments. * Values are
significantly different from no extracellular L-arginine
(P < 0.05).
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It is known that cNOS activity is dependent on calcium (10). Therefore,
the effects of altering extracellular calcium levels on alveolar
macrophage · NO production were determined. At the same time,
effects of extracellular magnesium were also studied, and the results
are shown in Table 2. Removal of both
calcium and magnesium from the incubation medium or removal of
magnesium alone has no effect on cellular · NO formation.
However, if extracellular calcium is removed and magnesium is present
in the medium, there is a 58% increase in alveolar macrophage
· NO production. To increase intracellular calcium levels,
the cells were exposed to the calcium ionophore A-23187. Incubation of
alveolar macrophages with A-23187 leads to a 50%
increase in · NO formation (Table
3). Inclusion of L-NAME in the
incubation medium blocks the A-23187-induced increase in · NO
formation; i.e., in the presence of A-23187 and L-NAME (1 mM), · NO production is 98 ± 9% of control
(n = 5). These results demonstrate
that alveolar macrophage · NO production is enhanced when the
medium contains magnesium and no calcium and when the internal calcium
concentration is increased by using a calcium ionophore.
In other cell types, a variety of compounds such as methacholine,
bradykinin, histamine, ATP, and ADP can stimulate release of
· NO from eNOS (9, 26). Therefore, we determined the effects of these compounds on · NO formation by alveolar macrophages, and the results are shown in Table 4.
Methacholine is the only compound we tested that stimulates
· NO production; i.e., it produces a twofold increase. The
methacholine-induced increase in · NO is blocked by
L-NAME; i.e., in the presence of methacholine and L-NAME (1 mM), · NO production is 108 ± 9%
of control (n = 5). Incubation of the
cells with three other compounds leads to inhibition of · NO
formation. ATP produces a 45% inhibition, whereas ADP and histamine
cause a 20% inhibition. Bradykinin has no effect. The results of these
experiments demonstrate that, with the exception of methacholine, these
compounds have different effects on · NO production in
alveolar macrophages and endothelial cells.
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Table 4.
Effects of methacholine, ATP, ADP, histamine, or bradykinin on
nitric oxide production by alveolar macrophages
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Alveolar macrophage ATP and lactate
contents. It has been shown by other investigators that
there is an increase in the rate of glycolysis when · NO
levels are elevated in rat peritoneal macrophages (1, 23). Therefore,
we performed experiments to determine if alveolar macrophage ATP and
lactate (the major end product of glycolysis) levels are affected when
the amounts of cell · NO are altered by using
L-NAME or L-arginine. The results are shown in
Table 5. Incubation of alveolar macrophages
with L-NAME or L-arginine leads to levels of
· NO production that are 68 and 145% of control,
respectively. However, there are no effects of either treatment on cell
ATP or lactate content. These results suggest that endogenous alveolar
macrophage · NO levels do not directly affect cellular energy
metabolism.
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Table 5.
Effects of L-NAME or L-arginine on alveolar
macrophage nitric oxide production and cell ATP and lactate content
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Effects of DPPC vesicles and lung surfactant on
· NO production. We have shown previously
that incubation of rat alveolar type II cells with DPPC vesicles leads
to an increase in · NO production from an eNOS (24). As a
result of this effect, there is also an increase in type II cell ATP
levels. Therefore, experiments were performed to determine the effects
of DPPC vesicles on alveolar macrophage · NO formation.
Incubation of the cells with DPPC vesicles (150 µg/ml, a
concentration that produces a maximal effect) leads to a time-dependent
increase in · NO production that is maximal after 2 h (Fig.
4). The concentration dependence for this
effect is shown in Fig. 5. · NO
production increases with the concentration of DPPC vesicles up to a
level of 150 µg/ml, where the effect is maximal. The DPPC-induced
production of · NO by alveolar macrophages is completely
blocked by L-NAME (Table 6).
There is no effect of DPPC on alveolar macrophage ATP levels as there
is in type II cells. Incubation of alveolar macrophages with DPPC (150 µg/ml) for 2 h leads to · NO levels that are 270 ± 40%
of control and ATP levels that are 108 ± 8% of control. Thus these
results demonstrate that incubation of alveolar macrophages with DPPC
vesicles leads to a time- and concentration-dependent increase in
cellular · NO formation.

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Fig. 4.
Time course for dipalmitoyl phosphatidylcholine (DPPC)-induced increase
in nitric oxide production by alveolar macrophages. Cells (4 × 106/ml) were incubated (37°C)
in phosphate-buffered medium containing calcium (1.8 mM), magnesium
(1.0 mM), and BSA (0.5%) for varying times up to 4 h in the absence
and presence of DPPC vesicles (150 µg/ml). After the appropriate
incubation period, cells were centrifuged, and the nitrate and nitrite
in the supernatants was measured as described in METHODS.
Points were obtained by subtracting nitric oxide production by control
cells from that by DPPC-exposed cells at each time point. Values are
means ± SE for 5 different experiments.
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Fig. 5.
Effects of different concentrations of DPPC vesicles on nitric oxide
production by alveolar macrophages. Cells (4 × 106/ml) were incubated (37°C)
in phosphate-buffered medium containing calcium (1.8 mM), magnesium
(1.0 mM), and BSA (0.5%) for 4 h in the absence (control) and presence
of various concentrations of DPPC vesicles. After the incubation
period, cells were centrifuged, and nitrate + nitrite in the
supernatants was measured as described in METHODS. Nitrate + nitrite production by control cells was 0.27 ± 0.03 nmol/106 cells. Values are means ± SE for 6 experiments.
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In our previous study (24), we determined that the effects of DPPC
vesicles were the same as the effects of lung surfactant on alveolar
type II cell · NO levels. Therefore, experiments were performed to compare the effects of lung surfactant and DPPC vesicles on alveolar macrophage · NO production. The results are shown in Table 7. During a 2-h incubation with
lung surfactant (150 µg DSPC/ml) or DPPC vesicles (150 µg/ml),
· NO levels are elevated by 326 and 257%, respectively. Thus
lung surfactant and DPPC vesicles have similar effects on alveolar
macrophage · NO formation.
Effect of L-NAME on DPPC degradation by
alveolar macrophages. We have shown previously that rat
alveolar macrophages degrade DPPC vesicles (and lung surfactant DSPC)
in vitro (25). Therefore, experiments were performed to determine if
· NO levels in alveolar macrophages affect the breakdown of
DPPC by these cells. The results are shown in Table
8. When alveolar macrophages are incubated with 50 µg DPPC/ml, an amount that we used previously (25), 23% of
the lipid is degraded in 4 h. This amount of degradation is similar to
that reported in our previous paper (25). However, the amount of DPPC
degraded is not affected by L-NAME. In these experiments,
· NO production over the 4-h incubation period was increased
by 200 ± 10% in the presence of DPPC and was not increased in the
presence of DPPC and L-NAME; i.e., in the presence of DPPC + L-NAME, · NO formation was 100 ± 6% of
control (n = 8). These results
demonstrate that, although DPPC-induced · NO production is
inhibited by L-NAME, there is no effect of the
inhibitor on DPPC breakdown. Therefore, alveolar macrophage
· NO levels do not appear to be related to DPPC degradation
by the cells.
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DISCUSSION |
The results of our experiments demonstrate that naive unstimulated rat
alveolar macrophages do produce · NO and that there is a
cNOS, which is an eNOS, present in the cells. In addition, we have
characterized the eNOS in terms of factors known to influence it in
other cell types. For example, alveolar macrophage · NO production is dependent on extracellular L-arginine. Only
one of the four NOS inhibitors tested inhibits · NO
formation. L-NAME inhibits cellular · NO
production by ~30%, but L-NMMA, L-NIO, and
aminoguanidine have no effect. NOS activity can also be influenced by
calcium and magnesium. When the incubation medium contains magnesium
and no calcium, · NO formation is ~60% greater than with
all other extracellular combinations of calcium and/or
magnesium. Furthermore, if the intracellular calcium level is increased
by using a calcium ionophore, · NO production is increased by
50%. Five compounds known to stimulate release of · NO from
eNOS in other cell types (9, 26) were studied. Only methacholine stimulates · NO production by alveolar macrophages. ATP, ADP, and histamine inhibit · NO formation, and bradykinin has no
effect.
It is interesting that the amounts of · NO produced by the
alveolar macrophage eNOS and many of its characteristics discussed above are different from those reported for eNOS activity in rat alveolar type II cells and bovine aortic endothelial cells. For example, the amount of · NO produced by alveolar macrophages
is less than that produced by other cell types. If the maximum rate of
· NO production, i.e., during the first hour of incubation, is calculated on the basis of alveolar macrophage protein for comparison with the other cell types, the value is ~12
pmol · mg
protein
1 · min
1.
This rate is less than that reported for alveolar type II cells (24) or
endothelial cells (5), which are 50 and 37 pmol · mg
protein
1 · min
1,
respectively. On a per cell basis, type II cells produce approximately five to six times more · NO during a 4-h incubation period
than do alveolar macrophages. There are also differences in the
dependence of · NO formation on extracellular
L-arginine. The eNOS activity in type II cells is
independent of extracellular L-arginine (19). The partially
purified eNOS from bovine aortic endothelial cells has been reported to
have a low requirement for L-arginine (Michaelis constant = 2.9 µM; see Ref. 31), although agonist-stimulated · NO
production by endothelial cells appears to require a small amount (1 µM) of extracellular L-arginine for maximal activity (5).
Therefore, it appears that endogenous levels of the substrate are
sufficient to support at least basal NOS activity in type II and
endothelial cells. However, alveolar macrophage · NO
production can be stimulated by extracellular L-arginine
(10-100 µM), possibly indicating some difference in the
substrate concentration dependence of the eNOS in these cells.
There are also differences in the response of these three cell types to
alterations in extracellular calcium levels. · NO production
by both alveolar type II cells (24) and bovine aortic endothelial cells
(5) is sensitive to extracellular calcium. Removal of calcium from the
incubation medium leads to reductions in the formation of
· NO by 50 and 35% in endothelial and type II cells,
respectively. Changes in extracellular magnesium have no effect in type
II cells and have not been studied, as far as we know, in endothelial
cells. However, alveolar macrophages respond differently.
· NO production is not altered in these cells if both calcium
and magnesium are removed from the incubation medium, but it is
increased by 58% if calcium is removed and magnesium remains in the
medium. The reason(s) for these responses of alveolar macrophages to
changes in extracellular calcium and magnesium is not known. It may be
that alveolar macrophages are much less permeable to calcium than are
endothelial or type II cells or that magnesium somehow alters internal
calcium stores in alveolar macrophages. The important thing, however,
is that all three cell types respond to an increase in intracellular
calcium in the same manner; i.e., · NO formation is increased
in alveolar macrophages, type II cells, and endothelial cells in the
presence of the calcium ionophore A-23187. This demonstrates the
calcium dependence of · NO production.
Alveolar macrophages also respond in a different manner to NOS
inhibitors and to compounds known to stimulate eNOS in other cell
types. Of the four NOS inhibitors we tested, only L-NAME inhibits · NO production and only by ~30%.
L-NAME has about the same effect in type II cells (24).
However, in type II cells, L-NMMA and L-NIO
demonstrate the most potent effects, inhibiting by 40-50%.
L-NMMA is also a good inhibitor in endothelial cells (5).
Thus L-NMMA inhibits · NO formation in type II
and endothelial cells but has no effect in alveolar macrophages. Of the
five compounds known to stimulate · NO production by eNOS (9,
26), only methacholine enhances NOS activity in alveolar macrophages.
Three other compounds, ATP, ADP, and histamine, actually inhibit
· NO formation in alveolar macrophages. Although the reasons
for the different responses of alveolar macrophages to these inhibitors and other compounds are not known, it may be that there are different eNOS isoforms in the various cell types.
The role that the eNOS plays in alveolar macrophage function is not
known. One possibility is that eNOS activity helps to regulate cellular
metabolism. For example, it is known that, when endogenous levels of
· NO are increased in alveolar type II cells, there is an
increase in the rate of cellular ATP synthesis and, therefore, an
increase in cell ATP levels (24). Also, there are at least two reports
of an increase in the rate of glycolysis when · NO levels are
elevated in rat peritoneal macrophages (1, 23). However, we were not
able to affect alveolar macrophage ATP or lactate levels by either
decreasing cellular · NO production with
L-NAME or increasing · NO formation
with L-arginine. In addition, a DPPC-induced increase in
· NO production of almost threefold does not affect cellular
ATP levels. Thus it appears that the eNOS in alveolar macrophages does
not affect cellular metabolism as it does in alveolar type II cells.
One of the most interesting findings from our study is the increase in
alveolar macrophage · NO production that occurs during incubation of the cells with DPPC vesicles or lung surfactant. Exposure
of the cells to either lung surfactant of DPPC vesicles leads to an
approximately threefold increase in · NO formation. Although
we are assuming that the surfactant-induced increase in · NO
levels is due to an increase in its formation, it is also possible that
there may be a reduction in · NO degradation. The DPPC-induced response is both time and concentration dependent and is
completely inhibited by L-NAME. We reported a similar
finding from our experiments with alveolar type II cells, i.e.,
exposure of these cells to DPPC or lung surfactant leads to increased
production of · NO (24). Because there is also an increase in
type II cell ATP synthesis, as well as the increase in · NO
formation, we postulated that the elevation in · NO levels in
type II cells may be related to surfactant metabolism, e.g., recycling
of surfactant. It is known that rat alveolar macrophages are capable of
degrading surfactant DSPC or DPPC vesicles in vitro (25). Therefore, it
is possible that the DPPC-induced increase in alveolar macrophage
· NO formation may be related to the degradation of DPPC.
However, we were able to inhibit the DPPC-induced · NO
formation with L-NAME, but there was no effect on DPPC
degradation. Thus it appears that there is no relationship between
· NO production and the degradation of lipids by alveolar
macrophages.
Although the physiological role of the DPPC/lung surfactant-induced
increase in alveolar macrophage · NO levels is not known, it
does not appear to be related to surfactant metabolism. Our results do
suggest that the endogenous · NO level of alveolar macrophages in the lungs is maintained at a higher level because the
cells are continually exposed to surfactant. It is known that · NO has some beneficial anti-inflammatory effects. For
example, · NO functions as an anti-inflammatory agent by
suppressing adhesion and/or activation of neutrophils (18).
Other anti-inflammatory effects of · NO include inhibition of
platelet aggregation (2), inhibition of cytokine-induced expression of
adhesion molecules (6), and inhibition of neutrophil respiratory burst
due to inhibition of NADPH oxidase (8). Therefore, one very important physiological role for the higher alveolar macrophage · NO
levels in the presence of surfactant (i.e., in vivo) may be the
maintenance of an anti-inflammatory state. Experiments are currently
being done to test this hypothesis.
In summary, naive unstimulated alveolar macrophages do produce
· NO. The NOS responsible appears to be an eNOS. The amount of · NO formed is much less than that produced by eNOS in
other cells, i.e., alveolar type II cells and endothelial cells. Some properties of the alveolar macrophage eNOS are similar to and some are
different from the eNOS in these other cell types. Alveolar macrophage
· NO levels do not seem to be related to cellular metabolism. · NO production is increased approximately threefold in the
presence of DPPC vesicles or lung surfactant. However, this increase in · NO formation is not related to surfactant or cellular
energy metabolism. It is possible that the DPPC/lung surfactant-induced increase in alveolar macrophage constitutive · NO levels is
important in maintaining an elevated anti-inflammatory state in vivo.
 |
FOOTNOTES |
Address for reprint requests: P. R. Miles, NIOSH, HELD/M/S 2015, 1095 Willowdale Rd., Morgantown, WV 26505.
Received 28 July 1997; accepted in final form 16 December 1997.
 |
REFERENCES |
1.
Albina, J. E.,
and
B. Mastrofrancesco.
Modulation of glucose metabolism in macrophages by products of nitric oxide synthase.
Am. J. Physiol.
264 (Cell Physiol. 33):
C1594-C1599,
1993[Abstract/Free Full Text].
2.
Alheid, U.,
J. C. Frolich,
and
U. Forstermann.
Endothelium-derived relaxing factor from cultured human endothelial cells inhibits aggregation of human platelets.
Thromb. Res.
47:
561-571,
1987[Medline].
3.
Bartlett, G. R.
Phosphorus assay in column chromatography.
J. Biol. Chem.
234:
466-468,
1959[Free Full Text].
4.
Belvisi, M. G.,
C. C. Stretton,
M. Yacoub,
and
P. J. Barnes.
Nitric oxide is the endogenous neurotransmitter of bronchodilator nerves in human airways and guinea-pig airways.
Eur. J. Pharmacol.
210:
221-222,
1992[Medline].
5.
Buckley, B. J.,
Z. Mirza,
and
A. R. Whorton.
Regulation of Ca2+-dependent nitric oxide synthase in bovine aortic endothelial cells.
Am. J. Physiol.
269 (Cell Physiol. 38):
C757-C765,
1995[Abstract].
6.
De Caterina, R.,
P. Libby,
H. B. Peng,
V. J. Thannickal,
T. B. Rajavashisth,
M. A. Gimbrone,
W. S. Shin,
and
J. K. Liao.
Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines.
J. Clin. Invest.
96:
60-68,
1995[Medline].
7.
Dinh-Xuan, A. T.,
T. W. Higenbottam,
and
A. C. Clelland.
Impairment of endothelium-dependent pulmonary artery relaxation in chronic obstructive lung disease.
N. Engl. J. Med.
324:
1539-1547,
1991[Abstract].
8.
Forslund, T.,
and
T. Sundqvist.
Nitric oxide reduces hydrogen peroxide production from human polymorphonuclear neutrophils.
Eur. J. Clin. Invest.
25:
9-14,
1995[Medline].
9.
Forstermann, U.,
J. S. Pollock,
and
M. Nakane.
Nitric oxide synthases in the cardiovascular system.
Trends Cardiovasc. Med.
3:
104-110,
1993.
10.
Gaston, B.,
J. M. Drazen,
J. Loscalzo,
and
J. S. Stamler.
The biology of nitrogen oxides in the airways.
Am. J. Respir. Crit. Care Med.
149:
538-551,
1994[Abstract].
11.
Green, L.,
D. Wagner,
J. Glogowski,
P. Skipper,
J. Wishnok,
and
S. Tannenbaum.
Analysis of nitrate, nitrite, and [14N]nitrite in biological fluids.
Anal. Biochem.
126:
131-138,
1982[Medline].
12.
Gutmann, I.,
and
A. W. Wahlefeld.
L-(+)-Lactate. Determination with lactate dehydrogenase and NAD.
In: Methods of Enzymatic Analysis (2nd ed.), edited by H. U. Bergmeyer. New York: Academic, 1974, vol. 3, p. 1464-1468.
13.
Ischiropoulos, H.,
L. Zhu,
and
J. S. Beckman.
Peroxynitrite formation from macrophage-derived nitric oxide.
Arch. Biochem. Biophys.
298:
446-451,
1992[Medline].
14.
Jorens, P. G.,
F. J. Van Overfield,
P. A. Vermiere,
H. Bult,
and
A. G. Herman.
Synergism between interleukin 1-
and the nitric oxide synthase inducer interferon-
in rat lung fibroblasts.
Eur. J. Pharmacol.
224:
7-12,
1992[Medline].
15.
King, R. J.,
and
J. A. Clements.
Surface active materials from dog lung. I. Method of isolation.
Am. J. Physiol.
223:
707-714,
1972[Medline].
16.
King, R. J.,
and
M. C. Macbeth.
Interaction of lipid and protein components of pulmonary surfactant. Role of phosphatidylglycerol and calcium.
Biochim. Biophys. Acta
647:
159-168,
1981[Medline].
17.
Kobzik, L.,
D. S. Bredt,
C. J. Lowenstein,
J. Drazen,
B. Gaston,
D. Sugarbaker,
and
J. S. Stamler.
Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization.
Am. J. Respir. Cell Mol. Biol.
9:
371-377,
1993[Medline].
18.
Kubes, P.,
M. Suzuki,
and
D. M. Granger.
Nitric oxide: an endogenous modulator of leukocyte adhesion.
Proc. Natl. Acad. Sci. USA
88:
4651-4655,
1991[Abstract].
19.
Lavnikova, N.,
J. C. Drapier,
and
D. L. Laskin.
A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity.
J. Leukoc. Biol.
54:
322-328,
1993[Abstract].
20.
Lorsbach, R. B.,
W. J. Murphy,
C. J. Lowenstein,
S. H. Snyder,
and
S. W. Russell.
Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing.
J. Biol. Chem.
268:
1908-1913,
1993[Abstract/Free Full Text].
21.
Lyons, C. R.,
G. J. Orloff,
and
J. M. Cunningham.
Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.
J. Biol. Chem.
267:
6370-6374,
1992[Abstract/Free Full Text].
22.
Mason, R. J.,
J. Nellenbogen,
and
J. A. Clements.
Isolation of disaturated phosphatidylcholines with osmium tetroxide.
J. Lipid Res.
17:
281-284,
1976[Abstract].
23.
Mateo, R. B.,
J. S. Reichner,
B. Mastrofrancesco,
D. Kraft-Stolar,
and
J. E. Albina.
Impact of nitric oxide in macrophage glucose metabolism and glyceraldehyde-3-phosphate dehydrogenase activity.
Am. J. Physiol.
268 (Cell Physiol. 37):
C669-C675,
1995[Abstract/Free Full Text].
24.
Miles, P. R.,
L. Bowman,
A Rengasamy,
and
L. Huffman.
Alveolar type II cell cNOS activity and ATP levels are increased by lung surfactant or DPPC vesicles.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L339-L346,
1997[Abstract/Free Full Text].
25.
Miles, P. R.,
J. Y. C. Ma,
and
L. Bowman.
Degradation of pulmonary surfactant disaturated phosphatidylcholines by alveolar macrophages.
J. Appl. Physiol.
64:
2474-2481,
1988[Abstract/Free Full Text].
26.
Moncada, S.,
R. M. J. Palmer,
and
E. A. Higgs.
NO: physiology, pathophysiology, and pharmacology.
Pharmacol. Rev.
43:
109-141,
1991[Medline].
27.
Myrvik, Q. N.,
E. S. Leake,
and
B. Fariss.
Lysozyme content of alveolar and peritoneal macrophages from the rabbit.
J. Immunol.
86:
133-136,
1961.
28.
Nakayama, D. K.,
D. A. Geller,
C. J. Lowenstein,
P. Davies,
B. R. Pitt,
R. L. Simmons,
and
T. R. Billiar.
Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat pulmonary artery smooth muscle.
Am. J. Respir. Cell Mol. Biol.
7:
471-476,
1992[Medline].
29.
Pendino, K. J.,
J. D. Laskin,
R. L. Shuler,
C J. Punjabi,
and
D. L. Laskin.
Enhanced production of nitric oxide by rat alveolar macrophages following inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase.
J. Immunol.
151:
7196-7205,
1993[Abstract/Free Full Text].
30.
Persoons, J. H. A.,
K. Schornagel,
J. Breve,
F. Berkenbasch,
and
G. Kraal.
Acute stress affects cytokines and nitric oxide production by alveolar macrophage differently.
Am. J. Respir. Crit. Care Med.
152:
619-624,
1995[Abstract].
31.
Pollock, J. S.,
U. Forstermann,
J. A. Mitchell,
T. D. Warner,
H. H. H. W. Schmidt,
M. Nakane,
and
F. Murad.
Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells.
Proc. Natl. Acad. Sci. USA
88:
10480-10484,
1991[Abstract].
32.
Wulff, K.,
and
W. Doppen.
Luminometric method.
In: Methods of Enzymatic Analysis (3rd ed.), edited by H. U. Bergmeyer. Deerfield Beach, FL: Verlag-Chemie, 1985, vol. VII, p. 357-364.
33.
Yui, Y.,
R. Hattori,
K. Kosuga,
H. Eizawa,
K. Hiki,
S. Ohkawa,
K. Ohnishi,
S. Terao,
and
C. Kawai.
Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils.
J. Biol. Chem.
266:
3369-3371,
1991[Abstract/Free Full Text].
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